Periodic bouncing of a plasmonic bubble in a binary liquid by competing solutal and thermal Marangoni forces

The physicochemical hydrodynamics of bubbles and droplets out of equilibrium, in particular with phase transitions, display surprisingly rich and often counterintuitive phenomena. Here we experimentally and theoretically study the nucleation and early evolution of plasmonic bubbles in a binary liquid consisting of water and ethanol. Remarkably, the submillimeter plasmonic bubble is found to be periodically attracted to and repelled from the nanoparticle-decorated substrate, with frequencies of around a few kilohertz. We identify the competition between solutal and thermal Marangoni forces as the origin of the periodic bouncing. The former arises due to the selective vaporization of ethanol at the substrate’s side of the bubble, leading to a solutal Marangoni flow toward the hot substrate, which pushes the bubble away. The latter arises due to the temperature gradient across the bubble, leading to a thermal Marangoni flow away from the substrate, which sucks the bubble toward it. We study the dependence of the frequency of the bouncing phenomenon from the control parameters of the system, namely the ethanol fraction and the laser power for the plasmonic heating. Our findings can be generalized to boiling and electrolytically or catalytically generated bubbles in multicomponent liquids.

Bubbles and bubble nucleation are ubiquitous in nature and technology, e.g., in boiling, electrolysis, and catalysis, where the phenomena connected with them have tremendous relevance for energy conversion, or in flotation, sonochemistry, cavitation, ultrasonic cleaning, and biomedical applications of ultrasound and bubbles. This also includes plasmonic bubbles, i.e., bubbles nucleating at liquid-immersed metal nanoparticles under laser irradiation, due to which an enormous amount of heat is produced because of a surface plasmon resonance (1–5). For an overview on the fundamentals of bubbles and their applications we refer to our recent review article (6). In general, in these applications the bubble nucleation does not occur in a pure liquid, but in multicomponent liquids. Because of that, various additional forces and effects come into play (7), which are not relevant in pure liquids. Examples are the Soret effect (8–10) or body forces arising due to density gradients. Once the multicomponent systems have interfaces, solutal Marangoni forces (11) become relevant. The phenomena become even richer once phase transitions occur in such systems, e.g., solidification (12, 13), evaporation (14–21), or dissolution of multicomponent droplets (22–25); or nucleation of a new phase such as in the so-called ouzo effect (26, 27); or in boiling (28), electrolysis (29, 30), or catalysis (31, 32). Similarly, also chemical reactions occurring at the interface in a multicomponent liquid lead to spectacular effects, such as swimming droplets (33, 34), phoretic self-propulsion (35–38), or pattern formation in electroconvection (39). The whole field could be summarized as physicochemical hydrodynamics, and although this is a classical subject (40), it received increasing attention in recent years due to its relevance for various applications, due to new experimental and numerical possibilities, and due to the beauty of the often surprising and counterintuitive phenomena. For recent reviews on physicochemical hydrodynamics, we refer to refs. 41 and 42.To exactly analyze the various competing forces playing a role in physicochemical hydrodynamical systems, one has to strive to have simple and clean geometries, allowing for precise measurements and a theoretical and numerical approach. For example, in refs. 43 and 44 we analyzed the competition between solutal Marangoni forces, gravity, and thermal diffusion by studying an oil droplet in a stably stratified liquid consisting of ethanol and water, imposing density and surface tension gradients on the droplet. Depending on the control parameters, the droplet was either stably levitating or jumping up and down, with a very low frequency of ∼0.02 Hz. Similar droplet and bubble oscillations originating from the competition between solutal Marangoni forces and gravity were observed in ref. 45.In this paper, we report and analyze another controlled physicochemical hydrodynamic bouncing phenomenon, even involving phase transitions, namely that of a nucleating plasmonic bubble (2, 4, 5), but now in an initially homogeneous binary liquid, for which the delay of bubble nucleation after turning on the laser depends on the composition of the binary liquid and the amount of dissolved gas (46) (next, of course, to the power of the employed laser). As in refs. 43 and 44, we will again see a bouncing behavior, but this time on a much faster timescale, corresponding to frequencies of ∼103 Hz. We will use this controlled physicochemical hydrodynamic system out of equilibrium to probe the competition between solutal and thermal Marangoni forces. That, in the presence of concentration gradients, the latter can compete with the former ones only is possible because of the very high-temperature gradients in the system of a nucleating plasmonic bubble. Under more standard conditions, such as for the evaporation of a binary droplet, the solutal Marangoni forces tend to be much stronger than the thermal ones (16).We note that plasmonic bubbles are in themselves very interesting with potential applications in biomedical diagnosis and therapy, micro- and nanomanipulation, and catalysis (1, 47–49). Also note that plasmonic bubbles directly after nucleation are pure vapor bubbles (50) originating from evaporation of the surrounding liquid, but during their expansion they are invaded by dissolved gas from the surrounding liquid (46, 51–53), which in the long term crucially determines their dynamics and lifetime.The key idea of this study here will build on the selective heating of the liquid surrounding the plasmonic bubble, namely on the side of the plasmonic nanoparticles. This leads to very strong temperature gradients across the bubble and thus to thermal Marangoni forces and at the same time to strong concentration gradients, as the evaporation of the surrounding binary liquid is selective, favoring the liquid with the lower boiling point. Thus, also solutal Marangoni forces along the bubble–liquid interface emerge. As we will see, which of these two different Marangoni forces is stronger depends on time and bubble position, leading to an oscillatory or bouncing bubble behavior.     

Sea spray aerosol concentration modulated by sea surface temperature

Natural aerosols in pristine regions form the baseline used to evaluate the impact of anthropogenic aerosols on climate. Sea spray aerosol (SSA) is a major component of natural aerosols. Despite its importance, the abundance of SSA is poorly constrained. It is generally accepted that wind-driven wave breaking is the principle governing SSA production. This mechanism alone, however, is insufficient to explain the variability of SSA concentration at given wind speed. The role of other parameters, such as sea surface temperature (SST), remains controversial. Here, we show that higher SST promotes SSA mass generation at a wide range of wind speed levels over the remote Pacific and Atlantic Oceans, in addition to demonstrating the wind-driven SSA production mechanism. The results are from a global scale dataset of airborne SSA measurements at 150 to 200 m above the ocean surface during the NASA Atmospheric Tomography Mission. Statistical analysis suggests that accounting for SST greatly enhances the predictability of the observed SSA concentration compared to using wind speed alone. Our results support implementing SST into SSA source functions in global models to better understand the atmospheric burdens of SSA.

Over two-thirds of the Earth is covered by the ocean. The material exchange between the ocean and the atmosphere affects the balance of the Earth’s energy on a global scale (1). Sea spray aerosol (SSA) is the major particulate material directly emitted from the ocean. Studies have shown that SSA dominates the aerosol mass in the marine boundary layer (MBL). Such dominance renders SSA an important player in climate change (2). However, the exact processes by which the SSA is introduced to the atmosphere still remains to be learned, making the SSA budget highly uncertain (3).It is generally established that SSA is produced by mechanical processes (4–6). Wind stress induces breaking waves that entrain bubbles into the surface ocean (7). Film and jet drops formed during bubble bursting are the main sources of SSA particles (8). The wind-driven mechanism is supported by the positive correlation between wind speed and SSA concentration from field observations (9, 10). Therefore, wind speed is used as the sole parameter to characterize SSA in many models (1, 4, 11).In addition to wind speed, sea surface temperature (SST) may play a large role in SSA production (12–15). SST affects the drop formation process by modifying the physical properties of the surface ocean water. An increase of SST reduces the kinematic viscosity and surface tension of the ocean, thereby enhancing the entrainment efficiency and rising speed of bubbles (12, 16). As a result, the number size distribution of the bubbles may change, leading to varying SSA properties (14, 15, 17).Limited laboratory and field studies regarding the effects of SST on SSA production have shown disparate results. Some argue that SSA production is independent of SST (18) or suppressed by increasing SST (14, 15, 19, 20) from 0 to 10 °C, while other laboratory (12, 21–23) and field measurements (3, 5) suggest that SSA production increases monotonically with water temperature. Furthermore, recent observations in the remote Atlantic Ocean shows that increasing SST enhances the modal mean diameter of SSA (24). On the other hand, model simulations have demonstrated that incorporating SST into SSA source functions generally improves the SSA prediction (3, 25, 26). The inconsistency in the previous work suggests that the impacts of SST on SSA formation remain unclear.In this study, we conducted unprecedented aircraft measurements of SSA concentration on a global scale during the Atmospheric Tomography Mission (ATom). These measurements consist of a series of flights spanning three seasons (summer, fall, and winter) over remote oceans (Fig. 1 and SI Appendix, Fig. S1). Our observations again confirm that wind speed is the dominant factor controlling the concentration of SSA. Further, we show that increasing SST enhances the mass concentration of SSA.Open in a separate windowFig. 1.Flight tracks during ATom2. The color indicates the flight altitude. The size of the markers represents the sea salt number fraction. The inset in the bottom right shows the vertical profile of sea salt number fraction. The flight tracks during ATom3 and ATom4 are similar to ATom2.     

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

In cardiomyocytes, Na_V1.5 channels mediate initiation and fast propagation of action potentials. The Ca²⁺-binding protein calmodulin (CaM) serves as a de facto subunit of Na_V1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the Na_V1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a “gain-of-function” defect, and Brugada syndrome, a “loss-of-function” phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on Na_V1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca²⁺-free CaM preassociation with Na_V1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent Na_V openings. Mechanistically, these effects arise from a CaM-dependent switch in the Na_V inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant Na_V1.5, local Ca²⁺ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of Na_V1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of Na_V1.5 channelopathies.

Voltage-gated sodium channels (Na_V) are responsible for the initiation and spatial propagation of action potentials (AP) in excitable cells (1, 2). Na_V channels undergo rapid activation that underlie the AP upstroke while ensuing inactivation permits AP repolarization. The Na_V1.5 channel constitutes the predominant isoform in cardiomyocytes, whose pore-forming α-subunit is encoded by the SCN5A gene. Na_V1.5 dysfunction underlies diverse forms of cardiac disease including cardiomyopathies, arrhythmias, and sudden death (3–6). Human mutations in Na_V1.5 are associated with two forms of inherited arrhythmias–congenital long QT syndrome 3 (LQTS3) and Brugada syndrome (BrS) (7). LQTS3 stems from delayed or incomplete inactivation of Na_V1.5 that causes persistent Na influx that prolongs AP repolarization—a “gain-of-function” phenotype (7–9). BrS predisposes patients to sudden death and is associated with a reduction in the peak Na current that may slow cardiac conduction or cause region-specific repolarization differences—a “loss-of-function” phenotype (10, 11). Genetic studies have identified an expanding array of mutations in multiple Na_V1.5 domains, including the channel carboxy-terminus (CT) that is a hotspot for mutations linked to both LQTS3 and BrS (12, 13). This domain interacts with the Ca²⁺-binding protein calmodulin (CaM), suggesting that altered CaM regulation of Na_V1.5 may be a common pathophysiological mechanism (12, 14–16). More broadly, human mutations in the homologous regions of neuronal Na_V1.1 (17, 18), Na_V1.2 (19, 20), and Na_V1.6 (21) as well as skeletal muscle Na_V1.4 (22) are linked to varied clinical phenotypes including epilepsy, autism spectrum disorder, neurodevelopmental delay, and myotonia (23). Taken together, a common Na_V mechanistic deficit—defective CaM regulation—may underlie these diverse diseases.CaM regulation of Na_V channels is complex, isoform specific, and mediated by multiple interfaces within the channel (14–16). The Na_V CT consists of a dual vestigial EF hand segment and a canonical CaM-binding “IQ” (isoleucine–glutamine) domain (24, 25) (Fig. 1A). The IQ domain of nearly all Na_V channels binds to both Ca²⁺-free CaM (apoCaM) and Ca²⁺/CaM, similar to Ca_V channels (26–31). As CaM is typically a Ca²⁺-dependent regulator, much attention has been focused on elucidating Ca²⁺-dependent changes in Na_V gating. For skeletal muscle Na_V1.4, transient elevation in cytosolic Ca²⁺ causes a dynamic reduction in the peak current, a process reminiscent of Ca²⁺/CaM-dependent inactivation of Ca_V channels (32). Cardiac Na_V1.5 by comparison exhibits no dynamic effect of Ca²⁺ on the peak current (32–34). Instead, sustained Ca²⁺ elevation has been shown to elicit a depolarizing shift in Na_V1.5 steady-state inactivation (SSI or h_∞), although the magnitude and the presence of a shift have been debated (32, 35). Additional CaM-binding sites have been identified in the channel amino terminus domain (36) and the III-IV linker near the isoleucine, phenylalanine, and methionine (IFM) motif that is well recognized for its role in fast inactivation (35, 37). However, recent cryogenic electron microscopy structures, biochemical, and functional analyses suggest that both the III-IV linker and the Domain IV voltage-sensing domain might instead interact with the channel CT in a state-dependent manner (38–43).Open in a separate windowFig. 1.Absence of dynamic Ca²⁺/CaM effects on WT Na_V1.5 SSI. (A, Left) Structure of Na_V1.5 transmembrane domain (6UZ3) (70) juxtaposed with that of Na_V1.5 CT–apoCaM complex (4OVN) (28). (Right) Arrhythmia-linked CT mutations highlighted in Na_V1.5 CT–apoCaM structure (LQTS3, blue; BrS, magenta; mixed syndrome, purple). (B) Dynamic Ca²⁺-dependent changes in Na_V1.5 SSI probed using Ca²⁺ photouncaging. Na currents specifying h_∞ at ∼100 nM (Left) and ∼4 μM Ca²⁺ step (Right). (C) Population data for Na_V1.5 SSI under low (black, Left) versus high (red, Right) intracellular Ca²⁺ reveal no differences (P = 0.55, paired t test). Dots and bars are mean ± SEM (n = 8 cells). (D) FRET two-hybrid analysis of Cerulean-tagged apoCaM interaction with various Venus-tagged Na_V1.5 CT (WT, black; IQ/AA, red; S[1904]L, blue). Each dot is FRET efficiency measured from a single cell. Solid line fits show 1:1 binding isotherm.Beyond Ca²⁺-dependent effects, the loss of apoCaM binding to the Na_V1.5 IQ domain increases persistent current (34, 44), suggesting that apoCaM itself may be pathophysiologically relevant. Indeed, Na_V1.5 mutations in the apoCaM-binding interface are associated with LQTS3 and atrial fibrillation (7), as well as a loss-of-function BrS phenotype and a mixed-syndrome phenotype whereby some patients present with BrS while others with LQTS3 (Fig. 1A) (13, 45). How alterations in CaM binding paradoxically elicits both gain-of-function and loss-of-function effects is not fully understood, though important to delineate pathophysiological mechanisms and for personalized therapies.Here, using single- and multichannel recordings, we show that apoCaM binding elicits two distinct effects on Na_V1.5 gating: 1) an increase in the peak channel open probability (P_O/peak) and 2) a reduction in the normalized persistent channel open probability (R_persist), consistent with previous studies (34, 44). The two effects may explain how mixed-syndrome mutations in the Na_V1.5 CT produce either BrS or LQTS3 phenotypes. On one hand, the loss of apoCaM association may diminish P_O/peak and induce BrS by shunting cardiac AP. On the other hand, increased R_persist may prevent normal AP repolarization, resulting in LQTS3. Analysis of elementary mechanisms suggests that these changes relate to a switch in the state dependence of channel inactivation. Furthermore, dynamic changes in Ca²⁺ can inhibit persistent current for certain mutant Na_V1.5 owing to enhanced Ca²⁺/CaM binding that occurs over the timescale of a cardiac AP. This effect may result in beat-to-beat variability in persistent Na current for some mutations. In all, these findings explain how a common deficit in CaM binding can contribute to distinct arrhythmogenic mechanisms.     

From the Cover: Evidence from South Africa for a protracted end-Permian extinction on land

Earth’s largest biotic crisis occurred during the Permo–Triassic Transition (PTT). On land, this event witnessed a turnover from synapsid- to archosauromorph-dominated assemblages and a restructuring of terrestrial ecosystems. However, understanding extinction patterns has been limited by a lack of high-precision fossil occurrence data to resolve events on submillion-year timescales. We analyzed a unique database of 588 fossil tetrapod specimens from South Africa’s Karoo Basin, spanning ∼4 My, and 13 stratigraphic bin intervals averaging 300,000 y each. Using sample-standardized methods, we characterized faunal assemblage dynamics during the PTT. High regional extinction rates occurred through a protracted interval of ∼1 Ma, initially co-occurring with low origination rates. This resulted in declining diversity up to the acme of extinction near the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary. Regional origination rates increased abruptly above this boundary, co-occurring with high extinction rates to drive rapid turnover and an assemblage of short-lived species symptomatic of ecosystem instability. The “disaster taxon” Lystrosaurus shows a long-term trend of increasing abundance initiated in the latest Permian. Lystrosaurus comprised 54% of all specimens by the onset of mass extinction and 70% in the extinction aftermath. This early Lystrosaurus abundance suggests its expansion was facilitated by environmental changes rather than by ecological opportunity following the extinctions of other species as commonly assumed for disaster taxa. Our findings conservatively place the Karoo extinction interval closer in time, but not coeval with, the more rapid marine event and reveal key differences between the PTT extinctions on land and in the oceans.

Mass extinctions are major perturbations of the biosphere resulting from a wide range of different causes including glaciations and sea level fall (1), large igneous provinces (2), and bolide impacts (3, 4). These events caused permanent changes to Earth’s ecosystems, altering the evolutionary trajectory of life (5). However, links between the broad causal factors of mass extinctions and the biological and ecological disturbances that lead to species extinctions have been difficult to characterize. This is because ecological disturbances unfold on timescales much shorter than the typical resolution of paleontological studies (6), particularly in the terrestrial record (6–8). Coarse-resolution studies have demonstrated key mass extinction phenomena including high extinction rates and lineage turnover (7, 9), changes in species richness (10), ecosystem instability (11), and the occurrence of disaster taxa (12). However, finer time resolutions are central to determining the association and relative timings of these effects, their potential causal factors, and their interrelationships. Achieving these goals represents a key advance in understanding the ecological mechanisms of mass extinctions.The end-Permian mass extinction (ca. 251.9 Ma) was Earth’s largest biotic crisis as measured by taxon last occurrences (13–15). Large outpourings from Siberian Trap volcanism (2) are the likely trigger of calamitous climatic changes, including a runaway greenhouse effect and ocean acidification, which had profound consequences for life on land and in the oceans (16–18). An estimated 81% of marine species (19) and 89% of tetrapod genera became extinct as established Permian ecosystems gave way to those of the Triassic. In the ocean, this included the complete extinction of reef-forming tabulate and rugose corals (20, 21) and significant losses in previously diverse ammonoid, brachiopod, and crinoid families (22). On land, many nonmammalian synapsids became extinct (16), and the glossopterid-dominated floras of Gondwana also disappeared (23). Stratigraphic sequences document a global “coral gap” and “coal gap” (24, 25), suggesting reef and forest ecosystems were rare or absent for up to 5 My after the event (26). Continuous fossil-bearing deposits documenting patterns of turnover across the Permian–Triassic transition (PTT) on land (27) and in the oceans (28) are geographically widespread (29, 30), including marine and continental successions that are known from China (31, 32) and India (33). Continental successions are known from Russia (34), Australia (35), Antarctica (36), and South Africa’s Karoo Basin (Fig. 1 and 37–40), the latter providing arguably the most densely sampled and taxonomically scrutinized (41–43) continental record of the PTT. The main extinction has been proposed to occur at the boundary between two biostratigraphic zones with distinctive faunal assemblages, the Daptocephalus and Lystrosaurus declivis assemblage zones (Fig. 1), which marks the traditional placement of the Permian–Triassic geologic boundary [(37) but see ref. 44]. Considerable research has attempted to understand the anatomy of the PTT in South Africa (38, 39, 45–52) and to place it in the context of biodiversity changes across southern Gondwana (53, 54) and globally (29, 31, 32, 44, 47, 55).Open in a separate windowFig. 1.Map of South Africa depicting the distribution of the four tetrapod fossil assemblage zones (Cistecephalus, Daptocephalus, Lystrosaurus declivis, Cynognathus) and our two study sites where fossils were collected in this study (sites A and B). Regional lithostratigraphy and biostratigraphy within the study interval are shown alongside isotope dilution–thermal ionization mass spectrometry dates retrieved by Rubidge et al., Botha et al., and Gastaldo et al. (37, 44, 80). The traditional (dashed red line) and associated PTB hypotheses for the Karoo Basin (37, 44) are also shown. Although traditionally associated with the PTB, the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary is defined by first appearances of co-occurring tetrapod assemblages, so its position relative to the three PTB hypotheses is unchanged. The Ripplemead member (*) has yet to be formalized by the South African Committee for Stratigraphy.Decades of research have demonstrated the richness of South Africa’s Karoo Basin fossil record, resulting in hundreds of stratigraphically well-documented tetrapod fossils across the PTT (37, 39, 56). This wealth of data has been used qualitatively to identify three extinction phases and an apparent early postextinction recovery phase (39, 45, 51). Furthermore, studies of Karoo community structure and function have elucidated the potential role of the extinction and subsequent recovery in breaking the incumbency of previously dominant clades, including synapsids (11, 57). Nevertheless, understanding patterns of faunal turnover and recovery during the PTT has been limited by the scarcity of quantitative investigations. Previous quantitative studies used coarsely sampled data (i.e., assemblage zone scale, 2 to 3 Ma time intervals) to identify low species richness immediately after the main extinction, potentially associated with multiple “boom and bust” cycles of primary productivity based on δ¹³C variation during the first 5 My of the Triassic (41, 58). However, many details of faunal dynamics in this interval remain unknown. Here, we investigate the dynamics of this major tetrapod extinction at an unprecedented time resolution (on the order of hundreds of thousands of years), using sample-standardized methods to quantify multiple aspects of regional change across the Cistecephalus, Daptocephalus, and Lystrosaurus declivis assemblage zones.     

Atomic-scale evidence for highly selective electrocatalytic N−N coupling on metallic MoS2

Molybdenum sulfide (MoS₂) is the most widely studied transition-metal dichalcogenide (TMDs) and phase engineering can markedly improve its electrocatalytic activity. However, the selectivity toward desired products remains poorly explored, limiting its application in complex chemical reactions. Here we report how phase engineering of MoS₂ significantly improves the selectivity for nitrite reduction to nitrous oxide, a critical process in biological denitrification, using continuous-wave and pulsed electron paramagnetic resonance spectroscopy. We reveal that metallic 1T-MoS₂ has a protonation site with a pK_a of ∼5.5, where the proton is located ∼3.26 Å from redox-active Mo site. This protonation site is unique to 1T-MoS₂ and induces sequential proton−electron transfer which inhibits ammonium formation while promoting nitrous oxide production, as confirmed by the pH-dependent selectivity and deuterium kinetic isotope effect. This is atomic-scale evidence of phase-dependent selectivity on MoS₂, expanding the application of TMDs to selective electrocatalysis.

Transition-metal dichalcogenides (TMDs) have gained considerable attention in recent years due to their variable crystal phases, which allow for precise tuning of their electronic, optical, magnetic, and catalytic properties (1, 2). For example, molybdenum sulfide (MoS₂), which is one of the most extensively studied TMDs, exists as different polymorphs depending on the orientation of sulfur atoms around the molybdenum center. In octahedral coordination (1T phase), MoS₂ exhibits metallic behavior, whereas the material acts as a semiconductor in trigonal prismatic coordination (2H phase) (3–6). In addition to higher conductivity, 1T-MoS₂ has enlarged layer spacing and more electrochemical active sites (7, 8), making it a promising next-generation material for batteries (9, 10), memristors (11, 12), capacitors (13, 14), and numerous other energy-related applications (15–17).In the field of electrocatalysis, phase engineering has mainly been used to enhance catalytic activity. For instance, exchanging 2H-MoS₂ for 1T-MoS₂ results in a marked increase toward the hydrogen evolution reaction (18, 19). Considering the advantage of TMDs being able to control the atomic-scale structure, phase engineering may also open possibilities to control the selectivity of multielectron/proton reactions with multiple possible products, such as CO₂ reduction (20–23), denitrification (NO₃⁻/NO₂⁻ reduction) (24–26), and the electrosynthesis of functional molecules (27–30). Selectivity is a critical requirement for cascade catalysis, one-pot reaction systems, and multistep catalytic processes, and strategies to guide the complex chemical reaction network toward the desired end product are necessary (31, 32). However, to the best of our knowledge, no studies have attempted to exploit the advantages of phase-engineered materials for selective electrocatalysis.One effective approach to explore phase-engineered MoS₂ for selectivity control is to utilize the newly proposed concept of sequential proton−electron transfer (SPET) (off-diagonal pathways, Fig. 1A) (33, 34). In contrast to the extensively studied concerted proton−electron transfer (CPET) pathway, the energy landscape of sequential (decoupled) proton−electron transfer (SPET) pathways is pH-dependent (Fig. 1B). This leads to pH-dependent reaction rates (Fig. 1C), where the maximum reaction rate can be obtained at a pH close to the pK_a of the reaction intermediate (33, 34). This was recently observed experimentally for nitrite reduction to dinitrogen – an artificial analog of biological denitrification – on partially oxygenated molybdenum sulfide (oxo-MoS_x), and the record high selectivity toward dinitrogen was achieved by simple pH optimization (35). In contrast, this pH dependence was absent in the case of crystalline 2H-MoS₂, demonstrating that the SPET pathway is a unique property of oxo-MoS_x and is therefore probably phase-dependent. However, the origin of the SPET behavior on this material remains unclear. Therefore, elucidating the mechanism at the atomic level would help rationalize the relationship between selectivity and crystal phases, thus providing significant insight into the newly proposed SPET mechanism (33, 34) to enhance the selectivity of multistep electrochemical processes.Open in a separate windowFig. 1.Selectivity control of MoS₂ based on SPET theory. (A) Diagram showing the possible pathways for proton−electron transfer on MoS₂. In the blue pathway (CPET), protons and electrons are transferred in a single elementary step. In contrast, stepwise pathways (SPET) generate an intermediate whose charge depends on whether the electron or proton transfers first (red and black pathways, respectively). (B) Diagram showing the energetic landscape of SPET. The landscape depends on the relationship between the pK_a of the reaction intermediate and the solution pH. (C) Influence of pH on reaction selectivity. The rates of SPET reactions (red lines) show a pH dependence with a maximum corresponding to the pK_a of the intermediate. Therefore, the relative rate of one reaction over another can be tuned by changing the pH. In contrast, the rate of CPET reactions are pH-independent, and therefore, their relative rates are also constant with respect to pH.Here, we identified the atomic-scale origin of SPET-driven selectivity on MoS₂ using continuous-wave electron paramagnetic resonance (CW-EPR), Raman, and pulsed ¹H/²H electron−nuclear double-resonance (ENDOR) spectroscopy. Specifically, a proton located at the first coordination sphere (∼3.26 Å) of a redox-active Mo center was found to have a pK_a value matching that involved in the pH-dependent electrocatalytic selectivity and H/D kinetic isotope effect (KIE). The observed pH-dependent behavior is specific to 1T-MoS₂, as oxo-MoS_x was assigned to the 1T phase using high-resolution transmission electron microscopy (HRTEM), Raman- and X-ray photoelectron spectroscopy (XPS). These results not only provide atomic-scale evidence of SPET in heterogeneous catalysis, but also demonstrate how the phase engineering of TMDs can be used to enhance their electrocatalytic selectivity.     

Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations

Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to μm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α₃D using atomic force microscopy (AFM)–based force spectroscopy. α₃D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α₃D’s configurational diffusion constant within the context of Kramers theory varies with pH. The resulting pH dependence provides a test for AFM-based force spectroscopy’s ability to track intrinsic changes in protein folding dynamics. Experimentally, however, α₃D is challenging. It unfolds at low force (<15 pN) and exhibits fast-folding kinetics. We therefore used focused ion beam–modified cantilevers that combine exceptional force precision, stability, and temporal resolution to detect state occupancies as brief as 1 ms. Notably, equilibrium and nonequilibrium force spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in destabilization mechanism. We reconstructed a one-dimensional free-energy landscape from dynamic data via an inverse Weierstrass transform. At both neutral and low pH, the resulting constant-force landscapes showed minimal differences (∼0.2 to 0.5 k_BT) in transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.

Single-molecule force spectroscopy (SMFS) has been remarkably successful across broad classes of biological molecules (RNA, DNA, and proteins) (1–5). A particularly fruitful data acquisition regime probes multiple back-and-forth folding/unfolding transitions at near-equilibrium and equilibrium conditions (6–9). This methodology efficiently yields numerous transitions and therefore a wealth of kinetic data, one-dimensional (1D) free-energy landscape parameters, and even a full 1D projection of the free-energy landscape along the stretching axis (10, 11). The standard SMFS assay has the molecule of interest tethered via a flexible linker to the force probe, such as an optically trapped bead or an atomic force microscopy (AFM) cantilever (Fig. 1A). These micrometer-sized force probes are the primary measurement (x_meas) but have finite response time and are therefore coupled to, but do not precisely track, molecular dynamics (x_prot) (Fig. 1B) (12–14). Additionally, the flexible linker’s compliance modifies this coupling between the molecule and the force probe. Linkers stretched at a finite force (F) can even create an entropic barrier not present in the absence of applied force (15, 16). More generally, there is an expanding set of theoretical and experimental studies (12–30) investigating how such instrumental and assay parameters affect the underlying biomolecular dynamics and whether the measured dynamics are dominated by the instrument used to measure them.Open in a separate windowFig. 1.Probing the folding and unfolding dynamics of a globular protein by SMFS. (A) Cartoon showing a polyprotein consisting of a single copy of α₃D (blue) and two copies of NuG2 (red) stretched with an atomic force microscope. At low forces, the mechanically labile α₃D repeatedly unfolds and refolds as detected by a change in cantilever deflection. (B) A conceptual two-dimensional free-energy landscape shows the underlying protein extension (x_prot) and the experimentally measured extension (x_meas). The macroscopic force probe has finite temporal resolution, and the application of force can introduce an entropic barrier between resolved states. (C) The sum of the equilibrium folding and unfolding rates for α₃D in a strong denaturant (5 to 6 M urea) as a function of pH as determined in a prior smFRET study (37). (D) A conceptual sketch of α₃D’s 1D free-energy landscape deduced by a combination of smFRET and molecular dynamics studies based on Ref. 37. The dramatic increase in α₃D’s kinetics at low pH shown in panel C was explained as increased configurational diffusion along a smooth rather than a rough energy landscape.AFM characterization of proteins is widely used (1–5) and therefore is an important experimental regime to explore, distinct from numerous studies investigating instrumental effects on nucleic acid hairpins measured with optical traps (17, 18, 23, 24, 26, 31). Historically, limited force precision and stability coupled with the slow response of the force probe has made it challenging to perform AFM-based equilibrium and near-equilibrium studies (32) and thereby difficult to quantify the role of instrumental artifacts. Recent work using a standard gold-coated cantilever concluded that the equilibrium dynamics of the fast-folding protein gpW were dominated by the dynamics of the cantilever diffusing on a force-induced entropic barrier (29). Such results raise significant concerns about interpreting rates or landscapes measured in AFM studies of globular protein folding and thereby motivate the following question: How do variations in intrinsic protein folding dynamics manifest in AFM-based studies, particularly in an experimental regime dominated by an instrument-induced entropic barrier?Here, we address this question by directly modulating a globular protein’s underlying folding dynamics without significantly changing the height of the barrier or the free-energy difference between the states. To do so, we studied α₃D using AFM-based force spectroscopy (Fig. 1A). The dynamics and energetics of α₃D, a computationally designed, fast-folding three-helix bundle of 73 amino acids (33, 34), have been studied by traditional ensemble (33) and single-molecule fluorescence resonance energy transfer (smFRET) (35–39) assays. Equilibrium smFRET studies in chemical denaturants showed accelerated folding/unfolding kinetics as pH was reduced (35). A subsequent landmark paper (37) combined state-of-the-art smFRET and microsecond-long, all-atom molecular dynamics simulations to show that this acceleration resulted from suppression of nonnative contacts changing the local roughness of the 1D landscape (Fig. 1 C and D) rather than a change in the height or the overall shape of the barrier between states. In the context of Kramers theory (40), this roughness manifests as a change in D, the conformational diffusion coefficient along the 1D landscape. The authors concluded that most, if not all, of the 14-fold change in folding kinetics came from an increase in D. This pH-dependent change in kinetics serves as a benchmark of α₃D’s dynamics in the absence of the force probe and associated linker. In other words, we will leverage conditions known to modulate the rate of folding along the molecular coordinate (x_prot) while measuring the consequence of that change on the measured coordinate (x_meas) (Fig. 1B).While α₃D provides a conceptually attractive means to modulate intrinsic molecular dynamics, it presents significant experimental challenges. Like gpW (28, 29), α₃D unfolds at a low force (< 15 pN) by AFM standards (2, 3, 41) and exhibits even faster folding kinetics under force. Thus, spatiotemporal resolution is critical, and instrumentation limitations are expected to be even more pronounced. Force drift is also a critical issue, particularly for extended assays (>1 to 100 s) because standard gold-coated cantilevers exhibit significant force drift (42); yet, equilibrium assays of structured RNA (6) and proteins (9) are sensitive to sub-pN changes in F. We therefore used focused ion beam (FIB)–modified cantilevers (32, 43) that combine sub-pN stability over 100 s (43, 44) with a ∼13-fold improvement in spatiotemporal precision compared with the standard cantilever used in the aforementioned AFM study characterizing gpW (29). This stability in conjunction with a newly designed polyprotein construct allowed us to measure an individual α₃D unfold and fold over 5,000 times and for periods up to 1 h using both constant velocity (v) and equilibrium (v = 0) data acquisition protocols. Rates derived from both the equilibrium and dynamic data recapitulated α₃D’s pH-dependent kinetics from smFRET. However, the reconstructed 1D folding-energy landscape was consistent with the predicted entropic barrier and therefore encodes no information about α₃D’s folding landscape beyond ΔG₀, the thermodynamic stability of α₃D. Importantly, rate analysis yielded the expected asymmetric distance to the transition state from the folded and unfolded state and revealed a significant increase in the distance to the unfolding transition state as pH was lowered. These studies demonstrate that AFM-force spectroscopy can track changes in intrinsic protein dynamics with high precision, even in mechanically labile, fast-folding systems.     

The evolution of red color vision is linked to coordinated rhodopsin tuning in lycaenid butterflies

Color vision has evolved multiple times in both vertebrates and invertebrates and is largely determined by the number and variation in spectral sensitivities of distinct opsin subclasses. However, because of the difficulty of expressing long-wavelength (LW) invertebrate opsins in vitro, our understanding of the molecular basis of functional shifts in opsin spectral sensitivities has been biased toward research primarily in vertebrates. This has restricted our ability to address whether invertebrate G_q protein-coupled opsins function in a novel or convergent way compared to vertebrate G_t opsins. Here we develop a robust heterologous expression system to purify invertebrate rhodopsins, identify specific amino acid changes responsible for adaptive spectral tuning, and pinpoint how molecular variation in invertebrate opsins underlie wavelength sensitivity shifts that enhance visual perception. By combining functional and optophysiological approaches, we disentangle the relative contributions of lateral filtering pigments from red-shifted LW and blue short-wavelength opsins expressed in distinct photoreceptor cells of individual ommatidia. We use in situ hybridization to visualize six ommatidial classes in the compound eye of a lycaenid butterfly with a four-opsin visual system. We show experimentally that certain key tuning residues underlying green spectral shifts in blue opsin paralogs have evolved repeatedly among short-wavelength opsin lineages. Taken together, our results demonstrate the interplay between regulatory and adaptive evolution at multiple G_q opsin loci, as well as how coordinated spectral shifts in LW and blue opsins can act together to enhance insect spectral sensitivity at blue and red wavelengths for visual performance adaptation.

Opsins belong to a diverse multigene family of G protein-coupled receptors that bind to a small nonprotein retinal moiety to form photosensitive rhodopsins and enable vision across animals (1–4). The tight relationship between opsin genotypes and spectral sensitivity phenotypes offers an ideal framework to analyze how specific molecular changes give rise to adaptations in visual behaviors (5). Notably, independent opsin gene gains and losses (6–13), genetic variation across opsins (14–16), spectral tuning mutations within opsins (17–21), and alterations in visual regulatory networks (22, 23) have contributed to opsin adaptation. Yet, the molecular and structural changes underlying the remarkable diversification of spectral sensitivity phenotypes identified in some invertebrates, including crustaceans and insects (24–27), are far less understood than those in vertebrate lineages (28–32).The diversity of opsin-based photoreceptors observed across animal visual systems is produced by distinct ciliary vertebrate c-opsin and invertebrate rhabdomeric based r-opsin subfamilies that mediate separate phototransduction cascades (31, 33–35). Vertebrate c-opsins function through the G protein transducing (G_t) signaling pathway, which activates cyclic nucleotide phosphodiesterase, ultimately resulting in a hyperpolarization response in photoreceptor cells through the opening of selective K⁺ channels (31, 36). By contrast, insect opsins transmit light stimuli through a G_q-type G protein (33, 37) with phosphoinositol (PLCβ) acting as an effector enzyme to achieve TRP channel depolarization in the invertebrate photoreceptor cell (34, 38).All vertebrate visual cone opsins derive from four gene families: short-wavelength-sensitive opsins SWS1 (or ultraviolet [UV]) with λ_max 344 to 445 nm and SWS2 with λ_max 400 to 470 nm, and longer-wavelength-sensitive opsins that specify the green MWS (or Rh2) pigments with λ_max 480 to 530 nm and red-sensitive LWS pigments with λ_max 500 to 570 nm (5, 30). Most birds and fish have retained the four ancestral opsin genes (39), with notable opsin expansions in cichlid fish opsins (23, 40), whereas SWS1 is extinct in monotremes, and SWS2 and M opsins are lost in marsupials and eutherian mammals (41). In primates, trichromatic vision is conferred through SWS1 (λ_max = 414 nm) and recent duplicate MWS (λ_max = 530 nm) and LWS opsins (λ_max = 560 nm) (42–44). In vertebrates, molecular evolutionary approaches and well-established in vitro opsin purification have identified the complex interplay between opsin duplications, regulatory and protein-coding mutations controlling opsin gene tuning, and spectral phenotypes notably in birds, fish, and mammals (45–47).Insect opsins are phylogenetically distinct but functionally analogous to those of vertebrates, and the ancestral opsin repertoire consists of three types of light-absorbing rhabdomeric G_q-type opsin specifying UV (350 nm), short-wavelength (blue, 440 nm) and long-wavelength pigments (LW, 530 nm) (48). Given the importance of color-guided behaviors and the remarkable photoreceptor spectral diversity observed in insects (26, 27), the dynamic opsin gene diversification found across lineages (Fig. 1) highlights their potentially central role in adaptation (27, 49, 50), yet the molecular basis of opsin functionality of rhabdomeric invertebrate G_q opsins remains understudied.Open in a separate windowFig. 1.Visual opsin gene evolution and spectral tuning mechanisms in insects. Visual opsin genes of the Atala hairstreak (E. atala, Lepidoptera, Lycaenidae) in comparison with those encoded in the genomes of diverse insects. The opsin types are highlighted in gray for UV, in blue for short wavelength (SW), and in green for long wavelength (LW). Numbers indicate multiple opsins, whereas no dot indicates gene loss. Colored circles indicate instances of shifted spectral sensitivities in at least one of the encoded opsins. The direction of shift is inferred from the opsin lambda max that departs from the typical range of absorbance in the opsin subfamily using wavelength boundaries for the various colors: UV <380 nm, violet 380 to 435 nm, blue 435 to 492 nm, green 492 to 530 nm, and red shifted >530 nm. Coleopteran lineages, and some hemipterans, lost the blue opsin locus and compensated for the loss of blue sensitivity via UV and/or LW gene duplications across lineages (11, 12). In butterflies, extended photosensitivity at short wavelengths is observed in Heliconius erato with two UV opsins at λ_max = 355 nm and 398 nm (10) and in P. rapae with two blue opsins with λ_max = 420 and 450 nm (17). A blue opsin duplication occurred independently in lycaenid butterflies (61). LW opsin duplications occurred independently in most major insect lineages (6, 16, 55) and confer a variable range of LW sensitivities with or without additional contributions from lateral filtering. In order to extend spectral sensitivity at longer wavelengths while sharpening blue acuity, some lycaenid butterflies have evolved a new color vision mechanism combining spectral shifts at a duplicate blue opsin and at the LW opsin. Images credit: Christopher Adams (illustrator).The recurrent evolution of red receptors in insects in particular suggests that perception of longer wavelengths can play an important role in the context of foraging, oviposition, and/or conspecific recognition (6, 27, 51–54). In butterflies, several mechanisms are likely to have provided extended spectral sensitivity to longer wavelengths. LW opsin duplications along with the evolution of lateral filtering between ommatidia has been demonstrated in two papilionids, Papilio xuthus (27) and Graphium sarpedon (55), as well as in a riodinid (Apodemia mormo) (6, 54). Lateral filtering pigments are relatively widespread across butterfly lineages, e.g., Heliconius (56), Pieris (57), Colias erate (58), and some moths [Adoxophyes orana (59) and Paysandisia archon (60)]. These pigments absorb short wavelengths and aid in shifting the sensitivity peak of green LW photoreceptors to longer wavelengths (27, 51, 56, 57, 61, 62). Despite creating distinct spectral types that can contribute to color vision, as identified in nymphalid (56), pierid (57), and lycaenid (62) species, all of which lack duplicated LW opsins (61, 63), lateral filtering alone cannot extend photoreceptor sensitivity toward the far red (700 to 750 nm) beyond the exponentially decaying long-wavelength rhodopsin absorbance spectrum (51). Thus, molecular variation of ancestral LW opsin genes is likely to have contributed an as yet underexplored mechanism to the diversification of long-wavelength photoreceptor spectral sensitivity. However, disentangling the relative contributions of lateral filtering and pure LW opsin properties has remained technically challenging using classical electrophysiological approaches (14, 64, although see, e.g., refs. 65, 66, 67) and has been limited by the lack of in vitro expression systems suitable for LW opsins.While opsin duplicates have been identified in numerous organisms, the spectral tuning mechanisms and interplay between new opsin photoreceptors in invertebrate visual system evolution are less well understood. Here we combine physiological, molecular, and heterologous approaches to start closing this gap in our knowledge of invertebrate G_q opsin evolution by investigating the functions, spectral tuning, and implications of evolving new combinations of short- and long-wavelength opsin types in lycaenid species. This butterfly group, comprising the famous blues, coppers, and hairstreaks, is the second largest family with about 5,200 (28%) of the some 18,770 described butterfly species (68). In light of their remarkable behavioral, ecological, and morphological diversity (69, 70), as well as pioneer studies in the Lycaena and Polyommatus genera supporting the rapid evolution of color vision in certain lineages (56, 61, 62), lycaenids provide an ideal candidate system for investigating opsin evolution and visual adaptations. Using the Atala hairstreak, Eumaeus atala, as a molecular and ecological model, we find coordinated spectral shifts at short- and long-wavelength G_q opsin loci and demonstrate that the combination of six ommatidial classes of photoreceptors in the compound eye uniquely extend spectral sensitivity at long wavelengths toward the far-red while concurrently sharpening acuity of multiple blue wavelengths. Together, these findings link the evolution of four-opsin visual systems to adaptation in the context of finely tuned color perception critical to the behavior of these butterflies.     

Heading perception depends on time-varying evolution of optic flow

There is considerable support for the hypothesis that perception of heading in the presence of rotation is mediated by instantaneous optic flow. This hypothesis, however, has never been tested. We introduce a method, termed “nonvarying phase motion,” for generating a stimulus that conveys a single instantaneous optic flow field, even though the stimulus is presented for an extended period of time. In this experiment, observers viewed stimulus videos and performed a forced-choice heading discrimination task. For nonvarying phase motion, observers made large errors in heading judgments. This suggests that instantaneous optic flow is insufficient for heading perception in the presence of rotation. These errors were mostly eliminated when the velocity of phase motion was varied over time to convey the evolving sequence of optic flow fields corresponding to a particular heading. This demonstrates that heading perception in the presence of rotation relies on the time-varying evolution of optic flow. We hypothesize that the visual system accurately computes heading, despite rotation, based on optic acceleration, the temporal derivative of optic flow.

James Gibson first remarked that the instantaneous motion of points on the retina (Fig. 1A) can be formally described as a two-dimensional (2D) field of velocity vectors called the “optic flow field” (or “optic flow”) (1). Such optic flow, caused by an observer’s movement relative to the environment, conveys information about self-motion and the structure of the visual scene (1–15). When an observer translates in a given direction along a straight path, the optic flow field radiates from a point in the image with zero velocity, or singularity, called the focus of expansion (Fig. 1B). It is well known that under such conditions, one can accurately estimate one’s “heading” (i.e., instantaneous direction of translation in retinocentric coordinates) by simply locating the focus of expansion (SI Appendix). However, if there is angular rotation in addition to translation (by moving along a curved path or by a head or eye movement), the singularity in the optic flow field will be displaced such that it no longer corresponds to the true heading (Fig. 1 C and D). In this case, if one estimates heading by locating the singularity, the estimate will be biased away from the true heading. This is known as the rotation problem (14).Open in a separate windowFig. 1.Projective geometry, the rotation problem, time-varying optic flow, and the optic acceleration hypothesis. (A) Viewer-centered coordinate frame and perspective projection. Because of motion between the viewpoint and the scene, a 3D surface point traverses a path in 3D space. Under perspective projection, the 3D path of this point projects onto a 2D path in the image plane (retina), the temporal derivative of which is called image velocity. The 2D velocities associated with all visible points define a dense 2D vector field called the optic flow field. (B–D) Illustration of the rotation problem. (B) Optic flow for pure translation (1.5-m/s translation speed, 0° heading, i.e., heading in the direction of gaze). Optic flow singularity (red circle) corresponds to heading (purple circle). (C) Pure rotation, for illustrative purposes only and not corresponding to any experimental condition (2°/s rightward rotation). (D) Translation + rotation (1.5 m/s translation speed, 0° heading, 2°/s rightward rotation). Optic flow singularity (red circle) is displaced away from heading (purple circle). (E) Three frames from a video depicting movement along a circular path with the line-of-sight initially perpendicular to a single fronto-parallel plane composed of black dots. (F) Time-varying evolution of optic flow. The first optic flow field reflects image motion between the first and second frames of the video. The second optic flow field reflects image motion between the second and third frames of the video. For this special case (circular path), the optic flow field evolves (and the optic flow singularity drifts) only due to the changing depth of the environment relative to the viewpoint. (G) Illustration of the optic acceleration hypothesis. Optic acceleration is the derivative of optic flow over time (here, approximated as the difference between the second and first optic flow fields). The singularity of the optic acceleration field corresponds to the heading direction. Acceleration vectors autoscaled for visibility.Computer vision researchers and vision scientists have developed a variety of algorithms that accurately and precisely extract observer translation and rotation from optic flow, thereby solving the rotation problem. Nearly all of these rely on instantaneous optic flow (i.e., a single optic flow field) (4, 9, 16–25) with few exceptions (26–29). However, it is unknown whether these algorithms are commensurate with the neural computations underlying heading perception.The consensus of opinion in the experimental literature is that human observers can estimate heading (30, 31) from instantaneous optic flow, in the absence of additional information (5, 10, 15, 32–34). Even so, there are reports of systematic biases in heading perception (11); the visual consequences of rotation (eye, head, and body) can bias heading judgments (10, 15, 35–37), with the amount of bias typically proportional to the magnitude of rotation. Other visual factors, such as stereo cues (38, 39), depth structure (8, 10, 40–43), and field of view (FOV) (33, 42–44) can modulate the strength of these biases. Errors in heading judgments have been reported to be greater when eye (35–37, 45, 46) or head movements (37) are simulated versus when they are real, which has been taken to mean that observers require extraretinal information, although there is also evidence to the contrary (10, 15, 33, 40, 41, 44, 47–50). Regardless, to date no one has tested whether heading perception (even with these biases) is based on instantaneous optic flow or on the information available in how the optic flow field evolves over time. Some have suggested that heading estimates rely on information accumulated over time (32, 44, 51), but no one has investigated the role of time-varying optic flow without confounding it with stimulus duration (i.e., the duration of evidence accumulation).In this study, we employed an application of an image processing technique that ensured that only a single optic flow field was available to observers, even though the stimulus was presented for an extended period of time. We called this condition “nonvarying phase motion” or “nonvarying”: The phases of two component gratings comprising each stationary stimulus patch shifted over time at a constant rate, causing a percept of motion in the absence of veridical movement (52). Phase motion also eliminated other cues that may otherwise have been used for heading judgments, including image point trajectories (15, 32) and their spatial compositions (i.e., looming) (53, 54). For nonvarying phase motion, observers exhibited large biases in heading judgments in the presence of rotation. A second condition, “time-varying phase motion,” or “time-varying,” included acceleration by varying the velocity of phase motion over time to match the evolution of a sequence of optic flow fields. Doing so allowed observers to compensate for the confounding effect of rotation on optic flow, making heading perception nearly veridical. This demonstrates that heading perception in the presence of rotation relies on the time-varying evolution of optic flow.     

A rate threshold mechanism regulates MAPK stress signaling and survival

Cells are exposed to changes in extracellular stimulus concentration that vary as a function of rate. However, how cells integrate information conveyed from stimulation rate along with concentration remains poorly understood. Here, we examined how varying the rate of stress application alters budding yeast mitogen-activated protein kinase (MAPK) signaling and cell behavior at the single-cell level. We show that signaling depends on a rate threshold that operates in conjunction with stimulus concentration to determine the timing of MAPK signaling during rate-varying stimulus treatments. We also discovered that the stimulation rate threshold and stimulation rate-dependent cell survival are sensitive to changes in the expression levels of the Ptp2 phosphatase, but not of another phosphatase that similarly regulates osmostress signaling during switch-like treatments. Our results demonstrate that stimulation rate is a regulated determinant of cell behavior and provide a paradigm to guide the dissection of major stimulation rate dependent mechanisms in other systems.

All cells employ signal transduction pathways to respond to physiologically relevant changes in extracellular stressors, nutrient levels, hormones, morphogens, and other stimuli that vary as functions of both concentration and rate in healthy and diseased states (1–7). Switch-like “instantaneous” changes in the concentrations of stimuli in the extracellular environment have been widely used to show that the strength of signaling and overall cellular response are dependent on the stimulus concentration, which in many cases needs to exceed a certain threshold (8, 9). Previous studies have shown that the rate of stimulation can also influence signaling output in a variety of pathways (10–17) and that stimulation profiles of varying rates can be used to probe underlying signaling pathway circuitry (4, 18, 19). However, it is still not clear how cells integrate information conveyed by changes in both the stimulation rate and concentration in determining signaling output. It is also not clear if cells require stimulation gradients to exceed a certain rate in order to commence signaling.Recent investigations have demonstrated that stimulation rate can be a determining factor in signal transduction. In contrast to switch-like perturbations, which trigger a broad set of stress-response pathways, slow stimulation rates activate a specific response to the stress applied in Bacillus subtilis cells (10). Meanwhile, shallow morphogen gradient stimulation fails to activate developmental pathways in mouse myoblast cells in culture, even when concentrations sufficient for activation during pulsed treatment are delivered (12). These observations raise the possibility that stimulation profiles must exceed a set minimum rate or rate threshold to achieve signaling activation. Although such rate thresholds would help cells decide if and how to respond to dynamic changes in stimulus concentration, the possibility of signaling regulation by a rate threshold has never been directly investigated in any system. Further, no study has experimentally examined how stimulation rate requirements impact cell phenotype or how cells molecularly regulate the stimulation rate required for signaling activation. As such, the biological significance of any existing rate threshold regulation of signaling remains unknown.The budding yeast Saccharomyces cerevisiae high osmolarity glycerol (HOG) pathway provides an ideal model system for addressing these issues (Fig. 1A). The evolutionarily conserved mitogen-activated protein kinase (MAPK) Hog1 serves as the central signaling mediator of this pathway (20–22). It is well established that instantaneous increases in osmotic stress concentration induce Hog1 phosphorylation, activation, and translocation to the nucleus (18, 21, 23–30). Activated Hog1 governs the majority of the cellular osmoadaptation response that enables cells to survive (23, 31, 32). Multiple apparently redundant MAPK phosphatases dephosphorylate and inactivate Hog1, which, along with the termination of upstream signaling after adaptation, results in its return to the cytosol (Fig. 1A) (23, 25, 26, 33–39). Because of this behavior, time-lapse analysis of Hog1 nuclear enrichment in single cells has proven an excellent and sensitive way to monitor signaling responses to dynamic stimulation patterns in real time (18, 27–30, 40, 41). Further, such assays have been readily combined with traditional growth and molecular genetic approaches to link observed signaling responses with cell behavior and signaling pathway architecture (27–29).Open in a separate windowFig. 1.Hog1 signaling and cell survival are sensitive to the rate of preconditioning osmotic stress application. (A) Schematic of the budding yeast HOG response. (B) Preconditioning protection assay workflow indicating the first stress treatments to a final concentration of 0.4 M NaCl (Left), high-stress exposure (Middle), and colony formation readout (Right). (C) High-stress survival as a function of each first treatment relative to the untreated first stress condition. Bars and errors are means and SD from three biological replicates. *Statistically significant by Kolmogorov–Smirnov test (P < 0.05). NS = not significant. (D) Treatment concentration over time. (E) Treatment rate over time for quadratic and pulse treatment. The rate for the pulse is briefly infinite (blue vertical line) before it drops to 0. (F) Hog1 nuclear localization during the treatments depicted in D and E. (Inset) Localization pattern in the quadratic-treated sample. Lines represent means and shaded error represents the SD from three to four biological replicates.Here, we use systematically designed osmotic stress treatments imposed at varying rates of increase to show that a rate threshold condition regulates yeast high-stress survival and Hog1 MAPK signaling. We demonstrate that only stimulus profiles that satisfy both this rate threshold condition and a concentration threshold condition result in robust signaling. We go on to show that the protein tyrosine phosphatase Ptp2, but not the related Ptp3 phosphatase, serves as a major rate threshold regulator. By expressing PTP2 under the control of a series of different enhancer–promoter DNA constructs, we demonstrate that changes in the level of Ptp2 expression can alter the stimulation rate required for signaling induction and survival. These findings establish rate thresholds as a critical and regulated component of signaling biology akin to concentration thresholds.     

Lipoteichoic acid polymer length is determined by competition between free starter units

Carbohydrate polymers exhibit incredible chemical and structural diversity, yet are produced by polymerases without a template to guide length and composition. As the length of carbohydrate polymers is critical for their biological functions, understanding the mechanisms that determine polymer length is an important area of investigation. Most Gram-positive bacteria produce anionic glycopolymers called lipoteichoic acids (LTA) that are synthesized by lipoteichoic acid synthase (LtaS) on a diglucosyl-diacylglycerol (Glc₂DAG) starter unit embedded in the extracellular leaflet of the cell membrane. LtaS can use phosphatidylglycerol (PG) as an alternative starter unit, but PG-anchored LTA polymers are significantly longer, and cells that make these abnormally long polymers exhibit major defects in cell growth and division. To determine how LTA polymer length is controlled, we reconstituted Staphylococcus aureus LtaS in vitro. We show that polymer length is an intrinsic property of LtaS that is directly regulated by the identity and concentration of lipid starter units. Polymerization is processive, and the overall reaction rate is substantially faster for the preferred Glc₂DAG starter unit, yet the use of Glc₂DAG leads to shorter polymers. We propose a simple mechanism to explain this surprising result: free starter units terminate polymerization by displacing the lipid anchor of the growing polymer from its binding site on the enzyme. Because LtaS is conserved across most Gram-positive bacteria and is important for survival, this reconstituted system should be useful for characterizing inhibitors of this key cell envelope enzyme.

All cell surfaces are rich in carbohydrate polymers that act as structural components, scaffolds for other molecules, and participants in signaling processes (1). The biological functions of a carbohydrate polymer are often greatly affected by its length. For example, depending on molecular weight, hyaluronic acid polymers can promote cell migration, differentiation, and inflammation or can inhibit these processes (2, 3). Similarly, the number of repeat units in bacterial O-antigen has a profound effect on complement activation and host cell uptake (4, 5). Unlike protein and nucleic acid polymers, which are assembled on a template that determines both length and composition, carbohydrate polymers are assembled without the use of a template. Template-independent length regulation is not as precise as template-directed polymerization, but physiological lengths of carbohydrate polymers typically fall into a defined range that is important for function (6). How different polymerases achieve length control is a fundamental question in the field.Several mechanisms for carbohydrate polymer length determination have been described. Some polymerases include a “molecular ruler” domain that measures the polymer against a portion of the enzyme (7), some use a dedicated “termination enzyme” to control length (8), and others rely on repeat unit concentration to control polymerization (9). These mechanisms are not mutually exclusive and can act together to control length (10, 11). The degree to which a polymerase is processive also influences product length. Processivity, a fundamental property of polymerases, refers to the number of elongation steps that occur without release of the growing polymer (12). A polymerase may be partially processive, in that more than one monomer addition occurs while the polymer is bound to the enzyme, but the polymer can be released and then rebind to continue elongation. A polymerase may also act in a distributive manner, where the growing polymer is released after each round of monomer addition. While some general mechanisms and aspects of length control for carbohydrate polymerases are known, here we describe a previously unknown mechanism for length regulation of a common type of lipoteichoic acid (LTA), a cell surface polymer that is crucially important to the physiology of most Gram-positive bacteria (13, 14).In the Gram-positive pathogen Staphylococcus aureus (Sa), LTA is a membrane-anchored poly(glycerol-phosphate) polymer involved in virulence (15–19), regulation of cell size and division (20–23), and osmotic stability (24, 25) (Fig. 1A). Sa LTA is assembled by the conserved lipoteichoic acid synthase (LtaS) on the cell surface using glucose(β1,6)-glucose(β1,3)-diacylglycerol (Glc₂DAG) as the membrane-anchored “starter unit” (20, 26). The polymer elongates in a process that involves the repeated transfer of phosphoglycerol units from phosphatidylglycerol (PG) to a catalytic threonine in LtaS (T300) and then to the tip of the growing polymer (Fig. 1B) (27–29). Repeat units may be modified by D-alanyl esters or, less commonly, GlcNAc moieties (24, 30). Because LTA is so important for Sa survival (13, 14, 21, 22), LtaS is a proposed target for antibiotics, and understanding its behavior may facilitate inhibitor development.Open in a separate windowFig. 1.LTA is a lipid-anchored polymer assembled from Glc₂DAG and PG on the bacterial cell surface. (A) Chemical structure of LTA from Sa. Phosphoglycerol repeat units may be modified with D-alanine esters or GlcNAc moieties. (B) Mechanism of LTA synthesis by LtaS. Phosphoglycerol units are transferred from PG to residue T300 to form a covalent intermediate, releasing DAG. Phosphoglycerol is then transferred to a Glc₂DAG starter unit to form GroP-Glc₂DAG. Additional repeat units are added to the glycerol tip of the polymer. (C) In Sa, PgcA and GtaB synthesize UDP-glucose from glucose-6-phosphate. UgtP uses UDP-glucose and DAG to make Glc₂DAG. LtaA exports Glc₂DAG to the cell surface. LtaS transfers phosphoglycerol units derived from PG to T300, releasing DAG for recycling. (D) Anti-LTA Western blot of Sa RN4220 wild-type (wt) or ΔugtP lysates. ΔugtP mutants lack Glc₂DAG, and LTA is instead polymerized directly on PG (20).Glc₂DAG, the starter unit for LTA polymerization, is biosynthesized on the cytoplasmic leaflet of the membrane by the sequential action of three enzymes: the phosphoglucose mutase PgcA, the UTP-glucose-1-phosphate uridylyltransferase GtaB, and the diacylglycerol β-glucosyltransferase UgtP (also called YpfP) (15, 20). Glc₂DAG is exported to the cell surface by the flippase LtaA (Fig. 1C) (15). An interesting feature of LtaS is that it can use PG as an alternative starter unit if Glc₂DAG synthesis or export is blocked (20). However, polymers formed on this alternative starter unit (PG-LTA) are significantly longer than polymers formed on Glc₂DAG (Glc₂DAG-LTA, Fig. 1D) (15, 23), and cells that make these longer polymers have cell division defects (20, 23), are much less virulent (15, 16), and are more sensitive to beta-lactam antibiotics and other cell envelope stresses (23). Whether the shorter polymers assembled on Glc₂DAG reflect the intrinsic behavior of LtaS or the action of other cellular factors is an important question that cannot be definitively answered with genetic approaches.Here we used in vitro reconstitution to test whether the identity of the LTA membrane anchor determines the length of the polymers that LtaS synthesizes. We show that the length differences observed between wild-type and mutant cells lacking Glc₂DAG are recapitulated in a proteoliposome system that contains only purified LtaS, PG, and either Glc₂DAG or an alternative anchor. Based on our studies, we propose a model for how polymer length can be controlled in polymerases that operate without a template.     

Cingulo-opercular control network and disused motor circuits joined in standby mode

Whole-brain resting-state functional MRI (rs-fMRI) during 2 wk of upper-limb casting revealed that disused motor regions became more strongly connected to the cingulo-opercular network (CON), an executive control network that includes regions of the dorsal anterior cingulate cortex (dACC) and insula. Disuse-driven increases in functional connectivity (FC) were specific to the CON and somatomotor networks and did not involve any other networks, such as the salience, frontoparietal, or default mode networks. Censoring and modeling analyses showed that FC increases during casting were mediated by large, spontaneous activity pulses that appeared in the disused motor regions and CON control regions. During limb constraint, disused motor circuits appear to enter a standby mode characterized by spontaneous activity pulses and strengthened connectivity to CON executive control regions.

Disuse is a powerful paradigm for inducing plasticity that has uncovered key organizing principles of the human brain (1–4). Monocular deprivation—prolonged covering of one eye—revealed that multiple afferent inputs can compete for representational territory in the primary visual cortex (1). Similar competition between afferents also shapes the somatomotor system. Manipulations such as peripheral nerve deafferentation, whisker trimming, and limb constraint all drive plasticity in the primary somatosensory and motor cortex (2–4). Most plasticity studies to date have used focal techniques, such as microelectrode recordings, to study local changes in brain function. As a result, little is known about how behavior and experience shape the brain-wide functional networks that support complex cognitive operations (5).The brain is composed of networks of regions that cooperate to perform specific cognitive functions (5–8). These functional networks show synchronized spontaneous activity while the brain is at rest, a phenomenon known as resting-state functional connectivity (FC) (9–11). FC can be measured noninvasively in humans using resting-state functional MRI (rs-fMRI) and has been used to parse the brain into canonical functional networks (12, 13), including visual, auditory, and somatomotor networks (14, 15); ventral and dorsal attention networks (8, 16); a default mode network with roles in internally directed cognition and episodic memory (7, 11); a salience network thought to assess the homeostatic relevance of external stimuli (17); a frontoparietal control network supporting error processing and moment-to-moment adjustments in behavior (18–20); and a cingulo-opercular control network (CON), which maintains executive control during goal-directed behavior (18, 19, 21). Each functional network likely carries out a variety of additional functions.A more recent advance in human neuroscience has been the recognition of individual variability in network organization (22–25). Most early rs-fMRI studies examined central tendencies in network organization using group-averaged FC measurements (10, 12, 13). Recent work has demonstrated that functional networks can be identified in an individual-specific manner if sufficient rs-fMRI data are acquired, an approach termed precision functional mapping (PFM) (22, 23, 26–30). PFM respects the unique functional anatomy of each person and avoids averaging together functionally distinct brain regions across individuals.We recently demonstrated that PFM can be used to follow the time course of disuse-driven plasticity in the human brain (31). Three adult participants (Nico, Ashley, and Omar) were scanned at the same time of day for 42 to 64 consecutive days (30 min of rs-fMRI per day) before, during, and after 2 wk of dominant upper-extremity casting (Fig. 1 A and B). Casting caused persistent disuse of the dominant upper extremity during daily behaviors and led to a marked loss of strength and fine motor skill in all participants. During casting, the upper-extremity regions of the left primary somatomotor cortex (L-SM1_ue) and right cerebellum (R-Cblm_ue) functionally disconnected from the remainder of the somatomotor network. Disused motor circuits also exhibited large, spontaneous pulses of activity (Fig. 1C). Disuse pulses did not occur prior to casting, started to occur frequently within 1 to 2 d of casting, and quickly waned after cast removal.Open in a separate windowFig. 1.Experimental design and spontaneous activity pulses. (A) Three participants (Nico, Ashley, and Omar) wore casts covering the entire dominant upper extremity for 2 wk. (B) Participants were scanned every day for 42 to 64 consecutive days before, during, and after casting. All scans included 30 min of resting-state functional MRI. (C) During the Cast period, disused somatomotor circuits exhibited large pulses of spontaneous activity. (C, Left) Whole-brain ANOVA showing which brain regions contained disuse-driven pulses. (C, Right) Time courses of all pulses recorded from the disused primary somatomotor cortex.Somatomotor circuits do not function in isolation. Action selection and motor control are thought to be governed by complex interactions between the somatomotor network and control networks, including the CON (18). Prior studies of disuse-driven plasticity, including our own, have focused solely on somatomotor circuits. Here, we leveraged the whole-brain coverage of rs-fMRI and the statistical power of PFM to examine disuse-driven plasticity throughout the human brain.     

Type III secretion system effector proteins are mechanically labile

Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins—such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)—impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy (ΔGunfold0) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published ΔGunfold0 for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force (F_unfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δx^‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust (F_unfold > 80 pN) and brittle (Δx^‡ < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

Type III secretion systems (T3SS) are large nanomachines utilized by both pathogenic and symbiotic bacteria to inject effector proteins directly into the cytoplasm of host cells (1–3). Once delivered, effector proteins facilitate host cell colonization through a variety of mechanisms (4–7), including down-regulation of the host immune response (8) and rearrangement of the cytoskeleton (9, 10). The T3SS apparatus, known as the injectisome, is a syringe-like structure with a hollow needle that spans the inner and outer bacterial membranes, the extracellular space, and the host membrane, enabling proteins to pass directly from bacteria to host cells (Fig. 1A) (2). Specialized bacterial chaperones often bind the N-terminal 50 to 100 amino acids (aa) of the effector proteins, known as the chaperone binding domain, and help maintain the effector N-terminal domain in an extended conformation. C-terminal to the chaperone binding domain, effector proteins contain one or more globular domains, which adopt their folded conformations even when in complex with their cognate chaperone (4, 11, 12). The effector proteins, or their chaperone complexes, are recognized by the base of the injectisome prior to secretion (13). At its narrowest point, the injectisome needle’s inner diameter is less than 2 nm (14–16). As a result, effector proteins must be mostly unfolded to be secreted (17–20). Secretion is thus thought to proceed by a “threading-the-needle mechanism,” where the N-terminal extended domain is released from the chaperone and fed to the injectisome, followed by unfolding of the C-terminal effector domain (21).Open in a separate windowFig. 1.Thermodynamic stability of T3SS effector proteins SptP_CD and SopE2_CD. (A) Schematic depiction of protein transport through the T3SS showing effector proteins, which are at least partially folded in the bacterial cytoplasm. Such effector proteins interact with an associated unfoldase to passage through the T3SS, which has an inner channel with a diameter <2 nm. Once inside the host cytoplasm, effector proteins refold to carry out their function. (B) Crystal structures of SptP_CD (Protein Data Bank [PDB] ID code 1G4U) and SopE2_CD (PDB ID code 1R9K). (C) Ellipticity from CD at λ = 222 nm plotted as a function of urea concentrations for SptP_CD (orange) and SopE2_CD (green). A fit of the data with Eq. 1 yielded the free energy of unfolding ΔGunfold0 for SptP_CD (6.9 ± 0.2 kcal/mol [mean ± fit error]) and SopE2_CD (6.0 ± 0.2 kcal/mol [mean ± fit error]). Data points are the result of at least three independent measurements. Error bars represent SD.Before proteins are secreted through the T3SS, they interact with a hexameric ATPase at the base of the T3SS that is capable of mediating chaperone release from effector proteins and effector-protein unfolding (15, 22). Indeed, most in vivo unfolding is catalyzed by unfoldases that work from one end of the substrate protein in stark contrast to the global effects of temperature, pH, or chemical denaturants. The most common examples of targeted protein unfolding are catalyzed by ATPases of the AAA(+) family that mechanically unfold their substrates (23, 24). For example, the AAA(+) ATPase ClpX forms a ring-shaped hexamer that mechanically pulls its substrates through its narrow central pore to unfold them (25). These are powerful unfoldases that can unfold even tightly packed proteins such as GFP, ubiquitin, and dihydrofolate reductase (DHFR) (23, 24, 26, 27). However, the T3SS ATPase does not belong to the AAA(+) family of ATPases. Instead, it is structurally similar to the catalytic β-subunit of the F₁F₀ ATP synthase, a rotary motor that normally couples proton gradient dissipation to ATP synthesis but can also run in reverse and hydrolyze ATP to do work (15, 28–30). The T3SS ATPase is not as powerful an unfoldase as the AAA(+) family, as fusions of effector proteins with GFP, ubiquitin, or DHFR stall in the injectisome and are poorly secreted (20, 22, 31, 32). These observations have led to the current model that T3SS effector proteins have low thermodynamic stability to facilitate their secretion (22, 31–33).While thermodynamic stability is the most common metric of protein stability, mechanical stability is a distinct metric that quantifies how easily a protein unfolds under force (F_unfold). Mechanical stability is typically measured by pulling across the N and C termini of single molecules via force spectroscopy using optical tweezers (34, 35) or an atomic force microscope (AFM) (36). Early force spectroscopy studies showed that thermodynamic stability does not correlate with mechanical stability (37–41). For example, titin’s I28 domain requires ∼20% more force to unfold than titin’s I27 domain [I85 and I91, respectively, in the new nomenclature (42)], despite I27 having approximately twofold higher thermodynamic stability (43). Importantly, AFM studies have shown that GFP (44), ubiquitin (45), and DHFR (46) are mechanically robust, requiring high forces to unfold despite their typical thermodynamic stabilities. These three proteins each stall the T3SS; thus, mechanical stability may be the physical determinant to proteins being secreted by the T3SS, rather than thermodynamic stability.Here, we determine the thermodynamic and mechanical stabilities of SptP and SopE2, two effector proteins from Salmonella enterica. These effectors are ideal candidates for this study as they have known crystal structures (10, 47), have characterized in vivo secretion kinetics (48), and represent effector proteins of different size and structure (Fig. 1B). We show that the catalytic domains of SptP and SopE2 have unremarkable thermodynamic stabilities, similar to many other previously characterized proteins, including GFP, ubiquitin, and DHFR. Conversely, our AFM-based force spectroscopy measurements demonstrate that SptP and SopE2 are among the most mechanically labile proteins studied to date by AFM. These two T3SS effector proteins are therefore mechanically labile while being thermodynamically stable, supporting the hypothesis that it is mechanical stability, not thermodynamic stability, that predicts efficient protein secretion by the T3SS.     

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl₂, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

The identity of a metal center often defines enzymatic activity, and swapping the native metal for an alternative one or introducing a new metal center has profound effects. More generally, the chemical utility of natural cofactors has inspired decades of study into synthetic analogs with distinct properties, and researchers have subsequently sought straightforward ways to put these novel cofactors back into proteins (1). Substituted metalloenzymes constitute one of the simplest cases. Changing the identity of the metal ion in metalloproteins has enabled powerful spectroscopic and functional studies of these proteins (2–10) in addition to new biocatalytic activities (11–20). However, most methods for producing such proteins with new-to-nature cofactors are limited by the inability to produce the novel protein–cofactor complex in vivo.Hemoproteins, in particular, have been studied through metal substitution because of their important biological functions and utility as biocatalysts. Heme is a ubiquitous and versatile cofactor in biology, and heme-dependent proteins serve essential gas sensing functions (21), metabolize an array of xenobiotic molecules (22), and perform synthetically useful oxygen activation and radical-based chemistry (23). Metal-substituted hemoproteins have enabled key spectroscopic studies of hemoprotein function and the development of biocatalysts with novel reactivity. For example, electron paramagnetic resonance (EPR) studies on cobalt-substituted sperm whale myoglobin (CoMb) enabled detailed characterization of the paramagnetic CoMbO₂ complex (3, 4, 24, 25). In analogous oxygen-binding studies in CoMb and cobalt-substituted hemoglobin (5, 6, 26), resonance Raman was used to identify the O–O stretching mode because cobalt-substituted proteins exhibit enhancement of this vibrational mode compared to the native iron proteins.Metal substitution has a profound effect on catalytic activity of hemoproteins, enabling numerous synthetic applications. Substitution of the native iron for cobalt in several hemoproteins, including a thermostable cytochrome c variant, enabled the reduction of water to H₂ under aerobic, aqueous conditions (27–29). Reconstitution of apoprotein with selected metalloporphyrins has been used to generate metal-substituted myoglobin and cytochrome P450s variants. These enzymes were effective as biocatalysts for C–H activation and carbene insertion reactions (11–14). In a tour de force of directed evolution, which required purification and cofactor reconstitution of each individual variant, Hartwig and coworkers generated a cytochrome P450 variant that utilizes a nonnative Ir(Me)mesoporphyrin cofactor to perform desirable C–H activation chemistry (14). These activities may not be unique to the Ir-substituted protein, as synthetic cobalt porphyrin complexes have been shown to mediate a variety of Co(III)-aminyl and -alkyl radical transformations, including C–H activation (30–32). Indeed, a number of cobalt porphyrin carbene complexes display significant carbon-centered radical character (33–35), whereas the corresponding Fe-porphyrin complexes are closed shell species (36, 37), indicating that cobalt porphyrins may possess distinct, complementary modes of reactivity (38–40).Inspired by these applications, researchers have sought strategies for generating metal-substituted hemoproteins. For many metalloproteins, metal substitution is carried out by removal of the native metal with a chelator and replacement with an alternate metal of similar coordination preference. This method is inapplicable to hemoproteins, as porphyrins do not readily exchange metal ions. Consequently, diverse methods have been employed to make metal-substituted hemoproteins (41–46). Early on, copper, cobalt, nickel, and manganese-substituted horseradish peroxidase (HRP) were prepared by a multistep process that subjected protein to strong acid and organic solvents (41, 42). Variations of this method have been used repeatedly (24, 43, 47–49). However, this method is applicable only to a narrow range of hemoproteins that tolerate the harsh treatment. With the advent of overexpression methods, significant improvement of metalloporphyrin-substituted protein yield was achieved by direct expression of the apoprotein and reconstitution with the desired metalloporphyrin in lysate prior to purification (50). Although this approach has many virtues, direct expression of apoprotein is ineffective for many hemoproteins, again limiting the utility of this method.As an alternative to the above in vitro approaches, researchers have pursued systems for direct in vivo expression of metal substituted hemoproteins. Two specialty strains of Escherichia coli (E. coli) were engineered to incorporate metalloporphyrin analogs from the growth medium into hemoproteins during protein expression. The engineered RP523 strain cannot biosynthesize heme and bears an uncharacterized heme permeability phenotype. Together, these two features enable this strain to assimilate and incorporate various metalloporphyrins into overexpressed hemoproteins with no background heme incorporation (44, 51–53). However, heme auxotrophy makes RP523 cells exceedingly sensitive to O₂, and, in many situations, RP523 cultures must be grown anaerobically. An alternative BL21(DE3)-based engineered strain harbors a plasmid bearing the heme transporter ChuA, which facilitates import of exogenous heme analogs (45). Production of metalloporphyrin-substituted protein with this ChuA-containing strain relies on growth in iron-limited minimal media, thereby diminishing heme biosynthesis. This method was used successfully to express metal-substituted versions of the heme domain of cytochrome P450 BM3 (45) and several myoglobin variants (11, 12). Because these cells biosynthesize a small quantity of their own heme, they are far more robust than the RP523 cells. Unfortunately, this advantage comes at the cost of increased heme contamination in the product protein (2 to 5%) (45).A set of intriguing papers reported the production of cobalt-substituted human cystathionine β-synthase (CoCBS) that relies on the de novo biosynthesis of CoPPIX from CoCl₂ and δ-aminolevulinic acid (δALA), a biosynthetic precursor to heme (46, 54). This method yielded significant amounts of CoCBS—albeit with modest heme contamination (7.4%)—sufficient for spectroscopic and functional characterization of the CoPPIX-substituted protein (8, 46). As cobalt is known to be toxic to E. coli, the researchers passaged the CBS expression strain through cobalt-containing minimal media for 12 d, enabling the cells to adapt to high concentrations of cobalt prior to protein expression. It is plausible that this serial passaging alters the E. coli cells, enabling the biosynthesis of CoPPIX and in vivo production of metal-substituted protein. The adaptation process is slow (>10 d), and it is unknown how genomic instability under these mutagenic conditions affects the reproducibility of this passaging approach.The possibility of facile CoPPIX production is particularly attractive for future biocatalysis efforts. As described above, synthetic cobalt porphyrins have been shown to perform a range of radical-mediated reactions. The ability to produce a CoPPIX center in vivo may enable engineering these unusual reactivities via directed evolution in addition to spectroscopic applications. We therefore set out to explore the unusual phenotype of CoPPIX production by E. coli and to ascertain whether it was possible to efficiently biosynthesize cobalt-containing hemoproteins in vivo from a single “generalist” cell line. Our goal was to achieve an efficient and facile method of cobalt-substituted hemoprotein production with minimal contamination of the native cofactor. Herein, we report the surprising discovery that native E. coli BL21(DE3) can biosynthesize a new-to-nature CoPPIX cofactor (Fig. 1). We use this insight to produce cobalt-substituted hemoproteins in vivo without requirement for complex expression methods or specialized strains.Open in a separate windowFig. 1.Chemical structures of iron protoporphyrin IX (FePPIX or heme b), cobalt protoporphyrin IX (CoPPIX), and free base protoporphyrin IX (H₂PPIX).     

Atomistic investigation of surface characteristics and electronic features at high-purity FeSi(110) presenting interfacial metallicity

Iron silicide (FeSi) is a fascinating material that has attracted extensive research efforts for decades, notably revealing unusual temperature-dependent electronic and magnetic characteristics, as well as a close resemblance to the Kondo insulators whereby a coherent picture of intrinsic properties and underlying physics remains to be fully developed. For a better understanding of this narrow-gap semiconductor, we prepared and examined FeSi(110) single-crystal surfaces of high quality. Combined insights from low-temperature scanning tunneling microscopy and density functional theory calculations (DFT) indicate an unreconstructed surface termination presenting rows of Fe–Si pairs. Using high-resolution tunneling spectroscopy (STS), we identify a distinct asymmetric electronic gap in the sub-10 K regime on defect-free terraces. Moreover, the STS data reveal a residual density of states in the gap regime whereby two in-gap states are recognized. The principal origin of these features is rationalized with the help of the DFT-calculated band structure. The computational modeling of a (110)-oriented slab notably evidences the existence of interfacial intragap bands accounting for a markedly increased density of states around the Fermi level. These findings support and provide further insight into the emergence of surface metallicity in the low-temperature regime.

Iron silicide (FeSi; cf. Fig. 1A) is an archetypical B20 compound (1, 2) featuring a cubic unit cell without an inversion center and a remarkable sevenfold coordination of the constituents. The ε-FeSi B20 phase presents unusual temperature-dependent physical properties explored by a multitude of seminal experimental (3–13) and groundbreaking theoretical investigations guided by different conceptual approaches (14–21). There is a general consensus to classify FeSi as a prototypical d-electron–based narrow-gap semiconductor (gap width Δ < 100 meV) whose intriguing material characteristics are strongly affected by electronic correlations and provide prospects for technological applications (21–23).Open in a separate windowFig. 1.Surface structure of the FeSi(110) single crystal. (A) FeSi B20 unit cell containing eight atoms. (B) Photo of the FeSi(110) single crystal (top view). (C) LEED pattern of FeSi(110) prepared in UHV (E_electron = 82 eV); reciprocal lattice vectors are indicated. (D) Large-scale STM image of the FeSi(110) surface (V_b = −100 mV, I_t = 500 pA). (Scale bar: 20 nm.) (E) High-resolution STM image of a flat terrace where atomic-like features are resolved (V_b = 1 V, I_t = 100 pA). (Scale bar: 45 Å.) (F) 2D-FFT of E featuring a rectangular reciprocal lattice in good agreement with the LEED observation. (Scale bar: 0.015 Å⁻¹.) (G) Zoomed-in STM image of the surface lattice (V_b = 300 mV, I_t = 500 pA). (H) Representative STM image showing adatoms and vacancies on the surface (V_b = −200 mV, I_t = 1 nA). (Scale bars in G and H: 10 Å.)The behavior of FeSi bulk specimens in the low-temperature (LT) regime proved particularly interesting and remains an enigmatic subject. The resistivity saturates or even slightly decreases at low temperatures and exhibits a metallization at elevated temperatures, well below those nominally to be expected from the gap size (7, 24–27). Based on estimates of impurity concentrations in the samples, defect or impurity states in the gap accounting for residual conductivity were hypothesized, though also arguments in favor of intrinsic behavior exist (21). Further evidence pointing toward the existence of in-gap states has been associated with spin-polaronic phenomena (28) or localized excitonic states (29). Quite recently, two groups independently reported the detection of an electron-dominated high-mobility surface conduction channel for T < ∼20 K via careful electrical transport measurements on high-purity samples of systematically varied geometric shape and Fe concentration, respectively (26, 27). Finding surface-related conduction channels would allow to reconcile the mixed metallic and semiconducting nature of FeSi at low temperatures in a simpler picture, that is, without violating the Ioffe-Regel criterion (30, 31).The physical properties of FeSi feature striking similarities with so-called heavy-fermion semiconductors or Kondo insulators (KI), as assessed notably for Ce₃Bi₄Pt₃ (21, 22) or SmB₆ (32). Accordingly, FeSi was repeatedly considered as a special member of this class, although the key feature of canonical f-electron KIs is the hybridization between the f band and a conduction band. The latter entails the formation of a hybridization gap between bands of mixed character. A long-standing puzzle in exemplary KIs is an LT resistivity saturation, which was ascribed to surface conductivity (33–37). Inspired by the discovery of topological insulators, it was also proposed that KIs can host topologically protected surface states (32, 38–40). The topological KI (TKI) concept provides a compelling explanation for robust metallic conduction channels that has been invoked in turn for FeSi (26), although earlier studies questioned the classification of FeSi as KI (12). In SmB₆, inversion of heavy quasiparticle bands at the Fermi level generates linearly dispersive heavy Dirac fermions residing in the narrow energy gap (32, 40), which were lately deduced for T < ∼5 K at the SmB₆(100)−(2 × 1) reconstructed surface, inducing characteristic changes in the local density of states within the gap region (41). Related findings exist for unreconstructed SmB₆(100) (42); however, there are notorious difficulties in the preparation of homogenous surfaces with this material, and certain aspects underlying the data interpretation are an unsettled matter of debate (43–45). Finally, a refined orbital-selective KI scenario was recently proposed for FeSi based on a dynamic mean-field theory analysis disentangling the eminent role of different Fe 3d components in the gap formation (21).This motivated us to get an exemplary high-quality FeSi single crystal surface under control for direct scrutiny. Despite numerous investigations of FeSi samples, an atomic-level characterization of the pertaining surface characteristics and related local electronic features is missing. Cleavage methods for B20 materials provide samples of limited quality. Accordingly, angle-resolved photoemission spectroscopy (ARPES) investigations emphasized the need of well-defined surfaces and appropriate preparation protocols (8, 12), albeit the atomistic nature of the investigated systems could hitherto not be probed. First electron tunneling spectroscopic measurements were carried out on cleaved samples without recognizing the surface structure, influence of impurities, surface defects, and morphology (46).Herein, we report on well-defined single crystalline FeSi surfaces with extended atomically flat terraces that were reproducibly prepared from polished high-quality (110)-oriented bulk samples via sputtering and annealing treatments. Employing LT-scanning tunneling microscopy (STM) as well as extensive computational modeling, we determined the surface atomic arrangement and termination with element-specific registry. Furthermore, by high-resolution scanning tunneling spectroscopy (STS) measurements, the narrow energy gap with distinct temperature-dependent characteristics is clearly resolved. For T < ∼10 K, it exhibits a markedly asymmetric shape with subtle features of in-gap states located on both sides of the Fermi level, where nonzero density of states (DOS) is present. Furthermore, the band structure obtained via density functional theory (DFT) calculations of a slab with (110) surface termination reproduces key features of STS spectra in a qualitative way, showing surface-related bands crossing the bulk energy gap. Our findings confirm that samples fabricated with the highest purity standards and examined with atomistic precision are a prerequisite for both developing a basic understanding of complex materials and making further progress toward harnessing their application potential. Specifically, we provide unambiguous evidence that the appearance of surface conductivity channels for the FeSi system in the LT regime can be attributed to interfacial symmetry breaking. Moreover, our findings reveal that the electronic properties of FeSi not only resemble that of the archetypical KI SmB₆ regarding bulk characteristics but also in the appearance of surface metallicity at low temperatures.     

Structure,self-assembly,and properties of a truncated reflectin variant

Naturally occurring and recombinant protein-based materials are frequently employed for the study of fundamental biological processes and are often leveraged for applications in areas as diverse as electronics, optics, bioengineering, medicine, and even fashion. Within this context, unique structural proteins known as reflectins have recently attracted substantial attention due to their key roles in the fascinating color-changing capabilities of cephalopods and their technological potential as biophotonic and bioelectronic materials. However, progress toward understanding reflectins has been hindered by their atypical aromatic and charged residue-enriched sequences, extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for aggregation. Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a straightforward mechanical agitation-based methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between the protein’s structural characteristics and intrinsic optical properties. Altogether, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and may inform new research directions across biochemistry, cellular biology, bioengineering, and optics.

Materials from naturally occurring and recombinant proteins are frequently employed for the study of fundamental biological processes and leveraged for applications in fields as diverse as electronics, optics, bioengineering, medicine, and fashion (1–13). Such broad utility is enabled by the numerous advantageous characteristics of protein-based materials, which include sequence modularity, controllable self-assembly, stimuli-responsiveness, straightforward processability, inherent biological compatibility, and customizable functionality (1–13). Within this context, unique structural proteins known as reflectins have recently attracted substantial attention because of their key roles in the fascinating color-changing capabilities of cephalopods, such as the squid shown in Fig. 1A, and have furthermore demonstrated their utility for unconventional biophotonic and bioelectronic technologies (11–40). For example, in vivo, Bragg stack-like ultrastructures from reflectin-based high refractive index lamellae (membrane-enclosed platelets) are responsible for the angle-dependent narrowband reflectance (iridescence) of squid iridophores, as shown in Fig. 1B (15–20). Analogously, folded membranes containing distributed reflectin-based particle arrangements within sheath cells lead to the mechanically actuated iridescence of squid chromatophore organs, as shown in Fig. 1C (15, 16, 21, 22). Moreover, in vitro, films processed from squid reflectins not only exhibit proton conductivities on par with some state-of-the-art artificial materials (23–27) but also support the growth of murine and human neural stem cells (28, 29). Additionally, morphologically variable coatings assembled from different reflectin isoforms can enable the functionality of chemically and electrically actuated color-changing devices, dynamic near-infrared camouflage platforms, and stimuli-responsive photonic architectures (27, 30–34). When considered together, these discoveries and demonstrations constitute compelling motivation for the continued exploration of reflectins as model biomaterials.Open in a separate windowFig. 1.(A) A camera image of a D. pealeii squid for which the skin contains light-reflecting cells called iridophores (bright spots) and pigmented organs called chromatophores (colored spots). Image credit: Roger T. Hanlon (photographer). (B) An illustration of an iridophore (Left), which shows internal Bragg stack-like ultrastructures from reflectin-based lamellae (i.e., membrane-enclosed platelets) (Inset). (C) An illustration of a chromatophore organ (Left), which shows arrangements of reflectin-based particles within the sheath cells (Inset). (D) The logo of the 28-residue-long N-terminal motif (RMN), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (E) The logo of the 28-residue-long internal motif (RMI), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (F) The logo of the 21-residue-long C-terminal motif (RMC), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (G) The amino acid sequence of full-length D. pealeii reflectin A1, which contains a single RMN motif (gray oval) and five RMI motifs (orange ovals). (H) An illustration of the selection of the prototypical truncated reflectin variant (denoted as RfA1TV) from full-length D. pealeii reflectin A1.Given reflectins’ demonstrated significance from both fundamental biology and applications perspectives, some research effort has been devoted to resolving their three-dimensional (3D) structures (30, 31, 35–39). For example, fibers drawn from full-length Euprymna scolopes reflectin 1a and films processed from truncated E. scolopes reflectin 1a were shown to possess secondary structural elements (i.e., α-helices or β-sheets) (30, 31). In addition, precipitated nanoparticles and drop-cast films from full-length Doryteuthis pealeii reflectin A1 have exhibited β-character, which was seemingly associated with their conserved motifs (35, 36). Moreover, nanoparticles assembled from both full-length and truncated Sepia officinalis reflectin 2 variants have demonstrated signatures consistent with β-sheet or α-helical secondary structure, albeit in the presence of surfactants (38). However, such studies were made exceedingly challenging by reflectins’ atypical primary sequences enriched in aromatic and charged residues, documented extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for poorly controlled aggregation (12, 14, 15, 30–32, 34–39). Consequently, the reported efforts have all suffered from multiple drawbacks, including the need for organic solvents or denaturants, the evaluation of only polydisperse or aggregated (rather than monomeric) proteins, a lack of consensus among different experimental techniques, inadequate resolution that precluded molecular-level insight, imperfect agreement between computational predictions and experimental observations, and/or the absence of conclusive correlations between structure and optical functionality. As such, there has emerged an exciting opportunity for investigating reflectins’ molecular structures, which remain poorly understood and the subject of some debate.Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a robust methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between its structural characteristics and optical properties. We first rationally select a prototypical reflectin variant expected to recapitulate the behavior of its parent protein by using a bioinformatics-guided approach. We next map the conformational and energetic landscape accessible to our selected protein by means of all-atom molecular dynamics (MD) simulations. We in turn produce our truncated reflectin variant with and without isotopic labeling, develop solution conditions that maintain the protein in a monomeric state, and characterize the variant’s size and shape with small-angle X-ray scattering (SAXS). We subsequently resolve our protein’s dynamic secondary and tertiary structures and evaluate its backbone conformational fluctuations with NMR spectroscopy. Finally, we demonstrate a straightforward mechanical agitation-based approach to controlling our truncated reflectin variant’s secondary structure, hierarchical self-assembly, and bulk refractive index distribution. Overall, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and appear poised to inform new directions across biochemistry, cellular biology, bioengineering, and optics.