Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

Binding of 3′,5′-cyclic adenosine monophosphate (cAMP) to hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels regulates their gating. cAMP binds to a conserved intracellular cyclic nucleotide-binding domain (CNBD) in the channel, increasing the rate and extent of activation of the channel and shifting activation to less hyperpolarized voltages. The structural mechanism underlying this regulation, however, is unknown. We used double electron–electron resonance (DEER) spectroscopy to directly map the conformational ensembles of the CNBD in the absence and presence of cAMP. Site-directed, double-cysteine mutants in a soluble CNBD fragment were spin-labeled, and interspin label distance distributions were determined using DEER. We found motions of up to 10 Å induced by the binding of cAMP. In addition, the distributions were narrower in the presence of cAMP. Continuous-wave electron paramagnetic resonance studies revealed changes in mobility associated with cAMP binding, indicating less conformational heterogeneity in the cAMP-bound state. From the measured DEER distributions, we constructed a coarse-grained elastic-network structural model of the cAMP-induced conformational transition. We find that binding of cAMP triggers a reorientation of several helices within the CNBD, including the C-helix closest to the cAMP-binding site. These results provide a basis for understanding how the binding of cAMP is coupled to channel opening in HCN and related channels.Ion channels are allosteric membrane proteins that open selective pores in response to various physiological stimuli, including binding of ligands and changes in transmembrane voltage (1). They are important for diverse physiological functions ranging from neurotransmission to muscle contraction. One such channel, the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel, underlies the current (termed I_h, I_f, or I_q) produced in response to hyperpolarization of cardiac pacemaker cells and neurons (2). In the heart, HCN channels are responsible for pace-making activity and may have a role in the autonomic regulation of the heart rate (3–5). In the brain, HCN channels are involved in repetitive firing of neurons and dendritic integration (6–8). Despite the important physiological roles of HCN channels, the structure of the channels and molecular mechanism of their function are not completely understood.HCN channels are part of the voltage-gated K⁺ channel superfamily (9). Like other members of this family, they are tetramers, with each subunit having a voltage-sensor domain of four transmembrane helices (S1–S4) and a pore-lining domain consisting of two transmembrane helices separated by a reentrant loop (S5-P-S6; Fig. 1A). However, HCN channels contain two key specializations that make them unique among the voltage-gated ion channels: (i) They are activated by membrane hyperpolarization instead of depolarization, and (ii) they are regulated by the direct binding of cyclic nucleotides, like the ubiquitous second messenger cAMP, to a cytoplasmic domain in the carboxyl-terminal region of the channel. The direct binding of the agonist cAMP to HCN channels increases the rate and extent of activation and shifts the voltage dependence of activation to more depolarizing voltages.Open in a separate windowFig. 1.Study of conformational changes in HCN2 using DEER. (A, Upper) Putative transmembrane topology of HCN2 channels highlighting the voltage sensor domain (S1–S4) and the pore domain (S5–S6). Only two subunits are shown. (A, Lower) Crystal structure [Protein Data Bank (PDB) ID code 3ETQ] of the cysteine-free cytoplasmic carboxyl-terminal domain of HCN2. One subunit of the tetramer is shown in color. (B) Schematic diagram showing the distance change between two cysteine-attached MTSL spin labels in a protein upon cAMP binding. In this example, the two positions are closer in the presence of cAMP. (C) Raw DEER time traces for HCN2_cys-free V537C,A624C labeled with MTSL are shown in black in the absence or presence of cAMP, as indicated. The colored curves are distance-distribution fits to the data. The oscillation frequency is higher in the presence of cAMP, indicating that the two positions are closer together in the ligand-bound form.The crystal structure of the carboxyl-terminal region bound to cAMP has been solved for several HCN channels (10–14). The nearly identical structures consist of fourfold symmetrical tetramers predicted to connect directly to the S6 segments that form the ion-conducting pore (Fig. 1A). Each of the subunits contains two domains: the cyclic nucleotide-binding domain (CNBD) and the C-linker domain. The CNBD exhibits strong structural similarity to the CNBDs of other cyclic nucleotide-binding proteins, including cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac, and the Escherichia coli catabolite gene activator protein (CAP) (15–19). The CNBD consists of an eight-stranded antiparallel β-roll, followed by two α-helices (B-helix and C-helix). cAMP binds in the anticonformation between the β-roll and the C-helix. The C-linker is a unique domain found only in HCN channels and their close homologs, cyclic nucleotide-gated (CNG) channels, and KCNH family K⁺ channels (14, 20, 21). It is situated between the CNBD and membrane-spanning domains of the channel, and is the site of virtually all intersubunit interactions in the structure (Fig. 1A). The C-linker has been found to play a key role in coupling conformational changes in the CNBD to opening of the pore (9, 22, 23).The ligand-induced movement of the C-helix is widely thought to initiate the conformational changes that lead to opening of the channel pore, but the structural evidence in support of this hypothesis is equivocal (10, 24–29). The crystal structure of the HCN2 carboxyl-terminal region in the absence of ligand shows little difference from the cyclic nucleotide-bound structure (12). The only significant differences between the two structures are observed in the F′-helix of the C-linker and in the C-helix. The proximal half of the C-helix is in the same position in the cAMP-bound and unbound structures, whereas the distal half is missing from the apo structure, indicating that it is disordered or can access multiple conformations. In contrast, studies on the soluble carboxyl-terminal fragment using transition metal ion FRET (tmFRET) demonstrate a relatively large movement (∼5 Å) at the proximal end of the C-helix upon binding of cAMP (12). The tmFRET studies also indicate a smaller movement at the distal end of the C-helix and increased disorder in the C-helix in the absence of cyclic nucleotides (12, 26).In this study, we examined the cAMP-induced conformational transition in the CNBD of HCN2 using double electron–electron resonance (DEER) spectroscopy. DEER is a pulse electron paramagnetic resonance (EPR) method that can determine distances and resolve distance distributions between pairs of sites within proteins separated by about 15–80 Å (30–33). In a typical DEER experiment, two sites in a protein are mutated to cysteines and labeled with small magnetic spin labels (Fig. 1B). DEER measures the pair’s magnetic through-space coupling via excitation of one label and probing of the other with a series of short microwave pulses. This method yields an oscillating signal whose frequency falls off with the third power of the distance between the labels (Fig. 1C). Crucially, DEER measures full-distance distributions, rather than just an average distance, providing quantitative information on structural heterogeneity and variability that is not accessible from X-ray crystal structures or ensemble FRET experiments. Using DEER, we found that the binding of cAMP to the isolated C-linker/CNBD of HCN2 causes the C-helix to move substantially toward the β-roll and decreases the conformational heterogeneity of the protein. These observations are the first step in understanding the mechanisms of ligand gating of HCN channels and the activation of other CNBD-containing proteins.     

Conserved properties of individual Ca2+-binding sites in calmodulin

Calmodulin (CaM) is a Ca²⁺-sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca²⁺ concentration into cellular signals by binding to a wide range of target proteins in a Ca²⁺-dependent manner. We do not fully understand how CaM performs its role as a high-fidelity signal transducer for more than 300 target proteins, but diversity among its four Ca²⁺-binding sites, called EF-hands, may contribute to CaM’s functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require CaM to decode Ca²⁺ signals using four specialized EF-hands, each with specific, conserved traits. In addition, we provide an extensive map of sites associated with target proteins and with human disease and correlate these with evolutionary sequence diversity. Our comprehensive evolutionary analysis provides a basis for understanding the sequence space associated with CaM function and should help guide future work on the relationship between structure, function, and disease.Eukaryotes use Ca²⁺ in numerous intracellular signaling pathways. Calmodulin (CaM) is a highly versatile Ca²⁺ signaling protein that is essential for at least dozens of cellular processes in eukaryotic cells. In humans it binds to more than 300 targets (1–3). Humans have three genes that encode identical CaM proteins, but mutations in just one of the three copies can cause disease (4–8), as can altered gene expression (9). Although CaM has been extensively studied, many details about its function are still poorly understood. The high evolutionary conservation along with the wide range of targets brings up the question of how a single Ca²⁺-binding protein displays both selectivity and flexibility in the context of its various signaling pathways.CaM binds Ca²⁺ at four, nonidentical sites that contain the structural motif called an EF-hand (10, 11), each of which contains an acidic Ca²⁺-coordinating loop, or “EF-loop” (Fig. 1A). The EF-loop spans 12 amino acids and provides at least six oxygen atoms for coordinating Ca²⁺ (12). The coordinating oxygen atoms are provided by the side chains at the first, third, fifth, and 12th positions of the EF-loop, and an oxygen from a main chain carbonyl group is provided at the seventh position (10). Water molecules participate in the Ca² coordination geometry (13). CaM functions as a sensor over a broad range of Ca²⁺ signals that vary in amplitude, duration, and location. Although biophysical and evolutionary sequence studies have resulted in a general understanding of the bulk properties of EF-hand–binding sites, the implications of differences in Ca²⁺ affinity among the four EF-hands deserves a thorough investigation.Open in a separate windowFig. 1.(A) Example of a Ca²⁺-bound EF-hand structure from PDBID 1CLL. A cartoon of an EF-hand peptide chain threads through a semitransparent representation of its molecular surface. The surface is the interface between molecular atoms and solvent rendered in PyMOL. Only atoms nearest the Ca²⁺ are shown and are depicted as spheres—green for Ca²⁺ and red for oxygens. A Ca²⁺-coordinating water is depicted as a semitransparent red sphere. Helices are gray, and the EF-loop is tan. (B) Maximum likelihood branch lengths of CaM and tubulin constrained to match the species tree in Torruella et al. (40). This tree covers much of eukaryotic diversity. Holozoa and Holomycota include animals and fungi, respectively, and their closely related protist lineages. SARPAE is described in the text. Both proteins are highly constrained, but whereas tubulin’s rate has been fairly consistent across eukaryotes, CaM underwent a dramatic speed-up in Ascomycete fungi, which include the model system S. cerevisiae.Previous reports showed that the large family of EF-hand proteins likely arose from a founder protein with a single EF-hand in the most recent common ancestor of all extant eukaryotes (11, 14–18). Different EF-hand–containing proteins bind Ca²⁺ with different affinities, suggesting that a protein with multiple EF-hands, such as CaM, may bind Ca²⁺ with a different affinity at each site (19–28). It has therefore been suggested that CaM’s four sites display different affinities and perhaps cooperativity (29, 30). We therefore hypothesized that CaM’s four, nonidentical loops may generate some of their functional flexibility by binding Ca²⁺ using different physical properties and explored whether such differences could be discerned in the evolutionary record.Evolutionary analyses can provide mechanistic insight into how CaM is used as a Ca²⁺ sensor across eukaryotes. Prior work showed that the protein sequence of CaM is evolving at a faster pace in fungal species (11, 31–33), reflecting the fact that although CaM is essential in Saccharomyces cerevisiae, the cells can survive with all four EF-hands ablated (34). However, previous evolutionary studies focused on a small subset of eukaryotes, either because few sequences were available at the time of publication or because the study was focused on a particular lineage. The vast expansion of taxonomic coverage in sequence databases, and the recent availability of new NMR and X-ray crystal structures of CaM, therefore demands a more comprehensive analysis. Unfortunately, CaM is a small, ancient, and highly conserved protein and therefore does not contain enough information to infer phylogenetic tree topologies. Kretsinger and Nakayama and coworkers (11, 16, 17, 35), for instance, found little correspondence between phylogenies inferred from protein, DNA, or intron–exon structure.To overcome this hurdle, we used a variety of techniques to explore sequence and structural conservation in CaM across eukaryotes. Our approach allows us to address several key questions: (i) How fast is CaM diverging in different phyla? (ii) How does the function of a site, or its association with disease, correlate with sequence conservation? (iii) What properties of the EF-hands are conserved over deep evolutionary time, and how might this correspond to functional plasticity?     

Energy landscape views for interplays among folding,binding, and allostery of calmodulin domains

Ligand binding modulates the energy landscape of proteins, thus altering their folding and allosteric conformational dynamics. To investigate such interplay, calmodulin has been a model protein. Despite much attention, fully resolved mechanisms of calmodulin folding/binding have not been elucidated. Here, by constructing a computational model that can integrate folding, binding, and allosteric motions, we studied in-depth folding of isolated calmodulin domains coupled with binding of two calcium ions and associated allosteric conformational changes. First, mechanically pulled simulations revealed coexistence of three distinct conformational states: the unfolded, the closed, and the open states, which is in accord with and augments structural understanding of recent single-molecule experiments. Second, near the denaturation temperature, we found the same three conformational states as well as three distinct binding states: zero, one, and two calcium ion bound states, leading to as many as nine states. Third, in terms of the nine-state representation, we found multiroute folding/binding pathways and shifts in their probabilities with the calcium concentration. At a lower calcium concentration, “combined spontaneous folding and induced fit” occurs, whereas at a higher concentration, “binding-induced folding” dominates. Even without calcium binding, we observed that the folding pathway of calmodulin domains can be modulated by the presence of metastable states. Finally, full-length calmodulin also exhibited an intriguing coupling between two domains when applying tension.Protein folding and conformational dynamics have often been characterized by the energy landscape of proteins (1–5). The energy landscape is dependent on the molecular physiochemistry and thus is modulated by many factors, such as chemical modification and ligand binding. Ligand binding, in turn, is dependent on the conformation of proteins. Thus, folding, binding, and allosteric conformational dynamics are mutually correlated. Despite their obvious correlation in concept, it has been very challenging to characterize how they are indeed coupled for any single proteins. Here, we address, in depth, how these three types of dynamics, folding, binding, and allosteric conformational dynamics, are coupled from the energy landscape perspective for a specific protein, calmodulin (CaM).CaM is a ubiquitous calcium-binding messenger protein involved in signal transduction (6) and, more importantly here, has been a model protein to investigate folding, binding, and allostery. Full-length CaM has two nearly symmetric globular domains connected by a flexible central helix (7, 8). Each domain is composed of paired EF hands containing two Ca²⁺-binding sites (Fig. 1A). Upon binding to Ca²⁺, each CaM domain undergoes substantial conformational change from a closed state to an open state, exposing a hydrophobic patch that can bind with target proteins and regulate downstream processes (9). CaM has been frequently used as a model in studying the folding of multidomain proteins (10, 11), allosteric transitions (12–14), slow conformational dynamics around physiological temperatures (15–18), metal ion binding (19, 20), and correlation between inherent flexibility and protein functions (21, 22). For example, using structure-based coarse-grained (CG) simulations, Chen and Hummer elucidated the coexistence of an unfolded state, a closed state, and an open state around physiological temperatures for the C-terminal domain of CaM (CaM-C) without Ca²⁺ binding (15), which reconciles some seemingly contradictory experimental observations on the slow conformational dynamics of CaM.Open in a separate windowFig. 1.(A) Three-dimensional structure of calmodulin domain at closed [Protein Data Bank (PDB) code: 1cfd] and open states (PDB code:1cll). Calcium ions are represented by yellow spheres. (B) Schematic of coupling among folding, calcium binding, and allosteric motions for the CaM domain. Due to the conformational transitions between open and closed states, in addition to the direct folding pathway (red solid arrow), folding to the most stable state may involve an alternative pathway via a metastable state (green arrow plus blue arrow). The calcium binding can modulate the relative stability of the conformational states and therefore the population of folding pathways. O, C, and U represent open, closed, and unfolded states, respectively.More recently, Rief and coworkers studied the Ca²⁺-dependent folding of CaM based on a new generation technique of single-molecule force spectroscopy, which can probe the reversible folding/unfolding transitions with near equilibrium conditions (10, 23, 24). Their results revealed that at high Ca²⁺ concentrations, the folding pathway of the CaM domain proceeds via a transition state capable of binding Ca²⁺ ions, demonstrating the coupling between Ca²⁺ binding and CaM folding. All these computational and experimental works provided unprecedented understanding of many aspects of the folding and allosteric transitions of CaM. However, a full picture of the coupling among folding, Ca²⁺ binding, and allosteric motions, as schematically shown in Fig. 1B, is still lacking. Particularly, two fundamental issues arising from the allostery and Ca²⁺-binding characteristics of CaM remain elusive: (i) How does the allosteric feature of the energy landscape contribute to the folding complexity? And (ii) how can the folding mechanism of CaM be modulated by Ca²⁺ binding?Motivated by previous computational and experimental studies (15, 23), in this work we investigated the folding coupled with Ca²⁺ binding and allosteric motions of the isolated CaM domains as well as the full-length CaM. To do so, we first integrated computational tools developed for folding, ligand binding, and allosteric motions together. The proposed CG protein model was used for the subsequent series of molecular dynamics (MD) simulations. First, corresponding to Rief’s experiments, we performed MD simulations of isolated CaM domains with pretensions, which gave consistent results with the experiments and, in addition, provided the direct structural assignment on the experimentally observed states. Second, at a higher temperature, without pretension we performed reversible folding/unfolding simulations for a wide range of Ca²⁺ concentrations. The conformational and ligand-binding energy landscape revealed as many as nine distinctive states. Then, we analyzed the binding-coupled folding reactions in terms of the nine states, finding multiple routes and their modulation by Ca²⁺ concentrations. Interestingly, as the Ca²⁺ concentration increases, the CaM domain folding mechanism switches from “combined spontaneous folding and induced fit” to “binding-induced folding,” which accords with the scenario deduced from single-molecule force spectroscopy experiments. Finally, the effects of tension on the conformational fluctuations of the full-length CaM are discussed.     

A synthetic porphyrin as an effective dual antidote against carbon monoxide and cyanide poisoning

Simultaneous poisoning by carbon monoxide (CO) and hydrogen cyanide is the major cause of mortality in fire gas accidents. Here, we report on the invention of an injectable antidote against CO and cyanide (CN^–) mixed poisoning. The solution contains four compounds: iron(III)porphyrin (Fe^IIITPPS, F), two methyl-β-cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) and imidazole (Im3CD, I), and a reducing agent (Na₂S₂O₄, S). When these compounds are dissolved in saline, the solution contains two synthetic heme models including a complex of F with P (hemoCD-P) and another one of F with I (hemoCD-I), both in their iron(II) state. hemoCD-P is stable in its iron(II) state and captures CO more strongly than native hemoproteins, while hemoCD-I is readily autoxidized to its iron(III) state to scavenge CN^– once injected into blood circulation. The mixed solution (hemoCD-Twins) exhibited remarkable protective effects against acute CO and CN^– mixed poisoning in mice (~85% survival vs. 0% controls). In a model using rats, exposure to CO and CN^– resulted in a significant decrease in heart rate and blood pressure, which were restored by hemoCD-Twins in association with decreased CO and CN^– levels in blood. Pharmacokinetic data revealed a fast urinary excretion of hemoCD-Twins with an elimination half-life of 47 min. Finally, to simulate a fire accident and translate our findings to a real-life scenario, we confirmed that combustion gas from acrylic cloth caused severe toxicity to mice and that injection of hemoCD-Twins significantly improved the survival rate, leading to a rapid recovery from the physical incapacitation.

Fire accidents frequently occur around the world and the consequent inhalation of combustion gases represents the leading cause of death under these circumstances (1–10). Typically, combustion gases consist, among others, of carbon dioxide, water (H₂O), carbon monoxide (CO), hydrogen cyanide (HCN), hydrochloric acid, nitrogen oxide, and sulfur oxide. Their amounts depend on the materials of combustion (3–10) and, among them, CO and HCN are the most dangerous due to their severe toxic effects in humans (3–4, 5–15). CO is generated during incomplete combustion of carbon materials while HCN derives from carbon- and nitrogen-containing materials. Thus, CO and HCN gases are simultaneously released when synthetic materials such as plastics, acrylic cloths, and urethanes are burned in buildings. Once inhaled, CO strongly binds to ferrous iron(II) heme proteins such as hemoglobin (Hb) in erythrocytes and myoglobin in muscles (16–19), thus compromising oxygen (O₂) transport/storage in the blood circulation and tissues. On the contrary, HCN binds as cyanide ion (CN^–) to ferric iron(III) heme, preferentially targeting cytochrome c oxidase (CcO) in mitochondria and interrupting O₂-dependent energy production (11, 20–22). Likewise, the toxic effects of CO on mitochondrial respiration are ascribed to its ability to bind to cytochrome c oxidase (23–25). Therefore, both CO and HCN can inhibit aerobic respiration mediated by several heme proteins leading to anoxia-induced lethal toxicity. As the molecular mechanisms of CO and HCN toxicity are closely related, additional or synergistic deleterious effects can be expected when these two gases are produced and inhaled at the same time (26–31). Therapeutic strategies to treat either CO or HCN poisoning have been developed independently (11, 13, 22, 32–37). However, to our knowledge, there is no medical intervention at present to neutralize simultaneously CO and HCN with an effective treatment in vivo.In the present study, we have developed an injectable antidote for CO and HCN mixed poisoning. This agent contains supramolecular compounds, termed hemoCDs, composed of a water-soluble iron(II/III)porphyrin and two cyclodextrin (CD) dimers. Based on our previous research on the synthesis and characterization of heme protein model structures (38–40), the gas-binding ability and the redox status of the iron center of the porphyrin can be regulated by the linker structure of the CD dimers englobing the porphyrin. The porphyrin complexed with a CD dimer having a pyridine linker (hemoCD-P, Fig. 1 A, Left) is stable in the iron(II) state in vivo and shows much higher CO-binding affinity compared to the affinities reported for native heme proteins (41–45). On the contrary, the porphyrin complexed with a CD dimer having an imidazole linker (hemoCD-I, Fig. 1 A, Right) is stable in the iron(III) state and shows a higher binding affinity to CN^– than native ferric met-hemoglobin (met-Hb) (46, 47). These two complexes do not bind to plasma proteins in the circulation when injected separately and are quickly excreted in urines without any chemical decomposition. Therefore, these compounds have the ability to capture either CO or CN^– to their iron(II/III) centers in vivo and expel these toxic ligands from the organism (41–47). Here, we have developed hemoCD-Twins that contains both hemoCD-P and hemoCD-I in saline for simultaneous removal of CO and HCN in vivo. This paper describes the primary pharmacological properties of hemoCD-Twins and its detoxification effects in animals intoxicated with CO and CN^–. To simulate a real-life fire accident, the burning of acrylic cloth with consequent release of combustion gases was also used as an experimental approach for antidotal tests. We found that hemoCD-Twins exhibits the following features: 1) The synthetic compounds are storable at room temperature over a year thanks to their chemical stability; 2) the solution can be quickly prepared without the need of special handlings; 3) a single administration of hemoCD-Twins shows immediate dual antidotal effect against CO and CN^–; and 4) the injected solution is quickly eliminated from the body without significant side effects. We are envisioning a scenario whereby a person who is accidentally exposed to fire gases containing CO and HCN can be promptly treated at the site by infusion with hemoCD-Twins.Open in a separate windowFig. 1.hemoCD-Twins as an antidote system against CO and CN^– mixed poisoning. (A) Biomimetic heme protein model compounds hemoCD-P and hemoCD-I that are used as CO and CN^– scavengers in vivo. (B) Chemical structures of the four components P, I, F, and S contained in hemoCD-Twins. (C) Preparation of hemoCD-Twins. Three powder compounds, F, P, and I, were dissolved in PBS in a 2:1:1 molar ratio, where the solution contains hemoCD-P and hemoCD-I in ferric iron(III) states. The solution is stable and storable at room temperature. The reducing agent S is added just before use. (D) Schematic illustration for the simultaneous removal of CO and CN^– by a single injection of hemoCD-Twins in vivo. CO and CN^– are removed from living organisms and excreted in the urine with hemoCD-P and hemoCD-I in ferrous iron(II) and ferric iron(III) complexes, respectively.     

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.Although proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein–protein interactions (2–4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein’s structural dynamics link to its functional mechanisms (12–21).A case in point is the nitric oxide synthase (NOS) enzymes (22–24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached “oxygenase” domain, which then enables NO synthesis to begin (22, 25–27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation (Fig. 1 A and B) (28, 30–36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42).Open in a separate windowFig. 1.(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31, 32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study. It has long been known that CaM binding alters NOSr structure such that, on average, it populates a more open conformation (43, 44). Recent equilibrium studies have detected a buildup of between two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26, 36, 37, 39, 40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34, 36, 45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.To address this gap, we used single-molecule fluorescence energy resonance transfer (FRET) spectroscopy to characterize individual molecules of nNOSr that had been labeled at two specific positions with Cyanine 3 (Cy3) donor and Cyanine 5 (Cy5) acceptor dye molecules, regarding their conformational states distribution and the associated conformational fluctuation dynamics, which in turn enabled us to determine how CaM binding impacts both parameters. This work provides a unique perspective and a novel study of the NOS enzymes and within the broader flavoprotein family, which includes the mammalian enzymes methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), and reveals how CaM’s control of the conformational behaviors may regulate the electron transfer reactions of NOS catalysis.     

An experimental test of the nicotinic hypothesis of COVID-19

The pathophysiological mechanisms underlying the constellation of symptoms that characterize COVID-19 are only incompletely understood. In an effort to fill these gaps, a “nicotinic hypothesis,” which posits that nicotinic acetylcholine receptors (AChRs) act as additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptors, has recently been put forth. A key feature of the proposal (with potential clinical ramifications) is the suggested competition between the virus’ spike protein and small-molecule cholinergic ligands for the receptor’s orthosteric binding sites. This notion is reminiscent of the well-established role of the muscle AChR during rabies virus infection. To address this hypothesis directly, we performed equilibrium-type ligand-binding competition assays using the homomeric human α7-AChR (expressed on intact cells) as the receptor, and radio-labeled α-bungarotoxin (α-BgTx) as the orthosteric-site competing ligand. We tested different SARS-CoV-2 spike protein peptides, the S1 domain, and the entire S1–S2 ectodomain, and found that none of them appreciably outcompete [¹²⁵I]-α-BgTx in a specific manner. Furthermore, patch-clamp recordings showed no clear effect of the S1 domain on α7-AChR–mediated currents. We conclude that the binding of the SARS-CoV-2 spike protein to the human α7-AChR’s orthosteric sites—and thus, its competition with ACh, choline, or nicotine—is unlikely to be a relevant aspect of this complex disease.

According to official reports, as of August 2022, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected nearly 600 million people and caused more than 6.5 million deaths worldwide (1). According to recent estimates by the World Health Organization that aim to capture deaths missed by national reporting systems, however, the pandemic’s true death toll is actually much higher: it amounts to ∼15 million (2). Despite intensive research, our understanding of the pathophysiological mechanisms underlying the broad range of respiratory, neurological, psychiatric, and cardiovascular symptoms that follow this viral infection remains limited (3–7). Although the angiotensin-converting enzyme 2 (ACE2) was identified as the main cell-entry receptor (8–10), other plasma membrane receptors (such as neuropilin-1) (11, 12) and cell-surface glycocalyx components (such as heparan sulfate) (13) were also reported to participate in the different facets of this disease.On the basis of amino acid sequence similarities between the SARS-CoV-2 spike protein and snake venom neurotoxins, it has recently been hypothesized that this coronavirus may also bind to nicotinic acetylcholine receptors (AChRs) (14–17). Moreover, it was suggested that the spike protein would bind to the receptor at a site that overlaps with the neurotransmitter-binding (“orthosteric”) sites, in such a way that neurotoxins, the spike protein, and small-molecule cholinergic ligands would all bind to the receptor in a mutually exclusive, competitive manner. On the spike protein, the regions that were hypothesized to bind to AChRs map to two separate sequences: S³⁷⁵TFKCYGVSPTKLNDL (S375–L390) (18), near the middle of the ACE2-binding domain (receptor-binding domain, RBD), and Y⁶⁷⁴QTQTNSPRRAR (Y674–R685) (14), at the furin-cleavage site between domains S1 and S2 (Fig. 1). Remarkably, this bold proposal received ample support from molecular-simulation studies that led to the identification of putative interatomic interactions bridging the AChR–spike protein-binding interface (18, 19). Importantly, these simulations also suggested that the Y674–R685 stretch of amino acids remains accessible—and thus, fully competent to bind to the AChR—in the context of the fully glycosylated, full-length spike protein. Furthermore, the interaction between the receptor and the Y674–R685 spike protein peptide was found to be highly dependent on the AChR subtype, the peptide seemingly acting as an antagonist of the α4β2-AChR and the fetal-muscle-type AChR, and probably, as an agonist of the α7-AChR (19). These differences suggest that the extrapolation of experimental results obtained with one type of AChR to another one need not be valid, despite the highly similar binding modes of neurotoxins to muscle-type and α7-AChRs (20, 21).Open in a separate windowFig. 1.Amino acid sequence of the spike protein of SARS-CoV-2 (GenBank: {"type":"entrez-protein","attrs":{"text":"QHD43416.1","term_id":"1791269090","term_text":"QHD43416.1"}}QHD43416.1). A PRRAR furin-cleavage site (a part of the Y674–R685 stretch, in cyan letters and underlined) separates the S1 domain from the S2 domain. The signal peptide is indicated with green letters; the ACE2-binding domain (RBD) is in orange; the S375–L390 stretch is in red and underlined; and the transmembrane segment is in magenta. The N terminus faces the extracellular milieu.An interaction between the spike protein and AChRs could have pathological consequences not only because it could provide an alternative pathway for the virus to attach to and enter cells, but also because it could disrupt physiological AChR-mediated signaling. Moreover, the notion that the binding of the spike protein to the AChR is competitive with that of small-molecule cholinergic ligands would suggest a novel mechanism by which nicotine consumption and smoking-cessation drugs could affect the course of the disease (15–17, 22–24), the better understood mechanisms being the direct effect of nicotine and its analogs on the α7-AChR–mediated antiinflammatory response to viral infection (25–29).However far-fetched these ideas may have seemed when first put forth, there is a well-known precedent: the rabies virus glycoprotein was reported to bind to the muscle-type (α1β1γδ) AChR in a manner that is mutually exclusive with the binding of α-bungarotoxin [a 74-amino acid neurotoxin from the Formosan banded krait; α-BgTx (30)] (31–35). This finding, along with other pieces of experimental evidence (e.g., refs. 36–38), has led to the well-established notion that the muscle AChR is one of the cell-attachment receptors for the rabies virus (39, 40). Quite notably, similar claims were made about the human-immunodeficiency virus-1 (HIV-1) glycoprotein 120 (gp120) and the muscle AChR (41, 42).Given this background, the suggestion of a binding interaction between the SARS-CoV-2 spike protein and several AChRs (α1β1γδ, α4β2, α7, and α9) (14, 18, 19) seemed most intriguing and worth investigating experimentally. To this end, we performed equilibrium-type ligand-binding competition studies using the homomeric human α7-AChR (expressed on intact cells) as the receptor, and radio-labeled α-BgTx (at a concentration that half-saturates the α7-AChR) as the competing ligand. We found that the two spike protein peptides (tested up to a concentration of ∼250 μM), the S1 domain (∼1.2 μM), and the entire S1–S2 ectodomain (∼375 nM) fail to displace bound α-BgTx from this receptor to any appreciable degree. Furthermore, we found that the S1 domain (∼20 nM) has no obvious effects on α7-AChR channel function. Thus, it seems inescapable to conclude that the binding of the SARS-CoV-2 spike protein to the human α7-AChR’s orthosteric sites—and more specifically, the competition with ACh, choline, or nicotine for so doing—is unlikely to be a relevant aspect of this complex disease.     

Conjugated polymers based on metalla-aromatic building blocks

Conjugated polymers usually require strategies to expand the range of wavelengths absorbed and increase solubility. Developing effective strategies to enhance both properties remains challenging. Herein, we report syntheses of conjugated polymers based on a family of metalla-aromatic building blocks via a polymerization method involving consecutive carbyne shuttling processes. The involvement of metal d orbitals in aromatic systems efficiently reduces band gaps and enriches the electron transition pathways of the chromogenic repeat unit. These enable metalla-aromatic conjugated polymers to exhibit broad and strong ultraviolet–visible (UV–Vis) absorption bands. Bulky ligands on the metal suppress π–π stacking of polymer chains and thus increase solubility. These conjugated polymers show robust stability toward light, heat, water, and air. Kinetic studies using NMR experiments and UV–Vis spectroscopy, coupled with the isolation of well-defined model oligomers, revealed the polymerization mechanism.

Conjugated polymers are macromolecules usually featuring a backbone chain with alternating double and single bonds (1–3). These characteristics allow the overlapping p-orbitals to form a system with highly delocalized π-electrons, thereby giving rise to intriguing chemical and physical properties (4–6). They have exhibited many applications in organic light-emitting diodes, organic thin film transistors, organic photovoltaic cells, chemical sensors, bioimaging and therapies, photocatalysis, and other technologies (7–10). To facilitate the use of solar energy, tremendous efforts have been devoted in recent decades to developing previously unidentified conjugated polymers exhibiting broad and strong absorption bands (11–13). The common strategies for increasing absorption involve extending π-conjugation by incorporating conjugated cyclic moieties, especially fused rings; modulating the strength of intramolecular charge transfer between donor and acceptor units (D–A effect); increasing the coplanarity of π conjugation through weak intramolecular interactions (e.g., hydrogen bonds); and introducing heteroatoms or heavy atoms into the repeat units of conjugated polymers (11–16). Additionally, appropriate solubility is a prerequisite for processing and using polymers and is usually achieved with the aid of long alkyl or alkoxy side chains (12, 17).Aromatic rings are among the most important building blocks for conjugated polymers. In addition to aromatic hydrocarbons, a variety of aromatic heterocycles composed of main-group elements have been used as fundamental components. These heteroatom-containing conjugated polymers show unique optical and electronic properties (4–10). However, while metalla-aromatic systems bearing a transition metal have been known since 1979 due to the pioneering work by Thorn and Hoffmann (18), none of them have been used as building blocks for conjugated polymers. The HOMO–LUMO gaps (E_g) of metalla-aromatics are generally narrower (Fig. 1) than those of their organic counterparts (19–22). We reasoned that this feature should broaden the absorption window if polymers stemming from metalla-aromatics are achievable.Open in a separate windowFig. 1.Comparison of traditional organic skeletons with metalla-aromatic building blocks (the computed energies are in eV). (A) HOMO–LUMO gaps of classic aromatic skeletons. (B) Carbolong frameworks as potential building blocks for novel conjugated polymers with broad absorption bands and improved solubility.In recent years, we have reported a series of readily accessible metal-bridged bicyclic/polycyclic aromatics, namely carbolong complexes, which are stable in air and moisture (23–25). The addition of osmium carbynes (in carbolong complexes) and alkynes gave rise to an intriguing family of d_π–p_π conjugated systems, which function as excellent electron transport layer materials in organic solar cells (26, 27). These observations raised the following question: Can this efficient addition reaction be used to access metalla-aromatic conjugated polymers? It is noteworthy that incorporation of metalla-aromatic units into conjugated polymers is hitherto unknown. In this contribution, we disclose a polymerization reaction involving M≡C analogs of C≡C bonds, which involves a unique carbyne shuttling strategy (Fig. 2A). This led to examples of metalla-aromatic conjugated polymers (polycarbolongs) featuring metal carbyne units in the main chain. On the other hand, the development of polymerization reactions plays a crucial role in involving certain building blocks in conjugated polymers (28–32). These efficient, specific, and feasible polymerizations could open an avenue for the synthesis of conjugated polymers.Open in a separate windowFig. 2.Design of polymers and synthesis of monomers. (A) Schematic illustration of the polymerization strategy. (B) Preparation of carbolong monomers. Insert: X-ray molecular structure for the cations of complex 3. Ellipsoids are shown at the 50% probability level; phenyl groups in PPh₃ are omitted for clarity.     

Repertoire-scale measures of antigen binding

Antibodies and T cell receptors (TCRs) are the fundamental building blocks of adaptive immunity. Repertoire-scale functionality derives from their epitope-binding properties, just as macroscopic properties like temperature derive from microscopic molecular properties. However, most approaches to repertoire-scale measurement, including sequence diversity and entropy, are not based on antibody or TCR function in this way. Thus, they potentially overlook key features of immunological function. Here we present a framework that describes repertoires in terms of the epitope-binding properties of their constituent antibodies and TCRs, based on analysis of thousands of antibody–antigen and TCR–peptide–major-histocompatibility-complex binding interactions and over 400 high-throughput repertoires. We show that repertoires consist of loose overlapping classes of antibodies and TCRs with similar binding properties. We demonstrate the potential of this framework to distinguish specific responses vs. bystander activation in influenza vaccinees, stratify cytomegalovirus (CMV)-infected cohorts, and identify potential immunological “super-agers.” Classes add a valuable dimension to the assessment of immune function.

Repertoires are routinely characterized according to the number and frequency of unique V(D)J-recombined antibody and T cell receptor (TCR) gene sequences they contain (henceforth “genes;” Fig. 1A). This is known as sequence diversity and is measured using a variety of sequence-based diversity indices, including (species) richness, Shannon entropy (1, 2), and others related to Hill’s ^qD-number framework (Fig. 1B) (3). Sequence-based diversity indices (henceforth “sequence diversity”) have shown promise as biomarkers, for example, as predictors of response to cancer immunotherapy (4) and as correlates of healthy aging (5–7). However, sequence diversity overlooks fundamental features of repertoire function. For example, sequence diversity cannot indicate whether a repertoire with a given number of different genes contains epitope-binding capacity (8) for many different epitopes or for only a few (Fig. 1 C and D), or how well antibodies or TCRs from a second repertoire might also bind a given set of epitopes (Fig. 1E). The reason for this shortcoming is that sequence diversity measures only the number of different antibodies or TCRs, but not their basic function: epitope binding.Open in a separate windowFig. 1.Sequence diversity vs. class diversity. Each circle represents a B or T cell; each color represents a unique antibody or TCR sequence. Similar colors encode antibodies or TCRs with similar epitope binding properties. Two repertoires, for example, repertoires 1 and 2 (A), that have the same total number of cells (A) and identical sequence frequency distributions (B), have identical sequence diversity (for all ^qD); Insets give the effective number versions (3, 50, 58) of entropy and BPI, ¹D = e^{Shannon entropy} and ^∞D = 1/BPI. Lower pairwise binding similarities in repertoire 2 (C) give repertoire 2 higher class diversity than repertoire 1; repertoire 2 can recognize more different epitopes (D). Color coding reflects optimal binding (e.g., red sequence, red epitope). The colors of the bars in E indicate the contributions of the antibody or TCR encoded by the sequence of that color. Similar colors bind better than different colors. Higher frequencies (B) can partially compensate for weaker binding.Epitope binding—of antibody to antigen or of TCR to peptide–major histocompatibility complex (pMHC)—is routinely measured using dissociation constants (K_d), for example, to determine which of several antibodies has the highest affinity for a given epitope (9, 10). (Another common measure is the half maximal inhibitory concentration [IC₅₀], used in inhibition experiments.) K_d is related to the Gibbs free energy of binding (ΔG) by the equation ΔG = −RTln(K_d), where R is the gas constant and T is the temperature, illustrating the relationship between K_d and thermodynamic first principles. In immunology, it is widely understood that antibodies or TCRs with similar gene sequences often have similar K_d for a given set of antigens or pMHCs (11–13), even as targeted substitutions of amino acids can change K_d enough to effectively abolish binding (14, 15) [binding is “error-tolerant but attack-prone” (16)]. Binding similarity among antibodies or TCRs (Fig. 1C) is the basis of phenomena fundamental to adaptive immunity, including polyspecificity/cross-reactivity and degeneracy/redundancy (17, 18). These phenomena are what allow so-called natural antibodies (IgM) to recognize many different antigens despite relatively low sequence diversity, with large numbers of antibodies of similar specificity compensating for individually weak K_ds (19, 20). Thus, in a qualitative sense, the idea that binding similarities between antibodies or TCRs can, in the aggregate, have important repertoire-scale effects is well established (Fig. 1 D and E) (21). We sought to develop this idea quantitatively, by developing quantitative repertoire-scale measures based on the binding properties of repertoires’ constituent antibodies and TCRs.     

Opacity and conductivity measurements in noble gases at conditions of planetary and stellar interiors

The noble gases are elements of broad importance across science and technology and are primary constituents of planetary and stellar atmospheres, where they segregate into droplets or layers that affect the thermal, chemical, and structural evolution of their host body. We have measured the optical properties of noble gases at relevant high pressures and temperatures in the laser-heated diamond anvil cell, observing insulator-to-conductor transformations in dense helium, neon, argon, and xenon at 4,000–15,000 K and pressures of 15–52 GPa. The thermal activation and frequency dependence of conduction reveal an optical character dominated by electrons of low mobility, as in an amorphous semiconductor or poor metal, rather than free electrons as is often assumed for such wide band gap insulators at high temperatures. White dwarf stars having helium outer atmospheres cool slower and may have different color than if atmospheric opacity were controlled by free electrons. Helium rain in Jupiter and Saturn becomes conducting at conditions well correlated with its increased solubility in metallic hydrogen, whereas a deep layer of insulating neon may inhibit core erosion in Saturn.Noble gases play important roles in the evolution and dynamics of planets and stars, especially where they appear in a condensed, purified state. In gas giant planets, helium and neon can precipitate as rain in metallic hydrogen envelopes, leading to planetary warming and specifically the anomalously slow cooling of Saturn (1–8). In white dwarf stars cooling can be especially fast due to the predicted low opacity of dense helium atmospheres, affecting the calibration of these objects as cosmological timekeepers (9–12). In these systems, the transformation of dense noble gases (particularly He) from optically transparent insulators to opaque electrical conductors is of special importance (2, 9, 11, 12).Dense noble gases are expected to show systematic similarities in their properties at extreme conditions (13–17); however, a general understanding of their insulator–conductor transformation remains to be established. Xe is observed to metallize near room temperature under pressures similar to those at Earth’s core–mantle boundary (18, 19). Ar and He are observed to conduct only at combined high pressure and temperature (12, 13, 17). Ne is predicted to have the highest metallization pressure of all known materials—10³ times that of Xe and 10 times that of He (14, 18, 20, 21)—and has never been documented outside of its insulating state. Experimental probes of extreme densities and temperatures in noble gases have previously relied on dynamic compression by shock waves (12, 13, 17, 22–24). However, in such adiabatic experiments, light and compressible noble gases heat up significantly and can ultimately reach density maxima (12, 13, 17, 21, 24, 25), so that conditions created often lie far from those deep within planets (7, 8) and stars (9).Here we report experiments in the laser-heated diamond anvil cell (15, 16, 26–29) on high-density and high-temperature states of the noble gases Xe, Ar, Ne, and He (Fig. 1). Rapid heating and cooling of compressed samples using pulsed laser heating (26, 27) is coupled with time domain spectroscopy of thermal emission (26) to determine sample temperature and transient absorption to establish corresponding sample optical properties (Figs. S1 and S2). A sequence of heat cycles to increasing temperature documents optical changes in these initially transparent insulators.Open in a separate windowFig. 1.Creating and probing extreme states of noble gases. (A) Configuration of laser heating and transient absorption probing of the diamond anvil cell, with probe beams transmitted through the cell into the detection system. (B) Microscopic view of the diamond cell cavity, which contains a noble gas sample and a metal foil (Ir) which converts laser radiation to heat and has small hole at the heated region through which probe beams are transmitted to test optical character of samples. (C) Finite element model (26) (Fig. S3) of the temperature distribution in heated Ar at 51 GPa (Fig. 2), with solid–melt (16) and insulator–conductor (α = 0.1 μm⁻¹) boundaries in the sample marked dashed and dotted, respectively. (D) Schematic of time domain probing during transient heating. Temperature is determined from thermal emission (red) and absorption from transmitted probe beams: a continuous laser (cw; green) and pulsed supercontinuum broadband (bb; blue).     

Electrophilic aromatic substitution reactions of compounds with Craig-Möbius aromaticity

Electrophilic aromatic substitution (EAS) reactions are widely regarded as characteristic reactions of aromatic species, but no comparable reaction has been reported for molecules with Craig-Möbius aromaticity. Here, we demonstrate successful EAS reactions of Craig-Möbius aromatics, osmapentalenes, and fused osmapentalenes. The highly reactive nature of osmapentalene makes it susceptible to electrophilic attack by halogens, thus osmapentalene, osmafuran-fused osmapentalene, and osmabenzene-fused osmapentalene can undergo typical EAS reactions. In addition, the selective formation of a series of halogen substituted metalla-aromatics via EAS reactions has revealed an unprecedented approach to otherwise elusive compounds such as the unsaturated cyclic chlorirenium ions. Density functional theory calculations were conducted to study the electronic effect on the regioselectivity of the EAS reactions.

Aromaticity, a core concept in chemistry, was initially introduced to account for the bonding, stability, reactivity, and other properties of many unsaturated organic compounds. There have been many elaborations and extensions of the concept of aromaticity (1, 2). The concepts of Hückel aromaticity and Möbius aromaticity are widely accepted (Fig. 1A). A π-aromatic molecule of the Hückel type is planar and has 4n + 2 conjugated π-electrons (n = 0 or an integer), whereas a Möbius aromatic molecule has one twist of the π-system, similar to that in a Möbius strip, and 4n π-electrons (3, 4). Since the discovery of naphthalene in 1821, aromatic chemistry has developed into a rich field and with a variety of subdisciplines over the course of its 200-y history, and the concept of aromaticity has been extended to other nontraditional structures with “cyclic delocalization of mobile electrons” (5). For example, benzene-like metallacycles—predicted by Hoffmann et al. as metallabenzenes—in which a metal replaces a C–H group in the benzene ring (6), have garnered extensive research interest from both experimentalists and theoreticians (7–12). As paradigms of the metalla-aromatic family, most complexes involving metallabenzene exhibit thermodynamic stability, kinetic persistence, and chemical reactivity associated with the classical aromaticity concept (13–15). Typically, like benzene, metallabenzene can undergo characteristic reactions of aromatics such as electrophilic aromatic substitution (EAS) reactions (16–18) (Fig. 1B, I) and nucleophilic aromatic substitution reactions (19–21).Open in a separate windowFig. 1.Schematic representations of aromaticity classification (A) and EAS reactions (B) of benzene, metallabenzene, and polycyclic metallacycles with Craig-Möbius aromaticity.The incorporation of transition metals has also led to an increase in the variety of the aromatic families (22–25). We have reported that stable and highly unusual bicyclic systems, metallapentalenes (osmapentalenes), benefit from Craig-Möbius aromaticity (26–30). In contrast to other reported Möbius aromatic compounds with twisted topologies, which are known as Heilbronner-Möbius aromatics (31–34), the involvement of transition metal d orbitals in π-conjugation switches the Hückel anti-aromaticity of pentalene into the planar Craig-Möbius aromaticity of metallapentalene (35–38) (Fig. 1A, III). Both the twisted topology and the planar Craig-Möbius aromaticity are well established and have been accepted as reasonable extensions of aromaticity (39–43). There has been no experimental evidence, however, as to whether these Möbius aromatic molecules can undergo classical aromatic substitution reactions, such as EAS reactions, instead of addition reactions. Given the key role of EAS in aromatic chemistry to obtain various derivatives, we sought to extend the understanding of the reactivity paradigm in the metalla-aromatic family.Our recent synthetic efforts associated with the metallapentalene system prompted us to investigate whether typical EAS reactions could proceed in these Craig-Möbius aromatics. If so, how could substitution be achieved in the same way that it is with traditional Hückel aromatics such as benzenes? In this paper, we present EAS reactions, mainly the halogenation of osmapentalene, osmafuran-fused osmapentalene, and osmabenzene-fused osmapentalene, which follow the classic EAS mechanistic scheme (Fig. 1B). With the aid of density functional theory (DFT) calculations, we characterized the effects on EAS reactivity and regioselectivity.     

Recent Advances in Archaeological Science Techniques Special Feature: A Neandertal dietary conundrum: Insights provided by tooth enamel Zn isotopes from Gabasa,Spain

The characterization of Neandertals’ diets has mostly relied on nitrogen isotope analyses of bone and tooth collagen. However, few nitrogen isotope data have been recovered from bones or teeth from Iberia due to poor collagen preservation at Paleolithic sites in the region. Zinc isotopes have been shown to be a reliable method for reconstructing trophic levels in the absence of organic matter preservation. Here, we present the results of zinc (Zn), strontium (Sr), carbon (C), and oxygen (O) isotope and trace element ratio analysis measured in dental enamel on a Pleistocene food web in Gabasa, Spain, to characterize the diet and ecology of a Middle Paleolithic Neandertal individual. Based on the extremely low δ⁶⁶Zn value observed in the Neandertal’s tooth enamel, our results support the interpretation of Neandertals as carnivores as already suggested by δ¹⁵N isotope values of specimens from other regions. Further work could help identify if such isotopic peculiarities (lowest δ⁶⁶Zn and highest δ¹⁵N of the food web) are due to a metabolic and/or dietary specificity of the Neandertals.

Over the last 30 years, analyses of nitrogen isotopes in collagen (δ¹⁵N_collagen) have provided direct evidence for Neandertal diets across Europe and Asia. These studies all indicate a carnivorous (1–12), or at least a meat-heavy, diet for European Neandertals. However, one peculiarity of Neandertal δ¹⁵N_collagen remains the subject of an ongoing debate. From the one Siberian and eight western European sites, where both Neandertal and associated fauna have been analyzed, nitrogen isotope ratios in Neandertal collagen are systematically higher than that of other carnivores (3, 6–8, 10, 11, 13, 14). An explanation for such elevated values could be the consumption of herbivores, such as mammoths, which themselves exhibit elevated δ¹⁵N values due to the consumption of plants growing on arid soils (1, 2, 7). While mammoth remains are usually scarce at Neandertal fossil localities, they were nonetheless occasionally consumed, as suggested by remains with cut marks and other human butchery signatures (15). The absence of mammoth remains at Middle Paleolithic sites could be a result of 1) Neandertals chose to leave large bone elements at the kill site and transport other edible carcass products, mainly meat, back to the habitation site (15), or 2) mammoths were not frequently consumed, and the δ¹⁵N peculiarity consequently reflects the consumption of other resources enriched in ¹⁵N.Alongside this δ¹⁵N peculiarity, one major obstacle to our knowledge of Neandertals’ subsistence patterns is that the preservation of organic matter limits the application of collagen-bound nitrogen isotope analysis to fossil specimens. Collagen degrades over time at a varying speed depending on climatic and environmental conditions (16). The oldest hominin specimen in which bone protein is preserved is that of Scladina (Belgium), which dates to 90,000 cal BP (calibrated years before the present) (17), but most studied specimens are younger than 50,000 cal BP (1–3, 6–8, 10–13, 18). Furthermore, these specimens are only from sites in northwestern and central Europe and Siberia, where climatic conditions favored collagen preservation. As a result, the variability of Neandertals’ diet over time and between regions may not accurately be reflected by the currently available isotope data. In Iberia, where the latest surviving Neandertals have been discovered (19, 20), collagen was successfully extracted for only one site (21). Therefore, our knowledge of Iberian Neandertal diets mostly relies on zooarcheological and dental calculus data, which show some inconsistencies (21–25). For instance, similar to other western European sites, zooarcheological studies emphasize the consumption of terrestrial mammals and birds (21). In contrast, analysis of dental calculus for microremains and ancient DNA metagenomic analysis (26–28) highlight the frequent consumption of plants and mushrooms. Indeed, Weyrich et al. (26) even suggest that Neandertals at El Sidrón (Fig. 1) rarely consumed meat but often ate mushrooms, which would also result in elevated δ¹⁵N values (29). The consumption of marine foods is also attested for coastal Neandertals, but its frequency cannot be truly assessed in the absence of isotope studies (21, 23–25, 30). Finally, cannibalism has been documented at two Iberian sites (El Sidrón and Zafarraya) (22, 31) (Fig. 1), though such practices appear limited and most likely occurred only during periods of nutritional stress (32). Therefore, it is challenging to confirm the homogeneity of Neandertals’ diets across time and space, calling into question a direct link between their subsistence strategy and disappearance.Open in a separate windowFig. 1.(A) Location of the Gabasa site as well as other Neandertal sites mentioned in the text. (B) Detailed map of the Gabasa region. San Estaban de Litera and Benabarre are nearby modern cities.This study aims to investigate if the Zn isotope proxy could help elucidate the dietary behaviors of Neandertals and the source of their δ¹⁵N peculiarity, specifically by studying a Late Pleistocene Iberian food web where the presence of mammoth has not been documented (33). The development of Zn isotope analysis (⁶⁶Zn/⁶⁴Zn, expressed as δ⁶⁶Zn) has proven that trophic level information can be retrieved from mammalian tooth enamel (δ⁶⁶Zn_enamel) (34, 35), including fossil samples from Pleistocene food webs where organic matter is typically not preserved (36, 37). Previous studies have demonstrated that δ⁶⁶Zn_enamel decreases by ca. 0.30 to 0.60 ‰ with each step in archeological and modern food webs (34–38) and that δ⁶⁶Zn values associated with breastfeeding are higher than postweaning-associated values (39). While the main source of variation of δ⁶⁶Zn_enamel values is diet, local geology can also likely influence the isotope ratio of a given animal (36, 39). To date, three modern assemblages from Koobi Fora (Kenya), Kruger Park, and the western Cape (South Africa) (40), a few animals from a historical site (Rennes, France) (41), and three Late Pleistocene sites (Tam Hay Marklot, Nam Lot, and Tam Pa Ling, Laos) (36, 37) are the only terrestrial food webs for which Zn isotope data in teeth and/or bones have been published (SI Appendix, Fig. S14). In the modern Koobi Fora savannah food web, δ⁶⁶Zn_enamel differences have been observed between browsers and grazers (35), but this pattern was not seen in any of the three Pleistocene Asian forest food webs (36, 37). Among modern and historical human populations, historically documented diets relying on plants are associated with higher δ⁶⁶Zn values than those that include a substantial quantity of animal products (41–44). Zinc isotopes of ancient hominins have been analyzed only in one Pleistocene Homo sapiens individual (37) from Southeast Asia, but not yet in any Neandertal specimen.This current study contributes significantly to our understanding of Iberian Neandertal diets by providing information on their trophic ecology for a region where traditional nitrogen isotope analyses are unfeasible due to the poor preservation of organic matter. We use the Zn isotopic tool as well as other mobility, ecological, and dietary proxies applied on tooth enamel from hominin and animal remains from the cave site Cueva de los Moros 1 (Gabasa, Pyrenees, Spain; Fig. 1). The site, excavated in the 1980s, is very well documented [for stratigraphic context, see Montes and Utrilla (45) and SI Appendix, Section S1]. All remains come from layers e, f, and g of a single stratigraphic layer directly above layer h dated to 143 ± 43 ka. Numerous carnivore remains were recovered along with Neandertal remains (layers e and f), allowing for comparison of the different meat-eating taxa. Coexisting herbivores from three different types of environmental contexts are homogeneously represented in layers e, f, and g: mountains (Iberian ibex [Capra pyrenaica], chamois [Rupicapra rupicapra]), forest (cervids including red deer [Cervus elaphus]), and open environments (horses [Equus ferus], European wild asses [Equus hydruntinus]).     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Chirality-matched catalyst-controlled macrocyclization reactions

Macrocycles, formally defined as compounds that contain a ring with 12 or more atoms, continue to attract great interest due to their important applications in physical, pharmacological, and environmental sciences. In syntheses of macrocyclic compounds, promoting intramolecular over intermolecular reactions in the ring-closing step is often a key challenge. Furthermore, syntheses of macrocycles with stereogenic elements confer an additional challenge, while access to such macrocycles are of great interest. Herein, we report the remarkable effect peptide-based catalysts can have in promoting efficient macrocyclization reactions. We show that the chirality of the catalyst is essential for promoting favorable, matched transition-state relationships that favor macrocyclization of substrates with preexisting stereogenic elements; curiously, the chirality of the catalyst is essential for successful reactions, even though no new static (i.e., not “dynamic”) stereogenic elements are created. Control experiments involving either achiral variants of the catalyst or the enantiomeric form of the catalyst fail to deliver the macrocycles in significant quantity in head-to-head comparisons. The generality of the phenomenon, demonstrated here with a number of substrates, stimulates analogies to enzymatic catalysts that produce naturally occurring macrocycles, presumably through related, catalyst-defined peripheral interactions with their acyclic substrates.

Macrocyclic compounds are known to perform a myriad of functions in the physical and biological sciences. From cyclodextrins that mediate analyte separations (1) to porphyrin cofactors that sit in enzyme active sites (2, 3) and to potent biologically active, macrocyclic natural products (4) and synthetic variants (5–7), these structures underpin a wide variety of molecular functions (Fig. 1A). In drug development, such compounds are highly coveted, as their conformationally restricted structures can lead to higher affinity for the desired target and often confer additional metabolic stability (8–13). Accordingly, there exists an entire synthetic chemistry enterprise focused on efficient formation and functionalization of macrocycles (14–18).Open in a separate windowFig. 1.(A) Examples of macrocyclic compounds with important applications. HCV, hepatitis C virus. (B) Use of chiral ligands in metal-catalyzed or mediated stereoselective macrocyclization reactions. (C) Remote desymmetrization using guanidinylated ligands via Ullmann coupling. (D) This work: use of copper/peptidyl complexes for macrocyclization and the exploration of matched and mismatched effect.In syntheses of macrocyclic compounds, the ring-closing step is often considered the most challenging step, as competing di- and oligomerization pathways must be overcome to favor the intramolecular reaction (14). High-dilution conditions are commonly employed to favor macrocyclization of linear precursors (19). Substrate preorganization can also play a key role in overcoming otherwise high entropic barriers associated with multiple conformational states that are not suited for ring formation. Such preorganization is most often achieved in synthetic chemistry through substrate design (14, 20–22). Catalyst or reagent controls that impose conformational benefits that favor ring formation are less well known. Yet, critical precedents include templating through metal-substrate complexation (23, 24), catalysis by foldamers (25) or enzymes (26–29), or, in rare instances, by small molecules (discussed below). Characterization of biosynthetic macrocyclization also points to related mechanistic issues and attributes for efficient macrocyclizations (30–34). Coupling macrocyclization reactions to the creation of stereogenic elements is also rare (35). Metal-mediated reactions have been applied toward stereoselective macrocyclizations wherein chiral ligands transmit stereochemical information to the products (Fig. 1B). For example, atroposelective ring closure via Heck coupling has been applied in the asymmetric total synthesis of isoplagiochin D by Speicher and coworkers (36–40). Similarly, atroposelective syntheses of (+)-galeon and other diarylether heptanoid natural products were achieved via Ullman coupling using N-methyl proline by Salih and Beaudry (41). Finally, Reddy and Corey reported the enantioselective syntheses of cyclic terpenes by In-catalyzed allylation utilizing a chiral prolinol-based ligand (42). While these examples collectively illustrate the utility of chiral ligands in stereoselective macrocyclizations, such examples remain limited.We envisioned a different role for chiral catalysts when addressing intrinsically disfavored macrocyclization reactions. When unfavorable macrocyclization reactions are confronted, we hypothesized that a catalyst–substrate interaction might provide transient conformational restriction that could promote macrocyclization. To address this question, we chose to explore whether or not a chiral catalyst-controlled macrocyclization might be possible with peptidyl copper complexes. In the context of the medicinally ubiquitous diarylmethane scaffold, we had previously demonstrated the capacity for remote asymmetric induction in a series of bimolecular desymmetrizations using bifunctional, tetramethylguanidinylated peptide ligands. For example, we showed that peptidyl copper complexes were able to differentiate between the two aryl bromides during C–C, C–O, and C–N cross-coupling reactions (Fig. 1C) (43–45). Moreover, in these intermolecular desymmetrizations, a correlation between enantioselectivity and conversion was observed, revealing the catalyst’s ability to perform not only enantiotopic group discrimination but also kinetic resolution on the monocoupled product as the reaction proceeds (44). This latter observation stimulated our speculation that if an internal nucleophile were present to undergo intramolecular cross-coupling to form a macrocycle, stereochemically sensitive interactions (so-called matched and mismatched effects) (46) could be observed (Fig. 1D). Ideally, we anticipated that transition state–stabilizing interactions might even prove decisive in matched cases, and the absence of catalyst–substrate stabilizing interactions might account for the absence of macrocyclization for these otherwise intrinsically unfavorable reactions. Herein, we disclose the explicit observation of these effects in chiral catalyst-controlled macrocyclization reactions.     

An image-computable model of how endogenous and exogenous attention differentially alter visual perception

Attention alters perception across the visual field. Typically, endogenous (voluntary) and exogenous (involuntary) attention similarly improve performance in many visual tasks, but they have differential effects in some tasks. Extant models of visual attention assume that the effects of these two types of attention are identical and consequently do not explain differences between them. Here, we develop a model of spatial resolution and attention that distinguishes between endogenous and exogenous attention. We focus on texture-based segmentation as a model system because it has revealed a clear dissociation between both attention types. For a texture for which performance peaks at parafoveal locations, endogenous attention improves performance across eccentricity, whereas exogenous attention improves performance where the resolution is low (peripheral locations) but impairs it where the resolution is high (foveal locations) for the scale of the texture. Our model emulates sensory encoding to segment figures from their background and predict behavioral performance. To explain attentional effects, endogenous and exogenous attention require separate operating regimes across visual detail (spatial frequency). Our model reproduces behavioral performance across several experiments and simultaneously resolves three unexplained phenomena: 1) the parafoveal advantage in segmentation, 2) the uniform improvements across eccentricity by endogenous attention, and 3) the peripheral improvements and foveal impairments by exogenous attention. Overall, we unveil a computational dissociation between each attention type and provide a generalizable framework for predicting their effects on perception across the visual field.

Endogenous and exogenous spatial attention prioritize subsets of visual information and facilitate their processing without concurrent eye movements (1–3). Selection by endogenous attention is goal-driven and adapts to task demands, whereas exogenous attention transiently and automatically orients to salient stimuli (1–3). In most visual tasks, both types of attention typically improve visual perception similarly [e.g., acuity (4–6), visual search (7, 8), perceived contrast (9–11)]. Consequently, models of visual attention do not distinguish between endogenous and exogenous attention (e.g., refs. 12–19). However, stark differences also exist. Each attention type differentially modulates neural responses (20, 21) and fundamental properties of visual processing, including temporal resolution (22, 23), texture sensitivity (24), sensory tuning (25), contrast sensitivity (26), and spatial resolution (27–34).The effects of endogenous and exogenous attention are dissociable during texture segmentation, a visual task constrained by spatial resolution [reviews (1–3)]. Whereas endogenous attention optimizes spatial resolution to improve the detection of an attended texture (32–34), exogenous attention reflexively enhances resolution even when detrimental to perception (27–31, 34). Extant models of attention do not explain these well-established effects.Two main hypotheses have been proposed to explain how attention alters spatial resolution. Psychophysical studies ascribe attentional effects to modulations of spatial frequency (SF) sensitivity (30, 33). Neurophysiological (13, 35, 36) and neuroimaging (37, 38) studies bolster the idea that attention modifies spatial profiles of neural receptive fields (RFs) (2). Both hypotheses provide qualitative predictions of attentional effects but do not specify their underlying neural computations.Differences between endogenous and exogenous attention are well established in segmentation tasks and thus provide an ideal model system to uncover their separate roles in altering perception. Texture-based segmentation is a fundamental process of midlevel vision that isolates regions of local structure to extract figures from their background (39–41). Successful segmentation hinges on the overlap between the visual system’s spatial resolution and the levels of detail (i.e., SF) encompassed by the texture (39, 41, 42). Consequently, the ability to distinguish between adjacent textures varies as resolution declines toward the periphery (43–46). Each attention type differentially alters texture segmentation, demonstrating that their effects shape spatial resolution [reviews (1–3)].Current models of texture segmentation do not explain performance across eccentricity and the distinct modulations by attention. Conventional models treat segmentation as a feedforward process that encodes the elementary features of an image (e.g., SF and orientation), transforms them to reflect the local structure (e.g., regions of similarly oriented bars), and then pools across space to emphasize texture-defined contours (39, 41, 47). Few of these models account for variations in resolution across eccentricity (46, 48, 49) or endogenous (but not exogenous) attentional modulations (18, 50). All others postulate that segmentation is a “preattentive” (42) operation whose underlying neural processing is impervious to attention (39, 41, 46–49).Here, we develop a computational model in which feedforward processing and attentional gain contribute to segmentation performance. We augment a conventional model of texture processing (39, 41, 47). Our model varies with eccentricity and includes contextual modulation within local regions in the stimulus via normalization (51), a canonical neural computation (52). The defining characteristic of normalization is that an individual neuron is (divisively) suppressed by the summed activity of neighboring neurons responsive to different aspects of a stimulus. We model attention as multiplicative gains [attentional gain factors (15)] that vary with eccentricity and SF. Attention shifts sensitivity toward fine or coarse spatial scales depending on the range of SFs enhanced.Our model is image-computable, which allowed us to reproduce behavior directly from grayscale images used in psychophysical experiments (6, 26, 27, 29–33). The model explains three signatures of texture segmentation hitherto unexplained within a single computational framework (Fig. 1): 1) the central performance drop (CPD) (27–34, 43–46) (Fig. 1A), that is, the parafoveal advantage of segmentation over the fovea; 2) the improvements in the periphery and impairments at foveal locations induced by exogenous attention (27–32, 34) (Fig. 1B); and 3) the equivalent improvements across eccentricity by endogenous attention (32–34) (Fig. 1C).Open in a separate windowFig. 1.Signatures of texture segmentation. (A) CPD. Shaded region depicts the magnitude of the CPD. Identical axis labels are omitted in B and C. (B) Exogenous attention modulation. Exogenous attention improves segmentation performance in the periphery and impairs it near the fovea. (C) Endogenous attention modulation. Endogenous attention improves segmentation performance across eccentricity.Whereas our analyses focused on texture segmentation, our model is general and can be applied to other visual phenomena. We show that the model predicts the effects of attention on contrast sensitivity and acuity, i.e., in tasks in which both endogenous and exogenous attention have similar or differential effects on performance. To preview our results, model comparisons revealed that normalization is necessary to elicit the CPD and that separate profiles of gain enhancement across SF (26) generate the effects of exogenous and endogenous attention on texture segmentation. A preferential high-SF enhancement reproduces the impairments by exogenous attention due to a shift in visual sensitivity toward details too fine to distinguish the target at foveal locations. The transition from impairments to improvements in the periphery results from exogenous attentional gain gradually shifting to lower SFs that are more amenable for target detection. Improvements by endogenous attention result from a uniform enhancement of SFs that encompass the target, optimizing visual sensitivity for the attended stimulus across eccentricity.     

Ion-binding properties of a K+ channel selectivity filter in different conformations

K⁺ channels are membrane proteins that selectively conduct K⁺ ions across lipid bilayers. Many voltage-gated K⁺ (K_V) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K⁺ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to K_V channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na⁺ or low [K⁺]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K⁺ ion binding to channels with an inactivated or conductive selectivity filter is different from K⁺ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.K⁺ channels are found in all three domains of life, where they selectively conduct K⁺ ions across cell membranes. Specific stimuli trigger the activation of K⁺ channels, which results in a hinged movement of the inner helix bundle (1–7). This opening on the intracellular side of the membrane initiates ion conduction across the membrane by allowing ions to enter into the channel. After a period, many channels spontaneously inactivate to attenuate the response (8–17). The inactivation process is a timer that terminates the flow of ions in the presence of an activator to help shape the response of the system. Two dominant types of inactivation have been characterized in voltage-dependent channels: N-type and C-type (18). N-type inactivation is fast and involves an N-terminal positively charged “ball” physically plugging the pore of the channel when the membrane is depolarized. C-type inactivation, on the other hand, is a slower process involving a conformational change in the selectivity filter that is initiated by a functional link between the intracellular gate and the selectivity filter (10, 19).Several experimental observations indicate a role for the selectivity filter in C-type inactivation. First, mutations in and around the selectivity filter can alter the kinetics of inactivation (20–23). Second, increasing concentrations of extracellular K⁺ ions decrease the rate of inactivation, as if the ions are stabilizing the conductive conformation of the channel to prevent a conformational change in the selectivity filter (14, 16, 17, 22). Finally, a loss of selectivity of K⁺ over Na⁺ has been observed during the inactivation process in Shaker channels, suggesting a role for the selectivity filter (24, 25). Together, these data indicate that channels in their inactivated and conductive conformations interact with K⁺ ions differently, and suggest that C-type inactivation involves a conformational change in the selectivity filter. Although several structures of K⁺ channels in their conductive state have been solved using X-ray crystallography, there is at present no universally accepted model for the C-type inactivated channel (1, 3–5, 9, 19, 26–28) (Fig. 1B).Open in a separate windowFig. 1.Macroscopic recordings and structural models of KcsA K⁺ channel. (A) Macroscopic currents of WT KcsA obtained by a pH jump from pH 8 to pH 4 reveal channel inactivation. Two models representing the conformation of the channel are shown below. (B) Conductive [Left, Protein Data Bank (PDB) ID code 1K4C] and constricted (Right, PDB ID code 1K4D) conformations of the selectivity filter are shown as sticks, and the ion-binding sites are indicated with green spheres. The thermodynamic properties of the conductive, constricted, and inactivated (Middle) conformations are the subject of this study.Inactivation in the K⁺ channel from Streptomyces lividians (KcsA) has many of the same functional properties of C-type inactivation, which has made it a model to understand its structural features (20). KcsA channels transition from their closed to open gate upon changing the intracellular pH from high to low (Fig. 1A). The rapid flux of ions through the channel is then attenuated by channel inactivation, where most open WT channels are not conducting, suggesting that crystal structures of open KcsA channels would reveal the inactivated channel. In some crystal structures of truncated WT KcsA solved with an open gate, the selectivity filter appears in the constricted conformation, similar to the conformation observed in structures of the KcsA channel determined in the presence of only Na⁺ ions or low concentrations of K⁺ ions (3, 10, 29, 30) (Fig. 1B). Solid-state and solution NMR also indicate that the selectivity filter of the KcsA channel is in the constricted conformation when the cytoplasmic gate is open (31–33).However, a recently published study shows that even when the constricted conformation of KcsA’s selectivity filter is prevented by a nonnatural amino acid substitution, the channel inactivates like WT channels, suggesting the constricted filter does not correspond to the functionally observed inactivation in KcsA (28). In this study, we use isothermal titration calorimetry (ITC) to quantify the ion-binding properties of WT and mutant KcsA K⁺ channels with their selectivity filters in different conformations and EPR spectroscopy to determine the conformation of the channels’ intracellular gates. A comparison of these ion-binding properties leads us to conclude that the conductive and inactivated filters are energetically more similar to each other than the constricted and inactivated filters.     

Coevolution can reverse predator–prey cycles

A hallmark of Lotka–Volterra models, and other ecological models of predator–prey interactions, is that in predator–prey cycles, peaks in prey abundance precede peaks in predator abundance. Such models typically assume that species life history traits are fixed over ecologically relevant time scales. However, the coevolution of predator and prey traits has been shown to alter the community dynamics of natural systems, leading to novel dynamics including antiphase and cryptic cycles. Here, using an eco-coevolutionary model, we show that predator–prey coevolution can also drive population cycles where the opposite of canonical Lotka–Volterra oscillations occurs: predator peaks precede prey peaks. These reversed cycles arise when selection favors extreme phenotypes, predator offense is costly, and prey defense is effective against low-offense predators. We present multiple datasets from phage–cholera, mink–muskrat, and gyrfalcon–rock ptarmigan systems that exhibit reversed-peak ordering. Our results suggest that such cycles are a potential signature of predator–prey coevolution and reveal unique ways in which predator–prey coevolution can shape, and possibly reverse, community dynamics.Population cycles, e.g., predator–prey cycles, and their ecological drivers have been of interest for the last 90 y (1–4). Classical models of predator–prey systems, developed first by Lotka (5) and Volterra (6), share a common prediction: Prey oscillations precede predator oscillations by up to a quarter of the cycle period (7). When plotted in the predator–prey phase plane, these cycles have a counterclockwise orientation (4). These cycles are driven by density-dependent interactions between the populations. When predators are scarce, prey increase in abundance. As their food source increases, predators increase in abundance. When the predators reach sufficiently high densities, the prey population is driven down to low numbers. With a scarcity of food, the predator population crashes and the cycle repeats.While many cycles, like the classic lynx–hare cycles (Fig. 1A) (3), exhibit the above characteristics, predator–prey cycles with different characteristics have also been observed. For example, antiphase cycles where predator oscillations lag behind prey oscillations by half of the cycle period (Fig. 1B) (8) and cryptic cycles where the predator population oscillates while the prey population remains effectively constant (Fig. 1C) (9) have been observed in experimental systems. This diversity of cycle types motivates the question, “Why do cycle characteristics differ across systems?”Open in a separate windowFig. 1.Examples of different kinds of predator–prey cycles. (A) Counterclockwise lynx–hare cycles (3). (B) Antiphase rotifer–algal cycles (8). (C) Cryptic phage-bacteria cycles (9). In all time series, red and blue correspond to predator and prey, respectively. See SI Text, section C for data sources.In Lotka–Volterra and other ecological models, predator and prey life history traits are assumed to be fixed. However, empirical studies across taxa have shown that prey (9–16) and predators (17–20) can evolve over ecological time scales. That is, changes in allele frequencies (and associated phenotypes) can occur at the same rate as changes in population densities or spatial distributions and alter the ecological processes driving the changes in population densities or distributions; this phenomenon has been termed “eco-evolutionary dynamics” (21, 22). Furthermore, predator–prey coevolution is important for driving community composition and dynamics (16, 19, 20, 23–26). This body of work suggests that the interaction between ecological and evolution processes has the potential to alter the ecological dynamics of communities.Experimental (8, 9, 13, 14) and theoretical studies (13, 27, 28) have shown that prey or predator evolution alone can alter the characteristics of predator–prey cycles and drive antiphase (Fig. 1B) and cryptic (Fig. 1C) cycles. Additional theoretical work has shown that predator–prey coevolution can also drive antiphase and cryptic cycles (29). Thus, evolution in one or both species is one mechanism through which antiphase or cryptic predator–prey cycles can arise. However, it is unclear if coevolution can drive additional kinds of cycles with characteristics different from those in Fig. 1.The main contribution of this study is to show that predator–prey coevolution can drive unique cycles where peaks in predator abundance precede peaks in prey abundance, the opposite of what is predicted by classical ecological models. We refer to these reversed cycles as “clockwise cycles.” The theoretical and empirical finding of clockwise cycles represents an example of how evolution over ecological time scales can alter community-level dynamics.     

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited-state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the photoexcited chromophore requires a spectroscopic technique capable of resolving atomic motions on the intrinsic timescale of femtosecond to picosecond. We use femtosecond stimulated Raman spectroscopy to study the excited-state conformational dynamics of a recently developed FP-calmodulin biosensor, GEM-GECO1, for calcium ion (Ca²⁺) sensing. This study reveals that, in the absence of Ca²⁺, the dominant skeletal motion is a ∼170 cm⁻¹ phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of ∼30 ps (6 times slower than wild-type GFP) to reach the green fluorescent state. The functional relevance of the motion is corroborated by molecular dynamics simulations. Upon Ca²⁺ binding, this in-plane rocking motion diminishes, and blue emission from a trapped photoexcited neutral chromophore dominates because ESPT is inhibited. Fluorescence properties of site-specific protein mutants lend further support to functional roles of key residues including proline 377 in modulating the H-bonding network and fluorescence outcome. These crucial structural dynamics insights will aid rational design in bioengineering to generate versatile, robust, and more sensitive optical sensors to detect Ca²⁺ in physiologically relevant environments.Green fluorescent protein (GFP) first emerged as a revolutionary tool for bioimaging and molecular and cellular biology about 20 years ago (1–3), and the quest to discover and engineer biosensors with improved and expanded functionality has yielded exciting advances. Recently, the color palette of genetically encoded Ca²⁺ sensors for optical imaging (the GECO series) has been expanded to include blue, improved green, red intensiometric, and emission ratiometric sensors (4–7). The GECO proteins belong to the GCaMP family of Ca²⁺ sensors that are chimeras of a circularly permutated (cp)GFP, calmodulin (CaM), and a peptide derived from myosin light chain kinase (M13) (8). The CaM unit undergoes large-scale structural changes upon Ca²⁺ binding as it wraps around M13. These changes, especially at the interfacial region where CaM interacts with cpGFP, allosterically alter the local environment of the tyrosine-derived chromophore and lead to dramatic fluorescence change in the presence of Ca²⁺ (9, 10). Because GCaMP and GECO proteins are genetically encodable, show sensitivity to physiologically relevant Ca²⁺ concentrations, and respond to Ca²⁺ concentration changes rapidly, they have gained increasing popularity for in vivo imaging of Ca²⁺ in neural and olfactory cells (11–13).Among the engineered GECO proteins, GEM-GECO1 is an intriguing case with the serine–tyrosine–glycine (SYG) derived chromophore (4). Upon ultraviolet (UV) excitation it fluoresces green in the absence of Ca²⁺ but blue upon Ca²⁺ binding with a K_d of 340 nM. This dual-emission behavior is unique among ratiometric Ca²⁺-sensing FPs as well as the few reported pH-dependent dual-emission GFP variants, which typically require a threonine–tyrosine–glycine (TYG) chromophore to keep the nearby glutamate largely protonated (14, 15). Dual emission is particularly useful for imaging in vivo because the signal color change is a direct consequence of analyte concentration. There remains room to further improve GEM-GECO1 because it has a low quantum yield and decreased dynamic range in vivo (5).The advanced imaging capabilities of the GECO series have been explored (4, 5, 7), but few spectroscopic studies exist for these unique Ca²⁺ sensors. In contrast, spectroscopy on wild-type (wt)GFP included infrared pump probe (16), time-resolved fluorescence (17–19), transient infrared (20, 21), and femtosecond Raman spectroscopy (22), as well as computational studies (23–25), providing a fairly complete picture of the photophysical and photochemical steps leading to green fluorescence (26, 27). In the electronic ground state (GS), wtGFP exists as a mixture of neutral chromophore (A, ∼400 nm peak absorbance) and a small population of anionic chromophore (B, ∼475 nm peak absorbance; Fig. 1B). Emission from the excited state of either form (A* at ∼460 nm and B* at ∼500 nm, respectively) is possible. The main emission pathway upon 400-nm excitation involves excited-state proton transfer (ESPT) from A* on a picosecond timescale to form the intermediate green fluorescent state (I*). We hypothesize that ESPT occurs in the Ca²⁺-free state of GEM-GECO1, but upon Ca²⁺ binding ESPT is disrupted and blue fluorescence occurs from A* (28). In this work, we aim to elucidate the ESPT mechanism of GEM-GECO1 as a function of the chromophore environment, using time-resolved femtosecond stimulated Raman spectroscopy (FSRS). Previous results (22) identified a low-frequency skeletal motion facilitating ESPT in wtGFP that provides guidance to unravel the chromophore dynamics in a flexible CaM–GFP complex. In the absence of a GEM-GECO1 crystal structure, we will compare spectroscopic signatures of the Ca²⁺-free/bound proteins at equilibrium in conjunction with site-specific mutagenesis and molecular dynamics simulations. We then infer how the local environment influences the chromophore structural evolution on the electronically excited state and leads to distinct fluorescence hues.Open in a separate windowFig. 1.Schematic structure and electronic spectroscopy of the M13-cpGFP-CaM chimera, GEM-GECO1. (A) The Ca²⁺-bound GCaMP2 structure (PDB ID 3EVR) for illustration purposes is shown with the GFP β-barrel in green, CaM in orange, the M13 peptide in magenta, and the Ca²⁺ bound to CaM in yellow. The black arrow indicates the β-barrel opening. The autocyclized SYG chromophore is highlighted by a black box with an asterisk and shown in Inset. (B) Normalized absorption and relative emission spectra of Ca²⁺-free (green solid and dashed lines) and Ca²⁺-bound GEM-GECO1 (blue solid and dashed lines, respectively). The corresponding spectra in wtGFP are shown in black. Blue fluorescence from Ca²⁺-bound protein (peak at 462 nm) is broad and asymmetric relative to the sharp green fluorescence of the Ca²⁺-free protein (peak at 511 nm).     

Base-enhanced catalytic water oxidation by a carboxylate–bipyridine Ru(II) complex

In aqueous solution above pH 2.4 with 4% (vol/vol) CH₃CN, the complex [Ru^II(bda)(isoq)₂] (bda is 2,2′-bipyridine-6,6′-dicarboxylate; isoq is isoquinoline) exists as the open-arm chelate, [Ru^II(CO₂-bpy-CO₂⁻)(isoq)₂(NCCH₃)], as shown by ¹H and ¹³C-NMR, X-ray crystallography, and pH titrations. Rates of water oxidation with the open-arm chelate are remarkably enhanced by added proton acceptor bases, as measured by cyclic voltammetry (CV). In 1.0 M PO₄^3–, the calculated half-time for water oxidation is ∼7 μs. The key to the rate accelerations with added bases is direct involvement of the buffer base in either atom–proton transfer (APT) or concerted electron–proton transfer (EPT) pathways.Metal-complex catalyzed water oxidation continues to evolve with new catalysts and new mechanistic insights (1–9). Studies on single-site Ru catalysts such as [Ru^II(Mebimpy)(bpy)(OH₂)]²⁺ [Mebimpy is 2,6-bis(1-methylbenzimidazol-2-yl)pyridine; bpy is 2,2′-bipyridine; Fig. 1], both in solution and on surfaces, reveal mechanisms in which stepwise oxidative activation of aqua precursors to Ru^V=O is followed by rate-limiting O–O bond formation (10–15). The results of kinetic and mechanistic studies have revealed the importance of concerted atom–proton transfer (APT) in the O–O bond-forming step. In APT, the O–O bond forms in concert with H⁺ transfer to water or to an added base (11, 12, 16–19). APT can promote dramatic rate enhancements. In a recent study on surface-bound [Ru(Mebimpy)(4,4′-((HO)₂OPCH₂)₂bpy)(OH₂)]²⁺ [4,4′-((HO)₂OPCH₂)₂bpy is 4,4′-bis-methlylenephosphonato-2,2′-bipyridine] stabilized by atomic layer deposition, a rate enhancement of ∼10⁶ was observed with 0.012 M added PO₄³⁻ at pH 12 compared with oxidation at pH 1 (20).Open in a separate windowFig. 1.Structures of [Ru^II(Mebimpy)(bpy)(OH₂)]²⁺ (Left) and [Ru^II(CO₂-bpy-CO₂)(isoq)₂] [1] (Right).Sun and coworkers (21, 22) have described the Ru single-site water oxidation catalysts, [Ru^II(bda)(L)₂] (H₂bda is 2,2′-bipyridine-6,6′-dicarboxylic acid, HCO₂-bpy-CO₂H; L is isoquinoline, 4-picoline, or phthalazine). They undergo rapid and sustained water oxidation catalysis with added Ce^IV. A mechanism has been proposed in which initial oxidation to seven coordinate Ru^IV is followed by further oxidation to Ru^V(O) with O–O coupling to give a peroxo-bridged intermediate, Ru^IVO–ORu^IV, which undergoes further oxidation and release of O₂ (21, 22). We report here the results of a rate and mechanistic study on electrochemical water oxidation by complex [1], [Ru^II(CO₂-bpy-CO₂)(isoq)₂] (isoq is isoquinoline) (Fig. 1). Evidence is presented for water oxidation by a chelate open form in acidic solutions. The chelate open form displays dramatic rate enhancements with added buffer bases, and the results of a detailed mechanistic study are reported here.