From the Cover: Electronic resonance with anticorrelated pigment vibrations drives photosynthetic energy transfer outside the adiabatic framework

The delocalized, anticorrelated component of pigment vibrations can drive nonadiabatic electronic energy transfer in photosynthetic light-harvesting antennas. In femtosecond experiments, this energy transfer mechanism leads to excitation of delocalized, anticorrelated vibrational wavepackets on the ground electronic state that exhibit not only 2D spectroscopic signatures attributed to electronic coherence and oscillatory quantum energy transport but also a cross-peak asymmetry not previously explained by theory. A number of antennas have electronic energy gaps matching a pigment vibrational frequency with a small vibrational coordinate change on electronic excitation. Such photosynthetic energy transfer steps resemble molecular internal conversion through a nested intermolecular funnel.     

The enzyme mechanism of nitrite reductase studied at single-molecule level

A generic method is described for the fluorescence “readout” of the activity of single redox enzyme molecules based on Förster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to single-molecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.     

Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix

Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Förster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics of poly-l-prolines with 1–10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.     

Single-molecule studies of group II intron ribozymes

Group II intron ribozymes fold into their native structure by a unique stepwise process that involves an initial slow compaction followed by fast formation of the native state in a Mg²⁺-dependent manner. Single-molecule fluorescence reveals three distinct on-pathway conformations in dynamic equilibrium connected by relatively small activation barriers. From a most stable near-native state, the unobserved catalytically active conformer is reached. This most compact conformer occurs only transiently above 20 mM Mg²⁺ and is stabilized by substrate binding, which together explain the slow cleavage of the ribozyme. Structural dynamics increase with increasing Mg²⁺ concentrations, enabling the enzyme to reach its active state.     

Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

Hsp70s, a family of chaperones that aids in protein folding, inhibits misfolding and aggregation, and transports proteins to their respective cellular destination, are among the most conserved proteins in the cell (1–4). They assist in the folding of nascent proteins co- or posttranslationally and prevent protein aggregation by specific interactions with protein sequences of five to seven hydrophobic amino acids (3, 4). Hsp70 chaperones are ubiquitously expressed in almost all organisms, including archaea and are found in many cellular compartments (5). Owing to their prokaryotic origin and uptake by endosymbiosis, they can be divided into cytosolic, endoplasmic reticulum (ER)-associated, mitochondrial, and plastid families. Hsp70s of these four families are more conserved throughout different phyla within one compartment than between the cellular organelles. They consist of a 44 kDa N-terminal nucleotide-binding domain (NBD) connected to a 28 kDa substrate-binding domain (SBD). The SBD itself is further divided into a β-sandwich domain consisting of two β-sheets with a hydrophobic cleft for substrate recognition and an α-helical lid. Despite their highly conserved sequence (48 to 58% sequence identity for the proteins investigated here), Hsp70s perform a multitude of additional functions in the different organelles with the aid of various cochaperones.To perform their functions, Hsp70 chaperones undergo a nucleotide-dependent conformational cycle. Multiple crystal structures, NMR, and single-pair Förster resonance energy transfer (FRET) experiments have elucidated the conformational states of the Hsp70s in their functional cycle (Fig. 1A) (6–12). When ATP is bound to the NBD, the domains contact each other and, for the SBD, the α-helical lid is found in an open conformation distant from the β-sheet (7–9, 11). Upon ATP hydrolysis, the NBD and SBD separate, and the lid closes upon the β-sheet of the SBD (6, 8–10). This conformational transition depends on the hydrolysis of ATP, which is intrinsically slow for Hsp70 chaperones (around 0.05 to 0.1/min at 30 °C) (13). The rate of ATP hydrolysis is increased by J-domain–containing heat-shock proteins 40 (Hsp40s, or J-proteins), which also confer specificity to Hsp70s for certain functionalities. J-domain–containing proteins play an important role in recruiting Hsp70s to their respective client proteins. In the ER, mitochondria, and plastids, Hsp70s are associated with the translocation of proteins across the respective organellar membrane. Mitochondria contain four different J-proteins conveying various functions to the mitochondrial Hsp70s. When interacting with membrane-anchored Pam16/18, they are involved in protein import (14–18), when binding to Jac1, they act as part of the iron–sulfur biogenesis (19), and, when interacting with Mdj1, they play a role in protein folding and prevent aggregation (20–23). The specialization by J-proteins is even more pronounced in the ER, where six J-proteins exist that modify the function of the Hsp70 (24). Protein folding occurs upon interaction of the Hsp70 BiP with ERdj3 and ERdj6 (25). SEC63 (or ERdj2) is important for protein translocation across the membrane (26), and ER-associated degradation is driven by binding to ERdj4 and ERdj5 (27–29).Open in a separate windowFig. 1.Conformational change of Hsp70 chaperones in the presence of different nucleotides observed by single-molecule 3C FRET using multiparameter fluorescence detection with pulsed interleaved excitation (MFD-PIE). (A) The conformational cycle of Hsp70s is shown schematically using the crystal structure of DnaK in its docked conformation with open lid (Protein Data Bank [PDB] accession code: 4B9Q) or with undocked domains and a closed lid (PDB accession code: 2KHO) (6, 7). The NBD is displayed in blue, the β-sheet of the SBD in green, and the α-helical lid in red. (B) A schematic representation of a single-molecule 3C-FRET experiment on DnaK (PDB accession code: 2KHO) showing the attachment sites of the fluorophores Atto488, Atto565, and Atto647N to the NBD and SBD of the molecule, respectively. (C–E) Histograms of the FRET efficiencies E_GR (Left), and E_BG (Right, dark shaded plots) and E_BR (Right, light shaded plots) of triple-labeled DnaK-PrK318-C425-C563 (Upper), Ssc1-PrK341-C448-C590 (Middle), and BiP-PrK167-C519-C638 (Lower) in their (C) apo form (gray, only measurable for BiP), (D) when bound to ADP (magenta), or (E) when bound to ATP (green).In prokaryotic cells, the major Hsp70 chaperone DnaK cooperates with trigger factor in the folding of nascent proteins upstream of the GroEL/GroES chaperonin complex (30, 31). Interestingly, DnaK only plays an essential role under stress conditions, such as elevated temperatures (3). Ssc1, the major Hsp70 of the mitochondrial matrix, on the other hand, has been shown to be essential for Saccharomyces cerevisiae growth, and its expression levels rise in response to stress through elevated temperatures (32, 33). It plays an important role in protein translocation into mitochondria, protein folding, and prevention of aggregation (32, 34). Unlike Ssc1 in mitochondria, BiP is the only Hsp70 member in the ER. It is involved in protein folding (25), protein translocation (26), ER-associated protein degradation (27–29), and the stress adaptability of the ER (35–37).Our comparative study focuses on intrinsic differences between Hsp70 chaperones regarding the allosteric interactions between the NBD and SBD throughout their functional cycle. As representative members of the different families, we chose the Hsp70s from the ER (BiP) and mitochondria (Ssc1) of eukaryotic cells and the well-characterized prokaryotic major Hsp70 DnaK (SI Appendix, Figs. S1 and S2 A–C). The selection of these three proteins allows us to compare Hsp70s from different cellular compartments and from different families of Hsp70s (11).To study the coordinated structural changes of these Hsp70s during their conformational cycle, we applied single-molecule three-color (3C) FRET. Single-molecule FRET with two fluorophores (also referred to as single-pair FRET) is a powerful tool to study conformational states of proteins in vitro but is limited to a single-distance readout. Three-color single-molecule FRET, on the other hand, gives access to three interdye distances simultaneously, allowing one to directly monitor the correlation of motions in different parts of the biomolecule. For solution-based, single-molecule FRET experiments, the recently developed 3C photon distribution analysis (3C-PDA) (38) enables a quantitative analysis of the underlying distance heterogeneity of the experimental system at hand. This methodology allows us to characterize the conformational landscape of the different Hsp70s with respect to the coordinated movement of the different domains.     

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

Molecular chaperones are an essential part of the machinery that avoids protein aggregation and misfolding in vivo. However, understanding the molecular basis of how chaperones prevent such undesirable interactions requires the conformational changes within substrate proteins to be probed during chaperone action. Here we use single-molecule fluorescence spectroscopy to investigate how the DnaJ–DnaK chaperone system alters the conformational distribution of the denatured substrate protein rhodanese. We find that in a first step the ATP-independent binding of DnaJ to denatured rhodanese results in a compact denatured ensemble of the substrate protein. The following ATP-dependent binding of multiple DnaK molecules, however, leads to a surprisingly large expansion of denatured rhodanese. Molecular simulations indicate that hard-core repulsion between the multiple DnaK molecules provides the underlying mechanism for disrupting even strong interactions within the substrate protein and preparing it for processing by downstream chaperone systems.Maintaining protein homeostasis in vivo requires a tight regulation of protein folding to prevent misfolding and aggregation. Molecular chaperones have evolved as an essential part of the cellular machinery that facilitates such processes in the complex and crowded environment of a living cell (1, 2). To assist protein folding, many chaperones proceed through complex conformational cycles in an ATP-dependent manner (3–5). For several chaperone systems, these cycles have been investigated in great detail by experiment and simulation (6–8). A remarkable example are the heat shock protein (Hsp) 70 chaperones, which are essential in prokaryotes and eukaryotes and are involved in co-translational folding, refolding of misfolded and aggregated proteins, protein translocation, and protein degradation (9). The Hsp70 chaperone DnaK from Escherichia coli together with its co-chaperone DnaJ and the nucleotide exchange factor GrpE form an ATP-driven catalytic reaction cycle (7) (Fig. 1A). Many denatured or misfolded substrate proteins are first captured by DnaJ and subsequently transferred to the DnaK–ATP complex, with DnaK in an open conformation. Substrate and DnaJ synergistically trigger DnaK’s ATPase activity, which leads to locking of the substrate in the DnaK–ADP complex, with DnaK in the closed conformation. Driven by the following GrpE-catalyzed ADP–ATP exchange, the DnaK–substrate complex dissociates (10). Since this ATP-driven cycle can even solubilize protein aggregates (11, 12), substantial forces must be transduced to the substrate protein (13–15). However, as for other chaperone systems (16), surprisingly little is known about how these forces and the resulting constraints of the underlying free energy surfaces affect the conformations of the denatured or misfolded substrate proteins. To better understand this important link between chaperone action and function, we probed the conformation of a substrate protein along the different stages of the chaperone cycle of DnaK with single-molecule Förster resonance energy transfer (smFRET), correlation spectroscopy, and microfluidic mixing.Open in a separate windowFig. 1.DnaK expands the denatured substrate protein. (A) Illustration of the DnaK–ATPase cycle. (B) Surface representation of rhodanese (PDB ID code 1RHS) with the subdomains indicated in different gray levels and the label positions of fluorescent dyes for single-molecule FRET measurements shown schematically. (C) FRET efficiency histograms of native rhodanese (gray) and denatured rhodanese under native conditions transiently populated in the microfluidic mixer (colored, measured 125 ms after dilution of rhodanese into native conditions). (D) FRET efficiency histograms of DnaJ–rhodanese complexes (0.5 µM DnaJ). (E) FRET efficiency histograms of DnaK–rhodanese complexes (0.5 µM DnaJ, 10 µM DnaK, and 1 mM ATP; DnaK and DnaJ were added simultaneously to rhodanese). Black lines indicate the DnaK–rhodanese complex population resulting from a fit that takes into account the residual population of refolded and DnaJ-bound rhodanese. The vertical lines in C–E indicate the positions of the FRET efficiency peaks of the native population of the respective rhodanese variants. The small populations at zero transfer efficiency in D (note the axis scaling and the small amplitudes of this population compared with E) originate from incomplete elimination of molecules with inactive acceptor fluorophores by pulsed interleaved excitation.     

Temperature dependence of protein folding kinetics in living cells

We measure the stability and folding rate of a mutant of the enzyme phosphoglycerate kinase (PGK) inside bone tissue cells as a function of temperature from 38 to 48 °C. To facilitate measurement in individual living cells, we developed a rapid laser temperature stepping method capable of measuring complete thermal melts and kinetic traces in about two min. We find that this method yields improved thermal melts compared to heating a sample chamber or microscope stage. By comparing results for six cells with in vitro data, we show that the protein is stabilized by about 6 kJ/mole in the cytoplasm, but the temperature dependence of folding kinetics is similar to in vitro. The main difference is a slightly steeper temperature dependence of the folding rate in some cells that can be rationalized in terms of temperature-dependent crowding, local viscosity, or hydrophobicity. The observed rate coefficients can be fitted within measurement uncertainty by an effective two-state model, even though PGK folds by a multistate mechanism. We validate the effective two-state model with a three-state free energy landscape of PGK to illustrate that the effective fitting parameters can represent a more complex underlying free energy landscape.     

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Phosphorylation of the T-cell receptor complex (TcR/CD3) mediates the survival and antigen-induced activation of T cells. TcR/CD3 phosphorylation is usually monitored using phospho-specific antibodies, which precludes dynamic measurements. Here, we have developed genetically encoded, live-cell reporters that enable simultaneous monitoring of the phosphorylation state and intracellular trafficking of CD3ζ, the major signal-transducing subunit of the TcR/CD3. We show that these reporters provide accurate readouts of TcR/CD3 phosphorylation and are sensitive to the local balance of kinase and phosphatase activities acting upon TcR/CD3. Using these reporters, we demonstrate that, in addition to the expected activation-dependent phosphorylation at the plasma membrane, tyrosine-phosphorylated CD3ζ accumulates on endosomal vesicles distinct from lysosomes. These results suggest that an intracellular pool of phosphorylated CD3ζ may help to sustain TcR/CD3 signaling after the receptor internalization.     

Phosphorylation-induced structural changes in smooth muscle myosin regulatory light chain

We have performed complementary time-resolved fluorescence resonance energy transfer (TR-FRET) experiments and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC) bound to myosin. PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin subfragment 1 (S1). TR-FRET resolved two simultaneously populated structural states of RLC, closed and open, in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mol fraction by ∼20%. MD simulations agree with experiments in remarkable detail, confirming the coexistence of two structural states, with phosphorylation shifting the system toward the more dynamic open structural state. This agreement between experiment and simulation validates the additional structural details provided by MD simulations: In the closed state, PD is bent onto the surface of the C-terminal lobe, stabilized by interdomain salt bridges. In the open state, PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by an intradomain salt bridge. The result is a vivid atomic-resolution visualization of the first step in the molecular mechanism by which phosphorylation activates smooth muscle.     

From the Cover: Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Fluorescent molecular switches have widespread potential for use as sensors, material applications in electro-optical data storages and displays, and superresolution fluorescence microscopy. We demonstrate that adjustment of fluorophore properties and environmental conditions allows the use of ordinary fluorescent dyes as efficient single-molecule switches that report sensitively on their local redox condition. Adding or removing reductant or oxidant, switches the fluorescence of oxazine dyes between stable fluorescent and nonfluorescent states. At low oxygen concentrations, the off-state that we ascribe to a radical anion is thermally stable with a lifetime in the minutes range. The molecular switches show a remarkable reliability with intriguing fatigue resistance at the single-molecule level: Depending on the switching rate, between 400 and 3,000 switching cycles are observed before irreversible photodestruction occurs. A detailed picture of the underlying photoinduced and redox reactions is elaborated. In the presence of both reductant and oxidant, continuous switching is manifested by “blinking” with independently controllable on- and off-state lifetimes in both deoxygenated and oxygenated environments. This “continuous switching mode” is advantageously used for imaging actin filament and actin filament bundles in fixed cells with subdiffraction-limited resolution.     

Resolving cadherin interactions and binding cooperativity at the single-molecule level

The cadherin family of Ca²⁺-dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions between cadherins that are at the core of cell–cell adhesion are a matter of considerable debate. A widely-accepted model is that cadherins adhere in 3 stages. First, the functional unit of cadherin adhesion is a cis dimer formed by the binding of the extracellular regions of 2 cadherins on the same cell surface. Second, formation of low-affinity trans interactions between cadherin cis dimers on opposing cell surfaces initiates cell–cell adhesion. Third, lateral clustering of cadherins cooperatively strengthens intercellular adhesion. Evidence of these cadherin binding states during adhesion is, however, contradictory, and evidence for cooperativity is lacking. We used single-molecule structural (fluorescence resonance energy transfer) and functional (atomic force microscopy) assays to demonstrate directly that cadherin monomers interact via their N-terminal EC1 domain to form trans adhesive complexes. We could not detect the formation of cadherin cis dimers, but found that increasing the density of cadherin monomers cooperatively increased the probability of trans adhesive binding.     

Novel Mutations of the ALMS1 Gene in Patients with Alström Syndrome

Objective Alström syndrome is an autosomal recessive genetic disease caused by a mutation in the ALMS1 gene. Alström syndrome is clinically characterized by multisystem involvement, including sensorineural deafness, cone-rod dystrophy, nystagmus, obesity, insulin resistance, type 2 diabetes and hypogonadism. The diagnosis is thus challenging for patients without this characteristic set of clinical symptoms. We explored the effectiveness of whole-exome sequencing in the diagnosis of Alström syndrome. Methods A girl with symptoms of Alström syndrome was tested and diagnosed with the disease by whole-exome sequencing. Results Whole-exome sequencing revealed two novel variants, c.6160_6161insAT: p.Lys2054Asnfs*21(exon 8) and c.10823_10824 delAG:p.Glu 3608Alafs*9 (exon16) in the ALMS1 gene, leading to premature termination codons and the domain of ALMS1 protein. Blood sample testing of her asymptomatic parents revealed them to be heterozygous carriers of the same mutations. Assembly showed that the mutations on both alleles were located in conserved sequences. A review of the ALMS1 gene nonsense mutation status was performed. Conclusion We herein report two novel variants of the ALMS1 gene discovered in a Chinese Alström syndrome patient that expand the mutational spectrum of ALMS1 and provided new insight into the molecular mechanism underlying Alström syndrome. Our findings add to the current knowledge concerning the diagnosis and treatment of Alström syndrome.     

Probing the resonance potential in the F atom reaction with hydrogen deuteride with spectroscopic accuracy

Reaction resonances are transiently trapped quantum states along the reaction coordinate in the transition state region of a chemical reaction that could have profound effects on the dynamics of the reaction. Obtaining an accurate reaction potential that holds these reaction resonance states and eventually modeling quantitatively the reaction resonance dynamics is still a great challenge. Up to now, the only viable way to obtain a resonance potential is through high-level ab initio calculations. Through highly accurate crossed-beam reactive scattering studies on isotope-substituted reactions, the accuracy of the resonance potential could be rigorously tested. Here we report a combined experimental and theoretical study on the resonance-mediated F + HD → HF + D reaction at the full quantum state resolved level, to probe the resonance potential in this benchmark system. The experimental result shows that isotope substitution has a dramatic effect on the resonance picture of this important system. Theoretical analyses suggest that the full-dimensional FH₂ ground potential surface, which was believed to be accurate in describing the resonance picture of the F + H₂ reaction, is found to be insufficiently accurate in predicting quantitatively the resonance picture for the F + HD → HF + D reaction. We constructed a global potential energy surface by using the CCSD(T) method that could predict the correct resonance peak positions as well as the dynamics for both F + H₂ → HF + H and F + HD → HF + D, providing an accurate resonance potential for this benchmark system with spectroscopic accuracy.     

Effects of Mono-Vacancies and Co-Vacancies of Nitrogen and Boron on the Energetics and Electronic Properties of Heterobilayer h-BN/graphene

A study is carried out which investigates the effects of the mono-vacancies of boron (VB) and nitrogen (VN) and the co-vacancies of nitrogen (N), and boron (B) on the energetics and the structural, electronic, and magnetic properties of an h-BN/graphene heterobilayer using first-principles calculations within the framework of the density functional theory (DFT). The heterobilayer is modelled using the periodic slab scheme. In the present case, a 4 × 4-(h-BN) monolayer is coupled to a 4 × 4-graphene monolayer, with a mismatch of 1.40%. In this coupling, the surface of interest is the 4 × 4-(h-BN) monolayer; the 4 × 4-graphene only represents the substrate that supports the 4 × 4-(h-BN) monolayer. From the calculations of the energy of formation of the 4 × 4-(h-BN)/4 × 4-graphene heterobilayer, with and without defects, it is established that, in both cases, the heterobilayers are energetically stable, from which it is inferred that these heterobilayers can be grown in the experiment. The formation of a mono-vacancy of boron (1 V_B), a mono-vacancy of nitrogen (1 V_N), and co-vacancies of boron and nitrogen (V_BN) induce, on the structural level: (a) for 1 V_B, a contraction n of the B-N bond lengths of ~2.46% and a slight change in the interfacial distance D (~0.096%) with respect to the heterobilayer free of defects (FD) are observed; (b) for 1 V_N, a slight contraction of the B-N of bond lengths of ~0.67% and an approach between the h-BN monolayer and the graphene of ~3.83% with respect to the FD heterobilayer are observed; (c) for V_BN, it can be seen that the N-N and B-B bond lengths (in the 1 V_B and 1 V_N regions, respectively) undergo an increase of ~2.00% and a decrease of ~3.83%, respectively. The calculations of the Löwdin charge for the FD heterobilayer and for those with defects (1 V_B, 1 V_N, and V_BN) show that the inclusion of this type of defect induces significant changes in the Löwdin charge redistribution of the neighboring atoms of VB and VN, causing chemically active regions that could favor the interaction of the heterobilayer with external atoms and/or molecules. On the basis of an analysis of the densities of states and the band structures, it is established that the heterobilayer with 1 V_B and V_BN take on a half-metallic and magnetic behavior. Due to all of these properties, the FD heterobilayer and those with 1 V_B, 1 V_N, and V_BN are candidates for possible adsorbent materials and possible materials that could be used for different spintronic applications.     

Cervical Root Enlargement in Segmental Zoster Paresis: A Study with Magnetic Resonance Imaging and Nerve Ultrasound

A 72-year-old woman presented with acute-progressive muscle weakness after a rash in the left upper limb. Muscle weakness was restricted to the left C5 innervated muscles. Short inversion time inversion recovery magnetic resonance imaging (MRI) showed a high-intensity signal in the left C5 nerve root, and nerve ultrasound showed its enlargement. She was diagnosed with segmental zoster paralysis (SZP) and treated with acyclovir and methylprednisolone. Her muscle strength gradually recovered, and the abnormal signal and enlargement in the left C5 nerve root improved. This is the first SZP case of confirmed improvement of abnormal findings on MRI and nerve ultrasound in association with muscle power recovery.     

Cryoglobulinemic glomerulonephritis with underlying occult HBV infection and Waldenström macroglobulinemia: A case report

Introduction:Occult hepatitis B virus (HBV) infection, defined as negative hepatitis B surface antigen (HBsAg) but detectable HBV DNA in serum and liver tissue, has very rarely been described in cryoglobulinemia (CG) patients. This case report sheds light on the possible link between occult HBV infection and CG.Patient concerns:A 76-year-old man presented with rapidly deteriorating renal function within 1 year.Diagnosis:Cryoglobulinemic glomerulonephritis was diagnosed through renal biopsy. Initially, the patient tested negative for HBsAg, but a low HBV viral load was later discovered, indicating an occult HBV infection. Further studies also revealed Waldenström macroglobulinemia (WM).Interventions:We treated the patient as WM using plasma exchange and rituximab-based immunosuppressive therapy.Outcomes:After 1 cycle of immunosuppressive treatment, there was no improvement of renal function. Shortly after, treatment was discontinued due to an episode of life-threatening pneumonia. Hemodialysis was ultimately required.Conclusion:Future studies are needed to explore the link between occult HBV infection and CG, to investigate the mediating role of lymphomagenesis, and to examine the effectiveness of anti-HBV drugs in treating the group of CG patients with occult HBV infection. We encourage clinicians to incorporate HBV viral load testing into the evaluation panel for CG patients especially in HBV-endemic areas, and to test HBV viral load for essential CG patients in whom CG cannot be attributed to any primary disease.     

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

Classical pharmacological models have incorporated an “intrinsic efficacy” parameter to capture system-independent effects of G protein–coupled receptor (GPCR) ligands. However, the nonlinear serial amplification of downstream signaling limits quantitation of ligand intrinsic efficacy. A recent biophysical study has characterized a ligand “molecular efficacy” that quantifies the influence of ligand-dependent receptor conformation on G protein activation. Nonetheless, the structural translation of ligand molecular efficacy into G protein activation remains unclear and forms the focus of this study. We first establish a robust, accessible, and sensitive assay to probe GPCR interaction with G protein and the Gα C terminus (G-peptide), an established structural determinant of G protein selectivity. We circumvent the need for extensive purification protocols by the single-step incorporation of receptor and G protein elements into giant plasma membrane vesicles (GPMVs). We use previously established SPASM FRET sensors to control the stoichiometry and effective concentration of receptor–G protein interactions. We demonstrate that GPMV-incorporated sensors (v-SPASM sensors) provide enhanced dynamic range, expression-insensitive readout, and a reagent level assay that yields single point measurements of ligand molecular efficacy. Leveraging this technology, we establish the receptor–G-peptide interaction as a sufficient structural determinant of this receptor-level parameter. Combining v-SPASM measurements with molecular dynamics (MD) simulations, we elucidate a two-stage receptor activation mechanism, wherein receptor–G-peptide interactions in an intermediate orientation alter the receptor conformational landscape to facilitate engagement of a fully coupled orientation that tunes G protein activation.

G protein–coupled receptors (GPCRs) are a superfamily of over 800 integral membrane proteins that are a major target of modern drugs (1, 2). GPCRs trigger cellular signaling cascades through the ligand-dependent activation of G proteins. The C terminus (last 27 amino acids) of the G protein α-subunit (G-peptide) is one of the important structural elements in the selective G protein activation by GPCRs (3, 4). An activated GPCR engages the G-peptide to relay GDP exchange and consequent G protein activation (5). Swapping residues in the G-peptide is a long-established strategy to drive promiscuous GPCR–G protein coupling, attesting to its role in cognate G protein recognition (6, 7). Despite its established function in selective G protein activation, the role of the G-peptide in determining agonist efficacy remains poorly understood and forms the focus of this study.Spectroscopic studies have highlighted dynamic conformational ensembles populated by the GPCR in response to ligand stimulation (8). In turn, single molecule Förster resonance energy transfer (FRET) studies of β2AR reveal that agonist binding causes a shift in the GPCR conformational ensemble, with corresponding changes in GDP–GTP exchange rates (9). The kinetics of conformational changes in the GPCR and consequent G protein activation were used to derive a “molecular efficacy” that quantifies the intrinsic effect that a ligand imposes directly onto its target GPCR. However, the structural basis of molecular efficacy and its association with the G-peptide requires further elucidation. Furthermore, high-resolution structures of GPCR–G protein complexes, stabilized by high efficacy agonists, reveal distinct orientations of the G-peptide bound to the receptor (10, 11). While these structures demonstrate that the transient GPCR–G protein interactions in cells are indeed dynamic, the relationship between these static snapshots and agonist molecular efficacy remains unclear.Delineating the structural dynamics of GPCR–G-peptide interactions is limited by the requirement of high concentrations of receptors and G proteins purified to homogeneity. To circumvent this limitation, we have previously employed GPCR systematic protein affinity strength modulation (SPASM) sensors to gain insights into the GPCR–G protein interaction in live cells. GPCR SPASM sensors contain a GPCR and a G-peptide tethered through an ER/K linker flanked by FRET probes. The ER/K linker controls the stoichiometry and effective concentration of the intramolecular interaction in live cells. SPASM sensors are designed to detect changes in the affinity of interacting proteins/peptides (12, 13). GPCR SPASM sensors have been extensively utilized to monitor the interaction strength between GPCR and G-peptides and have dissected the structural elements that modulate these interactions (14–16). Using a combination of GPCR SPASM sensors and molecular dynamics (MD) simulations, we have successfully gained insights into the structural dynamics of GPCR–G-peptide interactions (14–16).While GPCR SPASM sensors have been repeatedly used to probe the effects of full agonists (14–18), their narrow dynamic range, combined with the sensitivity of sensor measurements to live-cell expression levels, have limited reliable measurements with partial agonists. Here, we overcome these limitations by vesiculating GPCR SPASM sensors into giant plasma membrane vesicles (GPMVs) and use this system to probe into the structural and dynamic basis of agonist efficacy in GPCRs. GPMVs are large extracellular vesicles that can be generated from a variety of cell types via chemical vesiculation (19, 20). Isolated GPMVs that exhibit a size range of 0.1 to ∼15 μm are devoid of any detectable intracellular organelle signatures and are enriched in plasma membrane–associated proteins (21). GPMVs have been extensively used to study plasma membrane composition and architecture and provide a cell-free system to isolate plasma membrane–integrated proteins from the intracellular milieu. While GPMVs have been successfully utilized to study membrane proteins, such as receptors tyrosine kinases and ion channels (22, 23), their potential to investigate GPCR signaling remains unexplored.In the current study, we demonstrate the functional incorporation of β2AR-Gαs-peptide SPASM sensors into GPMVs (v-β2AR-Spep). The v-β2AR-Spep sensors show over a twofold increase in ligand-binding capacity and a 3.5-fold increase in sensor dynamic range compared to live cells. Unlike live cells, v-β2AR-Spep readout is not sensitive to cellular expression levels and shows robust reproducibility compared to membranes (z^’_GPMV = 0.50 to ∼0.61 versus z^’_membrane = −0.22 to ∼−1.28). The v-β2AR-Spep sensors can be frozen or preserved on ice for at least 12 d without compromising sensor readout. We leveraged the combined benefits of v-β2AR-Spep over live cell and membrane assays to profile the nuanced changes in GPCR–G-peptide interactions with 16 adrenergic receptor ligands of varying efficacy. The FRET intensity changes in v-β2AR-Spep for various agonists do not correlate with classical pharmacological parameters, including ligand binding affinity, half maximal effective concentration (EC₅₀), maximum response (E_max), and transducer ratio (τ). Instead, we find that the strength of the GPCR–G-peptide interaction shows perfect linear correlation between v-β2AR-Spep FRET sensor readout and both G protein activation and ligand molecular efficacy as reported using single molecule biophysical measurements (9). The partial agonists show reduced FRET intensity changes (ΔFRET) in v-β2AR-Spep compared to full agonists, revealing that the S-peptide interactions with partial-agonist–bound β2AR are weaker than with full-agonist–bound β2AR.Probing the structural dynamics of the β2AR-Spep interaction using a combination of computational analysis and v-β2AR-Spep mutagenesis experiments reveals that β2AR-Spep interactions unique to an intermediate orientation of the S-peptide (11) are essential in proceeding toward the fully active orientation. Using GPMV SPASM sensors not only for β2AR but also for Gαs-coupled β1 adrenergic receptor (β1AR) and dopamine receptor D1R, we demonstrate the broader significance of the intermediate orientation for multiple Gs-coupled GPCRs. While the intermediate orientation is essential for the receptor–G-peptide interaction, ligand efficacy correlates with the strength of the interaction in the fully active orientation. Furthermore, our MD simulations demonstrate a significantly lower interaction strength and stability for the intermediate compared to the fully coupled orientation. This disparity leads to strong allosteric communication between the ligand and G-peptide–binding sites only in the presence of full agonists and the fully coupled orientation. Taken together, our study 1) establishes a cell-free technology platform to profile the molecular efficacy of GPCR ligands, 2) provides insights into the relationship between agonist efficacy and strength of GPCR–G-peptide interaction, and 3) provides a two-stage model that accentuates the importance of intermediate conformations that are necessary but not sufficient for full activation of the GPCR–G protein complex.     

Coexistence of Sjögren syndrome in patients with synovitis,acne, pustulosis,hyperostosis, and osteitis syndrome: A retrospective observational study

To identify the prevalence and clinical characteristics of Sjögren syndrome (SS) in a Chinese single-center cohort of synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome.Patients diagnosed with SS were screened out from a cohort of 164 cases of SAHPO syndrome. Information regarding the patients’ gender, age at onset, clinical features, laboratory tests, bone scintigraphy, and treatment was reviewed.Five patients were screened out. The prevalence of SS in SAPHO patients was 3.05% The mean onset age of SS was 48.0 ± 12.0 years old and no apparent time order in the occurrence of SAPHO and SS was observed. Compared with the general SAPHO cohort, the 5 SS patients exhibited no significant difference in the SAPHO related clinical features or inflammatory markers, except for a higher prevalence of peripheral joints and bones involvement in bone scintigraphy. Objective evidence of dryness and positive salivary gland biopsy were found in all the patients. However, the positive rates of SSA and SSB antibody were only 20%. Anti-inflammatory treatment for SS was recorded in 3 patients (ESSDAI score: 3 in 2 patients; 12 in 1 patient) with extra-glandular manifestations, severe complications or poor response to the basic treatment.The prevalence of SS is higher in the SAPHO cohort than in the general Chinese population. Objective tests or biopsy might be more indicative than the antibody detection for SS diagnosis. Anti-inflammatory treatment should be prescribed in consideration of both the severity of SS and the demand for disease activity control of SAPHO.     

Relationship between serum uric acid and hypertension in patients with primary Sjögren's syndrome: A retrospective cohort study

Primary Sjögren''s syndrome (pSS) patients with hypertension (pSS‐HT) have a significantly increased risk of cardio‐cerebrovascular events. Serum uric acid (SUA), a potential inflammatory substance, is considered to be closely related to hypertension in the general population. Our aim is to assess the association between SUA and pSS‐HT. This is a retrospective cohort study. The diagnosis of pSS is based on the American European Consensus Classification criteria. Primary outcome was incident hypertension in pSS patients. Cox regression model was used to estimate the hazard ratios (HR) and 95% CI of SUA in pSS‐HT. The authors also plotted Kaplan–Meier plots to assess the cumulative risk of first hypertension in patients with hyperuricemia and normal uric acid. In addition, the dose‐response curve was also used to discuss the relationship between SUA and pSS‐HT. Finally, three hundred and fifty‐one pSS patients were enrolled from May 2011 to May 2020, of which 166 cases developed hypertension within a mean follow‐up of 3.91 years. Univariate Cox regression demonstrated that SUA was associated with the onset of hypertension in pSS (HR: 1.005 95%Cl: 1.002–1.009). After adjusting for the potential risk factors, the relationship remained unchanged (HR: 1.003, 95%Cl: 1.001–1.005). Kaplan‐Meier survival analysis showed a statistically significant difference of hypertension risk between hyperuricemia patients and normal uric acid patients (P = .026). There was also a significant dose‐effect relationship between SUA and hypertension in pSS in dose‐response model. In this study, the authors find that SUA may be closely associated with the development of hypertension in pSS, which is also confirmed by our dose‐response model. Therefore, SUA could be considered in the management of pSS‐HT.