Biophysical journal最新文献

{"title":"Ligand binding kinetics to evaluate the function and stability of A_2AR in nanodiscs.","authors":"John M Pettersen, Olivia McCracken, Anne Skaja Robinson","doi":"10.1016/j.bpj.2024.12.018","DOIUrl":"10.1016/j.bpj.2024.12.018","url":null,"abstract":"<p><p>G-protein-coupled receptors (GPCRs) represent one of the largest classes of therapeutic targets. However, developing successful therapeutics to target GPCRs is a challenging endeavor, with many molecules failing during in vivo clinical trials due to a lack of efficacy. The in vitro identification of drug-target residence time (1/k_off) has been suggested to improve predictions of in vivo success. Here, a ligand binding assay using fluorescence anisotropy was implemented to successfully determine on rates (k_on) and off rates (k_off) of labeled and unlabeled ligands binding to the adenosine A_2A receptor (A_2AR) purified into nanodiscs (A_2AR-NDs). The kinetic assay was used to determine the optimal storage conditions of A_2AR-NDs, where they were found to be stable for more than 6 months at -80°C. The binding assay was implemented to further understand receptor function by determining the effects of charged lipids on agonist binding kinetics, how sodium levels allosterically modulate A_2AR function, and how A_2AR protonation affects agonist binding.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142845757","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Driving forces of proton-pumping rhodopsins.","authors":"Akari Okuyama, Shoko Hososhima, Hideki Kandori, Satoshi P Tsunoda","doi":"10.1016/j.bpj.2024.09.007","DOIUrl":"10.1016/j.bpj.2024.09.007","url":null,"abstract":"<p><p>Proton-pumping rhodopsins are light-driven proton transporters that have been discovered from various microbiota. They are categorized into two groups: outward-directed and inward-directed proton pumps. Although the directions of transport are opposite, they are active proton transporters that create an H⁺ gradient across a membrane. Here, we aimed to study the driving force of the proton-pumping rhodopsins and the effect of ΔΨ and ΔpH on their pumping functions. We systematically characterized the H⁺ transport properties of nine different rhodopsins, six outward-directed H⁺ pumps and three inward-directed pumps, by patch-clamp measurements after expressing them in mammalian cells. The driving force of each pump was estimated from the slope of the current-voltage relations (I-V plot). Notably, among the tested rhodopsins, we found a large variation in driving forces, ranging from 83 to 399 mV. The driving force and decay rate of each pump current exhibited a good correlation. We determined driving forces under various pHs. pH dependency was less than predicted by the Nernst potential in most of the rhodopsins. Our study demonstrates that the H⁺-pumping rhodopsins from different organisms exhibit various pumping properties in terms of driving force, kinetics, and pH dependency, which could be evolutionarily derived from adaptations to their environments.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4274-4284"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142145034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"3D-aligned tetrameric ion channels with universal residue labels for comparative structural analysis.","authors":"Denis B Tikhonov, Vyacheslav S Korkosh, Boris S Zhorov","doi":"10.1016/j.bpj.2024.12.019","DOIUrl":"10.1016/j.bpj.2024.12.019","url":null,"abstract":"<p><p>Despite their large functional diversity and poor sequence similarity, tetrameric and pseudotetrameric potassium, sodium, calcium, and cyclic-nucleotide gated channels, as well as two-pore channels, transient receptor potential channels, and ionotropic glutamate receptor channels, share a common folding pattern of the transmembrane (TM) helices in the pore domain. In each subunit or repeat, two TM helices connected by a membrane-reentering P-loop contribute a quarter to the pore domain. The P-loop includes a membrane-descending helix, P1, which is structurally the most conserved element of these channels, and residues that contribute to the selectivity-filter region at the constriction of the ion-permeating pathway. In 24-TM channels, the pore domain is surrounded by four voltage-sensing domains, each with conserved folding of four TM helices. Hundreds of atomic-scale structures of these channels, referred to as \"P-loop channels,\" have been obtained through x-ray crystallography or cryoelectron microscopy. The number of experimental structures of P-loop channels deposited in the PDB is rapidly increasing. AlphaFold3, RoseTTAFold, and other computational tools can be used to generate three-dimensional (3D) models of P-loop channels that lack experimental structures. While comparative structural analysis of P-loop channels is desirable, it is hindered by variations in residue numbers and 3D orientations of the channels. To address this problem, we have developed a universal residue-labeling scheme for TM helices and P-loops. We further created a database of P-loop ion channels, PLIC: www.plic3da.com, which currently includes over 400 3D-aligned structures with relabeled residues. We use this database to compare multiple 3D structures of channels from different subfamilies. The comparison, which for the first time employs statistical methods, highlights conserved and variable elements in the channels' folding, reveals irregularities, and identifies outliers that warrant further analysis.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142852281","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hidden water's influence on rhodopsin activation.","authors":"Zachary T Bachler, Michael F Brown","doi":"10.1016/j.bpj.2024.11.012","DOIUrl":"10.1016/j.bpj.2024.11.012","url":null,"abstract":"<p><p>Structural biology relies on several powerful techniques, but these tend to be limited in their ability to characterize protein fluctuations and mobility. Overreliance on structural approaches can lead to omission of critical information regarding biological function. Currently there is a need for complementary biophysical methods to visualize these mobile aspects of protein function. Here, we review hydrostatic and osmotic pressure-based techniques to address this shortcoming for the paradigm of rhodopsin. Hydrostatic and osmotic pressure data contribute important examples, which are interpreted in terms of an energy landscape for hydration-mediated protein dynamics. We find that perturbations of rhodopsin conformational equilibria by force-based methods are not unrelated phenomena; rather they probe various hydration states involving functional proton reactions. Hydrostatic pressure acts on small numbers of strongly interacting structural or solvent-shell water molecules with relatively high energies, while osmotic pressure acts on large numbers of weakly interacting bulk-like water molecules with low energies. Local solvent fluctuations due to the hydration shell and collective water interactions affect hydrogen-bonded networks and domain motions that are explained by a hierarchical energy landscape model for protein dynamics.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4167-4179"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142643366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proton reactions: From basic science to biomedical applications.","authors":"Ana-Nicoleta Bondar, Thomas E DeCoursey","doi":"10.1016/j.bpj.2024.11.013","DOIUrl":"10.1016/j.bpj.2024.11.013","url":null,"abstract":"","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"E1-E5"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142791101","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Kinetic network modeling with molecular simulation inputs: A proton-coupled phosphate symporter.","authors":"Yu Liu, Chenghan Li, Meghna Gupta, Robert M Stroud, Gregory A Voth","doi":"10.1016/j.bpj.2024.03.035","DOIUrl":"10.1016/j.bpj.2024.03.035","url":null,"abstract":"<p><p>Phosphate, an essential metabolite involved in numerous cellular functions, is taken up by proton-coupled phosphate transporters of plants and fungi within the major facilitator family. Similar phosphate transporters have been identified across a diverse range of biological entities, including various protozoan parasites linked to human diseases, breast cancer cells with increased phosphate requirements, and osteoclast-like cells engaged in bone resorption. Prior studies have proposed an overview of the functional cycle of a proton-driven phosphate transporter (PiPT), yet a comprehensive understanding of the proposed reaction pathways necessitates a closer examination of each elementary reaction step within an overall kinetic framework. In this work, we leverage kinetic network modeling in conjunction with a \"bottom-up\" molecular dynamics approach to show how such an approach can characterize the proton-phosphate co-transport behavior of PiPT under different pH and phosphate concentration conditions. In turn, this allows us to reveal the prevailing reaction pathway within a high-affinity phosphate transporter under different experimental conditions and to uncover the molecular origin of the optimal pH condition of this transporter.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4191-4199"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140317742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Constant-pH MD simulations of the protonation-triggered conformational switching in diphtheria toxin translocation domain.","authors":"Nuno F B Oliveira, Alexey S Ladokhin, Miguel Machuqueiro","doi":"10.1016/j.bpj.2024.08.023","DOIUrl":"10.1016/j.bpj.2024.08.023","url":null,"abstract":"<p><p>Protonation of key residues in the diphtheria toxin translocation (T)-domain triggered by endosomal acidification is critical for inducing a series of conformational transitions critical for the cellular entry of the toxin. Previous experiments revealed the importance of histidine residues in modulating pH-dependent transitions. They suggested the presence of a \"safety latch\" preventing premature refolding of the T-domain by a yet poorly understood mechanism. Here, we used constant-pH molecular dynamics simulations to systematically investigate the protonation sequence in the wild-type T-domain and the following mutants: H223Q, H257Q, E259Q, and H223Q/H257Q. Comparison of these computational results with previous experimental data on T-domain stability and activity with the H-to-Q replacements confirms the role of H223 (pK_a = 6.5) in delaying the protonation of the main trigger, H257 (pK_a = 2.2 in the WT and pK_a = 4.9 in H223Q). Our calculations also reveal a very low pK_a for a neighboring acidic residue E259, which does not get protonated even during simulations at pH 3. This residue also contributes to the formation of the safety latch, with the pK_a of H257 increasing from 2.2 to 5.1 upon E259Q replacement. In contrast, the latter replacement has virtually no effect on the protonation of the H223. Thus, we conclude that the interplay of the protonation in the H223/H257/E259 triad has evolved to prevent triggering the accidental refolding of the T-domain by a fluctuation in the protonation of the main trigger at neutral pH, before the incorporation of the toxin inside the endosome. Subsequent acidification of the endosome overcomes the safety latch and triggers conformational switching via repulsion of H223⁺ and H257⁺. This protonation/conformation relationship corroborates experimental findings and offers a detailed stepwise molecular description of the transition mechanism, which can be instrumental in optimizing the potential applications of the T-domain for targeted delivery of therapies to tumors and other diseased acidic tissues.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4266-4273"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142104005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proton diffusion on the surface of mixed lipid membranes highlights the role of membrane composition.","authors":"Ambili Ramanthrikkovil Variyam, Mateusz Rzycki, Anna Yucknovsky, Alexei A Stuchebrukhov, Dominik Drabik, Nadav Amdursky","doi":"10.1016/j.bpj.2024.07.002","DOIUrl":"10.1016/j.bpj.2024.07.002","url":null,"abstract":"<p><p>Proton circuits within biological membranes, the foundation of natural bioenergetic systems, are significantly influenced by the lipid compositions of different biological membranes. In this study, we investigate the influence of mixed lipid membrane composition on the proton transfer (PT) properties on the surface of the membrane. We track the excited-state PT (ESPT) process from a tethered probe to the membrane with timescales and length scales of PT relevant to bioenergetic systems. Two processes can happen during ESPT: the initial PT from the probe to the membrane at short timescales, followed by diffusion of dissociated protons around the probe on the membrane, and the possible geminate recombination with the probe at longer timescales. Here, we use membranes composed of mixtures of phosphatidylcholine (PC) and phosphatidic acid (PA). We show that the changes in the ESPT properties are not monotonous with the concentration of the lipid mixture; at a low concentration of PA in PC, we find that the membrane is a poor proton acceptor. Molecular dynamics simulations indicate that the membrane is more structured at this specific lipid mixture, with the least number of defects. Accordingly, we suggest that the structure of the membrane is an important factor in facilitating PT. We further show that the composition of the membrane affects the geminate proton diffusion around the probe, whereas, on a timescale of tens of nanoseconds, the dissociated proton is mostly lateral restricted to the membrane plane in PA membranes, while in PC, the diffusion is less restricted by the membrane.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4200-4210"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141496991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Interior pH-sensing residue of human voltage-gated proton channel H_v1 is histidine 168.","authors":"Mingzhe Shen, Yandong Huang, Zhitao Cai, Vladimir V Cherny, Thomas E DeCoursey, Jana Shen","doi":"10.1016/j.bpj.2024.07.027","DOIUrl":"10.1016/j.bpj.2024.07.027","url":null,"abstract":"<p><p>The molecular mechanisms governing the human voltage-gated proton channel hH_v1 remain elusive. Here, we used membrane-enabled hybrid-solvent continuous constant pH molecular dynamics (CpHMD) simulations with pH replica exchange to further evaluate the structural models of hH_v1 in the closed (hyperpolarized) and open (depolarized) states recently obtained with MD simulations and explore potential pH-sensing residues. The CpHMD titration at a set of symmetric pH conditions revealed three residues that can gain or lose protons upon channel depolarization. Among them, residue H168 at the intracellular end of the S3 helix switches from the deprotonated to the protonated state and its protonation is correlated with the increased tilting of the S3 helix during the transition from the closed to the open state. Thus, the simulation data suggest H168 as an interior pH sensor, in support of a recent finding based on electrophysiological experiments of H_v1 mutants. We propose that protonation of H168 acts as a key that unlocks the closed channel configuration by increasing the flexibility of the S2-S3 linker, which increases the tilt angle of S3 and enhances the mobility of the S4 helix, thus promoting channel opening. Our work represents an important step toward deciphering the pH-dependent gating mechanism of hH_v1.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4211-4220"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141756970","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Searching for proton transfer channels in respiratory complex I.","authors":"Panyue Wang, Jackson Demaray, Stanislav Moroz, Alexei A Stuchebrukhov","doi":"10.1016/j.bpj.2024.07.041","DOIUrl":"10.1016/j.bpj.2024.07.041","url":null,"abstract":"<p><p>We have explored a strategy to identify potential proton transfer channels using computational analysis of a protein structure based on Voronoi partitioning and applied it for the analysis of proton transfer pathways in redox-driven proton-pumping respiratory complex I. The analysis results in a network of connected voids/channels, which represent the dual structure of the protein; we then hydrated the identified channels using our water placement program Dowser++. Many theoretical water molecules found in the channels perfectly match the observed experimental water molecules in the structure; some other predicted water molecules have not been resolved in the experiments. The channels are of varying cross sections. Some channels are big enough to accommodate water molecules that are suitable to conduct protons; others are too narrow to hold water but require only minor conformational changes to accommodate proton transfer. We provide a preliminary analysis of the proton conductivity of the network channels, classifying the proton transfer channels as open, closed, and partially open, and discuss possible conformational changes that can modulate, i.e., open and close, the channels.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"4233-4244"},"PeriodicalIF":3.2,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141878305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}