Biophysical journal最新文献

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.

The cochlea's mechanical response to sound stimulation is nonlinear, likely due to saturation of the mechanoelectric transduction current that is part of an electromechanical feedback loop. The ability of a second tone or tones to reduce the response to a probe tone is one manifestation of nonlinearity, termed suppression. Using optical coherence tomography to measure motion within the organ of Corti, regional motion variations have been observed. Here, we report on the suppression that occurs within the organ of Corti when a high-sound-level, low-frequency suppressor tone was delivered along with a sweep of discreet single tones. Responses were measured in the base of the gerbil cochlea at two best frequency locations, with two different directions of observation relative to the sensory tissue's anatomical axes. Suppression extended over a wide frequency range in the outer hair cell region, whereas it was typically limited to the best frequency peak in the reticular lamina region and at the basilar membrane. Aspects of the observed suppression were consistent with the effect of a saturating nonlinearity. Recent measurements have noted the three-dimensional nature of organ of Corti motion. The effects of suppression observed here could be due to a combination of reduced motion amplitude and altered vibration axis.

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.

The HIV-1 capsid is an irregularly shaped protein complex containing the viral genome and several proteins needed for integration into the host cell genome. Small molecules, such as the drug-like compound PF-3450074 (PF74) and the anionic sugar inositolhexakisphosphate (IP6), are known to impact capsid stability, although the mechanisms through which they do so remain unknown. In this study, we employed atomistic molecular dynamics simulations to study the impact of molecules bound to hexamers at the central pore (IP6) and the FG-binding site (PF74) on the interface between capsid oligomers. We found that the IP6 cofactor stabilizes a pair of neighboring hexamers in their flattest configurations, whereas PF74 introduces a strong preference for intermediate tilt angles. These results suggest that the tilt angle between neighboring hexamers is a primary mechanism for the modulation of capsid stability. In addition, hexamer-pentamer interfaces were highly stable, suggesting that pentamers are likely not the locus of disassembly.

Physical spatiotemporal characteristics of cellular cortex dominate cell functions and even determine cell fate. The cellular cortex is able to reorganize to a dynamic steady status with changed stiffnesses once stimulated, and thus alter the physiological and pathological activities of almost all types of cells. TGF-β2, a potent pleiotropic growth factor, plays important roles in cartilage development, endochondral ossification, and cartilage diseases. However, it is not yet known whether TGF-β2 would alter the physical spatiotemporal characteristics of the cell cortex such as cortex stiffness, thereby affecting the function of chondrocytes. In this study, we investigated the influence of TGF-β2 on cellular cortex stiffness of chondrocytes and the underlying mechanism. We firstly detected TGF-β2-induced changes in cytoskeleton and focal adhesion plaque, which were closely related to cellular cortex stiffness. We then characterized the landscape of nanoscale cortex stiffness in individual chondrocytes induced by TGF-β2 via atomic force microscopy. By using inhibitors, latrunculin A and blebbistatin, we verified the importance of cytoskeleton-focal adhesion plaque axis on cellular cortex stiffness of chondrocytes induced by TGF-β2. We finally elucidated that TGF-β2 enhanced the phosphorylation of Smad3 and facilitated the nuclear accumulation of p-Smad3. The p-Smad3 aggregated in the nuclei enhanced the cytoskeleton and focal adhesion plaque at transcriptional level, thereby mediating changes in cell cortex stiffness. Taken together, these results provide an understanding about the role of TGF-β2 on physical spatiotemporal properties of cell cortex in chondrocytes, and might provide cues for interpretation of cartilage development and interventions to cartilage diseases.

In eukaryotic cells, the phospholipid cardiolipin (CL) is a crucial component that influences the function and organization of the mitochondrial inner membrane. In this study, we examined its potential role in passive proton transmembrane flux using unilamellar vesicles composed of natural egg phosphatidylcholine (PC) alone or with the inclusion of 18 or 34 mol % CL. A membrane potential was induced by a potassium gradient, and oxonol VI dye was used to monitor membrane potential dissipation resulting from proton transmembrane efflux. Increasing the CL content led to a net increase in proton efflux, which was also dependent on the magnitude of the membrane potential. The same increase in proton efflux was measured in the presence of the equally negatively charged phosphatidylglycerol, indicating that the charge of CL plays a more important role than its structure in this mechanism. When varying the proton membrane permeability (p_H) using the protonophore CCCP, we observed that unlike PC liposomes, where a small amount of CCCP was sufficient to achieve maximum flux, a significantly larger amount of protonophore was required in the presence of CL. Conversely, increasing the buffer capacity increased proton flux, indicating that proton availability, rather than membrane permeability, may be the limiting factor for proton leak. Our findings demonstrated that a higher proton content associated with the membrane was correlated with an increasing leak in the presence of CL. Additionally, smaller liposome diameters appeared to favor proton leak. Taken together, our results suggest that the presence of negatively charged CL in a membrane traps protons and increases their leakage, potentially in a manner dependent on membrane curvature. We discuss the possible mechanisms and implications of these findings for mitochondrial respiration function.

The nuclear pore complex (NPC) is responsible for the selective transport of biomolecules in and out of the nucleus. This selective feature is achieved through intrinsically disordered proteins, FG-Nups, that are anchored to the inner wall of the NPC. Cargo smaller than approximately 5 nm can rapidly diffuse through the NPC whereas larger cargo is increasingly slowed down. Larger cargos bound to chaperone proteins (from the karyopherin or Kap family) can still be transported due to nonspecific interactions with the FG-Nups. Although various mechanisms for the transport of Kaps have been proposed, a consensus has still to be reached. Here, we conducted a coarse-grained molecular dynamics study to shed light on Kap translocation through NPCs. We investigated the effect of Kap surface charge and hydrophobicity on the transport rate. We found that the negative charge of the Kaps is essential for transport whereas Kap hydrophobicity of the transport particle aids in the translocation. Interestingly, our results indicate that the positive net charge of the nuclear Nups (especially Nup1) is instrumental for the transport of Kaps, revealing a (previously proposed) gradient of increasing binding affinity of the Kaps with FG-Nups from the cytoplasm to the nucleus.

Microtubule stability is known to be governed by a stabilizing GTP/GDP-Pi cap, but the exact relation between growth velocity, GTP hydrolysis, and catastrophes remains unclear. We investigate the dynamics of the stabilizing cap through in vitro reconstitution of microtubule dynamics in contact with microfabricated barriers, using the plus-end binding protein GFP-EB3 as a marker for the nucleotide state of the tip. The interaction of growing microtubules with steric objects is known to slow down microtubule growth and accelerate catastrophes. We show that the lifetime distributions of stalled microtubules, as well as the corresponding lifetime distributions of freely growing microtubules, can be fully described with a simple phenomenological 1D model based on noisy microtubule growth and a single EB3-dependent hydrolysis rate. This same model is furthermore capable of explaining both the previously reported mild catastrophe dependence on microtubule growth rates and the catastrophe statistics during tubulin washout experiments.

Cyanobacteria are responsible for up to 80% of aquatic carbon dioxide fixation and have evolved a specialized carbon concentrating mechanism to increase photosynthetic yield. As such, cyanobacteria are attractive targets for synthetic biology and engineering approaches to address the demands of global energy security, food production, and climate change for an increasing world's population. The bicarbonate transporter BicA is a sodium-dependent, low-affinity, high-flux bicarbonate symporter expressed in the plasma membrane of cyanobacteria. Despite extensive biochemical characterization of BicA, including the resolution of the BicA crystal structure, the dynamic understanding of the bicarbonate transport mechanism remains elusive. To this end, we have collected over 1 ms of all-atom molecular dynamics simulation data of the BicA dimer to elucidate the structural rearrangements involved in the substrate transport process. We further characterized the energetics of the transition of BicA protomers and investigated potential mutations that are shown to decrease the free energy barrier of conformational transitions. In all, our study illuminates a detailed mechanistic understanding of the conformational dynamics of bicarbonate transporters and provides atomistic insights to engineering these transporters for enhanced photosynthetic production.

NOTCH, a single-pass transmembrane protein, plays a crucial role in cell fate determination through cell-to-cell communication. It interacts with two canonical ligands, Delta-like (DLL) and Jagged (JAG), located on neighboring cells to regulate diverse cellular processes. Despite extensive studies on the functional roles of NOTCH and its ligands in cellular growth, the structural details of full-length NOTCH and its ligands remain poorly understood. In this study, we employed fragment-based modeling and multiscale simulations to study the full-length structure of the human NOTCH ectodomain, comprising 1756 amino acids. We performed coarse-grained dynamics simulations of NOTCH in both glycosylated and nonglycosylated forms to investigate the role of glycosylation in modulating its conformational dynamics. In apo form, coarse-grained simulations revealed that glycosylated NOTCH protein can transition from an elongated structure of ∼86 nm from the membrane surface to a semicompact state (∼23.81 ± 9.98 nm), which aligns with cryo-EM data. To transition from the apo form to ligand-bound forms of NOTCH, we followed an atomistic and integrative modeling approach to model the interactions between NOTCH-DLL4 and NOTCH-JAG1. Atomistic simulations of the smaller bound fragment EGF8-13 patch revealed conformational plasticity critical for NOTCH binding, while integrative modeling of full-length complexes suggested a larger binding surface than reported previously. Simulations of pathogenic mutations revealed that E360K and R448Q disrupted the NOTCH-ligand interaction surfaces, causing dissociation. In contrast, C1133Y in the Abruptex domain compromised protein stability by disrupting the domain's interaction with the ligand-binding domain in the apo form of NOTCH-ECD. These findings provide a detailed molecular understanding of NOTCH and its ligands, offering insights that could enable the development of novel therapeutic approaches to selectively target pathogenic NOTCH signaling.