Biophysical journal最新文献

The development of methods that allow a structural interpretation of linear and nonlinear vibrational spectra is of great importance, both for spectroscopy and for optimizing force field quality. The experimentally measured signals are ensemble averages over all accessible configurations, which complicates spectral calculations. To account for this, we present a recipe for calculating vibrational amide-I spectra of proteins based on metadynamics molecular dynamics simulations. For each frame, a one-exciton Hamiltonian is set up for the backbone amide groups, in which the couplings are estimated with the transition-charge coupling model for nonnearest neighbors, and with a parametrized map of ab initio calculations that give the coupling as a function of the dihedral angles for nearest neighbors. The local-mode frequency variations due to environmental factors such as hydrogen bonds are modeled by exploiting the linear relationship between the amide C-O bond length and the amide-I frequency. The spectra are subsequently calculated while taking into account the equilibrium statistical weights of the frames that are determined using a previously published reweighting procedure. By implementing all these steps in an efficient Fortran code, the spectra can be averaged over very large amounts of structures, thereby extensively covering the phase space of proteins. Using this recipe, the spectral responses of 2.5 million frames of a metadynamics simulation of the miniprotein Trp-cage are averaged to reproduce the experimental temperature-dependent IR spectra very well. The spectral calculations provide new insight into the origin of the various spectral signatures (which are typically challenging to disentangle in the congested amide-I region), and allow for a direct structural interpretation of the experimental spectra and for validation of the molecular dynamics simulations of ensembles.

Desensitization is a prominent feature of nearly all ligand-gated ion channels. Acid-sensing ion channels (ASICs) undergo desensitization within hundreds of milliseconds to seconds upon continual extracellular acidification. The ASIC mechanism of desensitization is primarily due to the isomerization or "flipping" of a short linker joining the 11th and 12th β sheets in the extracellular domain. In the resting and active states this β11-12 linker adopts an "upward" conformation while in the desensitized conformation the linker assumes a "downward" state. It is unclear if a single linker adopting the downward state is sufficient to desensitize the entire channel, or if all three are needed or some more complex scheme. To accommodate this downward state, specific peptide bonds within the linker adopt either trans-like or cis-like conformations. Since proline-containing peptide bonds undergo cis-trans isomerization very slowly, we hypothesized that introducing proline residues in the linker may slow or even abolish ASIC desensitization, potentially providing a valuable research tool. Proline substitutions in the chicken ASIC1 β11-12 linker (L414P and Y416P) slowed desensitization decays approximately 100- to 1000-fold as measured in excised patches. Both L414P and Y416P shifted the steady-state desensitization curves to more acidic pH values while activation curves and ion selectivity were largely unaffected (except for a left-shifted activation pH₅₀ of L414P). To investigate the functional stoichiometry of desensitization in the trimeric ASIC, we created families of L414P and Y416P concatemers with zero, one, two, or three proline substitutions in all possible configurations. Introducing one or two L414P or Y416P substitutions only slightly attenuated desensitization, suggesting that conformational changes in the single remaining faster wild-type subunits were sufficient to desensitize the channel. These data highlight the unusual cis-trans isomerization mechanism of ASIC desensitization and support a model where ASIC desensitization requires only a single subunit.

We propose a model for the feedback control processes that underlie the robustness and high sensitivity of mechanosensory hair cells. Our model encompasses self-tuning active processes intrinsic to these cells, which drive the amplification of mechanical stimuli by consuming metabolic energy, and a neural input process that protects these cells from damage caused by powerful stimuli. We explore the effects of these two feedback mechanisms on mechanical self-oscillations of the sense cells and their response to external forcing.

We elucidate the mechanism underpinning a recently discovered phenomenon in which cells respond to MHz-order mechanostimuli. Deformations induced along the plasma membrane under these external mechanical cues are observed to decrease the membrane tension, which, in turn, drives transient and reversible remodelling of its lipid structure. In particular, the increase and consequent coalescence of ordered lipid microdomains leads to closer proximity to mechanosensitive ion channels-Piezo1, in particular-that due to crowding, results in their activation to mobilise influx of calcium (Ca²⁺) ions into the cell. It is such modulation of this second messenger that is responsible for the downstream signalling and cell fates that ensue. Additionally, we show that such spatiotemporal control over the membrane microdomains in cells-without necessitating biochemical factors-facilitates aggregation and association of intrinsically-disordered tau proteins in neuroblastoma cells, and their transformation to pathological conditions implicated in neurodegenerative diseases, thereby paving the way for the development of therapeutic intervention strategies.

The C9orf72 gene associated with amyotrophic lateral sclerosis/frontotemporal dementia is translated to five dipeptide repeat proteins, among which poly-proline-arginine (PR) is the most toxic in cell and animal models, contributing to a variety of cellular defects. It has been proposed that polyPR disrupts nucleocytoplasmic transport (NCT) through several mechanisms including accumulation in the nuclear pore complex (NPC), accumulation in the nucleolus, and direct interactions with transport receptors. The NPC, which is the key regulator of transport between the cytoplasm and nucleus, plays a central role in these suggested mechanisms. Exploring polyPR interaction with the NPC provides valuable insight into the molecular details of polyPR-mediated NCT defects. To address this, we use coarse-grained molecular dynamics models of polyPR and the yeast NPC lined with intrinsically disordered FG-nucleoporins (FG-Nups). Our findings indicate no aggregation of polyPR within the NPC or permanent binding to FG-Nups. Instead, polyPR translocates through the NPC, following a trajectory through the central low-density region of the pore. In the case of longer polyPRs, we observe a higher energy barrier for translocation and a narrower translocation channel. Our study shows that polyPR and FG-Nups are mainly engaged in steric interactions inside the NPC with only a small contribution of specific cation-pi, hydrophobic, and electrostatic interactions, allowing polyPR to overcome the entropic barrier of the NPC in a size-dependent manner.

Many bacteria are protected by different types of polysaccharide capsules, structures formed of long repetitive glycan chains that are sometimes free and sometimes anchored to the outer membrane via lipid tails. One type, called group 4 capsule, results from the expression of the gfcABCDE-etp-etk operon in Escherichia coli. Of the proteins encoded in this operon, GfcE is thought to provide the export pore for free polysaccharide chains, but none of the proteins has been implicated in the export of chains carrying a lipid anchor. For this function, GfcD has been a focus of attention as the only outer membrane β-barrel encoded in the operon. AlphaFold predicts two β-barrel domains in GfcD, a canonical N-terminal one of 12 strands and an unusual C-terminal one of 13 strands, which features a large lateral aperture between strands β1 and β13. This immediately suggests a lateral exit gate for hydrophobic molecules into the membrane, analogous to the one proposed for the lipopolysaccharide export pore LptD. Here, we report an unsteered molecular dynamics study of GfcD embedded in the bacterial outer membrane, with the common polysaccharide anchor, lipid A, inserted in the pore of the C-terminal barrel. Our results show that the lateral aperture does not collapse during simulations and membrane lipids nevertheless do not penetrate the barrel but the lipid chains of the lipid A molecule readily exit into the membrane.

Perforation of the outer mitochondrial membrane triggered by BAX and facilitated by its main activator cBID is a fundamental process in cell apoptosis. Here, we employ a newly designed correlative approach based on a combination of a fluorescence cross correlation binding with a calcein permeabilization assay to understand the involvement of BAX in pore formation under oxidative stress conditions. To mimic the oxidative stress, we enriched liposomal membranes by phosphatidylcholines with truncated sn-2 acyl chains terminated by a carboxyl or aldehyde moiety. Our observations revealed that oxidative stress enhances proapoptotic conditions involving accelerated pore-opening kinetics. This enhancement is achieved through increased recruitment of BAX to the membrane and facilitation of BAX membrane insertion. Despite these effects, the fundamental mechanism of pore formation remained unchanged, suggesting an all-or-none mechanism. In line with this mechanism, we demonstrated that the minimal number of BAX molecules at the membrane necessary for pore formation remains constant regardless of BAX activation by cBID or the presence of oxidized lipids. Overall, our findings give a comprehensive picture of the molecular mechanisms underlying apoptotic pore formation and highlight the selective amplifying role of oxidized lipids in triggering formation of membrane pores.