Biophysical journal最新文献

Caveolins are lipid-binding proteins that can organize membrane remodeling and oligomerize into the 8S complex. The CAV1-8S complex comprises a disk-like structure, about 15 nm in diameter, with a central beta barrel. Further oligomerization of 8S complexes remodels the membrane into caveolae vessels, with a dependence on cholesterol concentration. However, the molecular mechanisms behind membrane remodeling and cholesterol filtering are still not understood. Performing atomistic molecular dynamics simulations in combination with advanced sampling techniques, we describe how the CAV1-8S complex bends the membrane and accumulates cholesterol. Here, our simulations show an enhancing effect by the palmitoylations of CAV1, and we predict that the CAV1-8S complex can extract cholesterol molecules from the lipid bilayer and accommodate them in its beta barrel. Through backmapping to the all-atom level, we also conclude that the Martini v.2 coarse-grained force field overestimates membrane bending, as the atomistic simulations exhibit only very localized bending.

Polyethylene terephthalate (PET) is one of the most widely produced man-made polymers and is a significant contributor to microplastics pollution. The environmental and human health impacts of microplastics pollution have motivated a concerted effort to develop microbe- and enzyme-based strategies to degrade PET and similar plastics. A PETase derived from the bacteria Ideonella sakaiensis was previously shown to enzymatically degrade PET, triggering multidisciplinary efforts to improve the robustness and activity of this and other PETases. However, because these enzymes only erode the surface of the insoluble PET substrate, it is difficult to measure standard kinetic parameters, such as k_on, k_off, and k_cat, complicating interpretation of the activity of mutants using traditional enzyme kinetics frameworks. To address this challenge, we developed a single-molecule microscopy assay that quantifies the landing rate and binding duration of quantum dot-labeled PETase enzymes interacting with a surface-immobilized PET film. Wild-type PETase binding durations were well fit by a biexponential with a fast population having a 2.7 s time constant, interpreted as active binding events, and a slow population interpreted as nonspecific binding interactions that last tens of seconds. A previously described hyperactive mutant, S238F/W159H had both a faster apparent on-rate and a slower off-rate than wild-type PETase, potentially explaining its enhanced activity. Because this single-molecule approach provides a more detailed mechanistic picture of PETase enzymatic activity than standard bulk assays, it should aid future efforts to engineer more robust and active PETases to combat global microplastics pollution.

In this work, a new modeling approach is presented to obtain a two-dimensional transport lattice of a biological cellular system for the calculation of the potential distribution throughout the system and investigation of the corresponding membrane electroporation. The presented model has been obtained by a modified bilayer model of the cell membrane. This modified membrane model allows for an effective inclusion of the shape of the cell membrane in the potential calculation. The results of the model have shown good agreement with the results of the well-known Schwan equation and COMSOL Multiphysics for the circular cell. The simulation results show that both membranes of a mitochondrion can be simultaneously electroporated by an alternating voltage source with frequencies between 1 MHz and 1 GHz.

We present phalloidin-based points accumulation for imaging in nanoscale topography (phalloidin-PAINT), enabling quantitative superresolution imaging of filamentous actin (F-actin) in the cell body and delicate membrane protrusions. We demonstrate that the intrinsic phalloidin dissociation enables PAINT superresolution microscopy in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-PAINT are its ability to consistently quantify F-actin at the nanoscale throughout the entire cell and its enhanced preservation of fragile cellular structures. In a proof-of-concept study, we employed phalloidin-PAINT to superresolve F-actin structures in U2OS and dendritic cells (DCs). We demonstrate more consistent F-actin quantification in the cell body and structurally delicate membrane protrusions of DCs compared with direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse DCs as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane protrusions on the culture glass surface after lipopolysaccharide exposure. The concept of our work opens new possibilities for quantitative protein-specific PAINT using commercially available reagents.

There are increasing numbers of ion channel structures featuring heteromeric subunit assembly, exemplified by synaptic α1β_B glycine and α4β2 nicotinic receptors. These structures exhibit inherent pore asymmetry, but the relevance of this to function is unknown. Furthermore, molecular dynamics simulations performed on symmetrical homomeric channels often lead to thermal distortion whereby conformations of the resulting ensemble are also asymmetrical. When functionally annotating ion channels, researchers often rely on minimal constrictions determined via radius-profile calculations performed with computer programs, such as HOLE or CHAP, coupled with an assessment of pore hydrophobicity. However, such tools typically employ spherical probe particles, limiting their ability to accurately capture pore asymmetry. Here, we introduce an algorithm that employs ellipsoidal probe particles, enabling a more comprehensive representation of the pore geometry. Our analysis reveals that the use of nonspherical ellipsoids for pore characterization provides a more accurate and easily interpretable depiction of conductance. To quantify the implications of pore asymmetry on conductance, we systematically investigated carbon nanotubes with varying degrees of pore asymmetry as model systems. The conductance through these channels shows surprising effects that would otherwise not be predicted with spherical probes. The results have broad implications not only for the functional annotation of biological ion channels but also for the design of synthetic channel systems for use in areas such as water filtration. Furthermore, we make use of the more accurate characterization of channel pores to refine a physical conductance model to obtain a heuristic estimate for single-channel conductance. The code is freely available, obtainable as pip-installable python package and provided as a web service.

Prolyl oligopeptidases from psychrophilic, mesophilic, and thermophilic organisms found in a range of natural environments are studied using a combination of protein structure prediction, atomistic molecular dynamics, and trajectory analysis to determine how the S9 protease family adapts to extreme thermal conditions. We compare our results with hypotheses from the literature regarding structural adaptations that allow proteins to maintain structure and function at extreme temperatures, and we find that, in the case of prolyl oligopeptidases, only a subset of proposed adaptations are employed for maintaining stability. The catalytic and propeller domains are highly structured, limiting the range of mutations that can be made to enhance hydrophobicity or form disulfide bonds without disrupting the formation of necessary secondary structure. Rather, we observe a pattern in which overall prevalence of bound interactions (salt bridges and hydrogen bonds) is conserved by using increasing numbers of increasingly short-lived interactions as temperature increases. This suggests a role for an entropic rather than energetic strategy for thermal adaptation in this protein family.