The Dynamic Orientation of Membrane-Bound Peptides: Bridging Simulations and Experiments

AbstractThe structural organization in a peptide/membrane supramolecular complex is best described by knowledge of the peptide orientation plus its time-dependent and spatial fluctuations. The static orientation, defined by the peptide tilt and a rotation about its molecular axis, is accessible through a number of spectroscopic methods. However, peptide dynamics, although relevant to understand the functionality of these systems, remains largely unexplored. Here, we describe the orientation and dynamics of Trp-flanked and Lys-flanked hydrophobic peptides in a lipid bilayer from molecular dynamics simulations. A novel view is revealed, where collective nontrivial distributions of time-evolving and ensemble peptide orientations closely represent the systems as studied experimentally. Such global distributions are broad and unveil the existence of orientational states, which depend on the anchoring mode of interfacial residues. We show that this dynamics modulates 2H quadrupolar splittings and introduces ambiguity in the analysis of NMR data. These findings demonstrate that structural descriptions of peptide/membrane complexes are incomplete, and in cases even imprecise, without knowledge of dynamics

Pores Formed by Baxα5 Relax to a Smaller Size and Keep at Equilibrium

AbstractPores made by amphipathic cationic peptides (e.g., antimicrobials and fragments of pore-forming proteins) are typically studied by examining the kinetics of vesicle leakage after peptide addition or obtaining structural measurements in reconstituted peptide-lipid systems. In the first case, the pores have been considered transient phenomena that allow the relaxation of the peptide-membrane system. In the second, they correspond to equilibrium structures at minimum free energy. Here we reconcile both approaches by investigating the pore activity of the α5 fragment from the proapoptotic protein Bax (Baxα5) before and after equilibrium of peptide/vesicle complexes. Quenching assays on suspensions of large unilamellar vesicles suggest that in the presence of Baxα5, the vesicles maintain a leaky state for hours under equilibrium conditions. We proved and analyzed stable pores on single giant unilamellar vesicles (GUVs) in detail by monitoring the entrance of dyes added at different times after incubation with the peptide. When the GUVs came in contact with Baxα5, leakage started stochastically, was delayed for various periods of time, and in the majority of cases proceeded rapidly to completion. After hours in the presence of the peptide, the same individual GUVs that refilled completely at first instance maintained a porated state, which could be observed in subsequent leak-in events for serially added dyes. However, these long-term pores were smaller in size than the initial equilibration pores. Stable pores were also detected in GUVs made in the presence of Baxα5. The latter pores can be considered equilibrium states and may correspond to structures measured previously in bilayer stacks. Although pore formation may occur as a kinetic process, equilibrium pores may also be functionally relevant structures, especially in highly regulated systems such as the apoptotic mitochondrial pores induced by Bax

Understanding biomolecular motion, recognition, and allostery by use of conformational ensembles

We review the role conformational ensembles can play in the analysis of biomolecular dynamics, molecular recognition, and allostery. We introduce currently available methods for generating ensembles of biomolecules and illustrate their application with relevant examples from the literature. We show how, for binding, conformational ensembles provide a way of distinguishing the competing models of induced fit and conformational selection. For allostery we review the classic models and show how conformational ensembles can play a role in unravelling the intricate pathways of communication that enable allostery to occur. Finally, we discuss the limitations of conformational ensembles and highlight some potential applications for the future

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Solid state NMR analysis of peptides in membranes: Influence of dynamics and labeling scheme

AbstractThe functional state of a membrane-active peptide is often defined by its conformation, molecular orientation, and its oligomeric state in the lipid bilayer. These “static” structural properties can be routinely studied by solid state NMR using isotope-labeled peptides. In the highly dynamic environment of a liquid crystalline biomembrane, however, the whole-body fluctuations of a peptide are also of paramount importance, although difficult to address and most often ignored. Yet it turns out that disregarding such motional averaging in calculating the molecular alignment from orientational NMR-constraints may give a misleading, if not false picture of the system. Here, we demonstrate that the reliability of a simplified static or an advanced dynamic data analysis depends critically on the choice of isotope labeling scheme used. Two distinctly different scenarios have to be considered. When the labels are placed on the side chains of a helical peptide (such as a CD3- or CF3-group attached to the CαCβ bond), their nuclear spin interaction tensors are very sensitive to motional averaging. If this effect is not properly accounted for, the helix tilt angle tends to be severely underestimated. At the same time, the analysis of labels in the side chains allows to extract valuable dynamical information about whole-body fluctuations of the peptide helix in the membrane. On the other hand, the alternative labeling scheme where 15N-labels are accommodated within the peptide backbone, will yield nearly correct helix tilt angles, irrespective as to whether dynamics are taken into account or not

Stability of Asymmetric Lipid Bilayers Assessed by Molecular Dynamics Simulations

The asymmetric insertion of amphiphiles into biological membranes compromises the balance between the inner and outer monolayers. As a result, area expansion of the receiving leaflet and curvature strain may lead to membrane permeation, shape changes, or membrane fusion events. We have conducted both atomistic and coarse-grained molecular dynamics simulations of dipalmitoyl-phosphatidylcholine (DPPC) bilayers to study the effect of an asymmetric distribution of lipids between the two monolayers on membrane stability. Highly asymmetric lipid bilayers were found to be surprisingly stable within the submicrosecond time span of the simulations. Even the limiting case of a monolayer immersed in water ruptured spontaneously only after at least 20 ns simulation. A thermal shock could destabilize these kinetically trapped states. We also studied mixed systems composed of DPPC and short tail diC8PC lipids, showing that the presence of the cone-shaped short tail lipid facilitates the release of tension in the asymmetric systems via formation of a transmembrane pore. Thus, asymmetric area expansion and curvature stress cooperate to yield bilayer disruption. It appears that, although asymmetric area expansion destabilizes the bilayer structure, the activation energy for transmonolayer lipid re-equilibration is increased. Such a large kinetic barrier can be reduced by lipids with positive spontaneous curvature. These effects are important at the onset of bilayer destabilization phenomena, such as lipid pore formation and membrane fusion, and should be considered for the mechanism of induction of such processes by peptides and proteins.