Biophysics of Large Membrane Channels

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

I. Global and local effects in lipid-mediated interactions between peripheral and integral membrane proteins. Amphitropic proteins (APs) are a subfamily of water-soluble peripherally membrane-bound proteins that interact directly with the lipid membrane rather than with intrinsic membrane proteins. They are therefore strongly influenced by membrane properties. The notion that lipids are not merely passive fillers of the space between membrane proteins, but actively interact with them, controlling protein conformational transitions and impacting their function, is now well-accepted. When an AP interacts with a membrane containing an integral membrane protein, a ternary protein-lipid-protein system is created. Even in the absence of direct interactions between the amphitropic and integral proteins, the two proteins can affect each other by modifying lipid membrane properties, either at the global (i.e., whole-membrane) or local (i.e., confined to a small area around the bound or integrated protein) scale. These lipid-mediated protein-protein interactions are indirect and, therefore, difficult to elucidate; independent experimental data are required to report on each individual interaction to understand the whole system. Examples for which comprehensive data are available are remarkably rare. In the present study, we describe how these difficulties could be surmounted by using the channel-forming integral membrane protein gramicidin A (grA) reconstituted in a planar lipid membrane and exposed to the amphitropic proteins – dimeric tubulin or α-synuclein. Importantly, there are no known direct interactions between these APs and grA, thus revealing the role of the lipid membrane. We leverage our detailed understanding of the tubulin-lipid interaction mechanism, as well as the self-reporting properties of the grA channel, to understand how tubulin affects the properties of the lipid membrane. Here, grA serves a dual role. First, it reports on the global properties of the lipid membrane; grA results, combined with the well-understood tubulin-lipid interaction, yield a complete picture of the mutual effect of tubulin binding on the lipid membrane. Second, the presence of the grA conducting dimer alters the local membrane curvature and creates binding sites for tubulin in an otherwise inert membrane composition. Similarly, we also find that α-synuclein increases grA channel lifetime in a dose-dependent manner. The practical importance of our study is based on the observation that many membrane-altering small molecules, such as anesthetics, tricyclic antidepressants, and psychedelics, have membrane-modifying properties similar to APs. Thus, the recognition of lipid-mediated protein-protein interactions could be instrumental in understanding the off-target action of some of these drugs on cell and organelle membranes and, therefore, on membrane proteins residing in, or peripherally associated with, those membranes. II. Conformational plasticity of mitochondrial VDAC2 controls the kinetics of its interaction with cytosolic proteins. The voltage-dependent anion channel (VDAC) is the most abundant integral protein of the mitochondrial outer membrane. It is the major pathway for water-soluble metabolites and small ions to cross this membrane. In mammals, there are three VDAC isoforms: VDAC1, VDAC2, and VDAC3. Despite ~70% sequence similarity between the isoforms and the ability of them all to form large conductive anionic channels (~4 nS in 1 M KCl at room temperature), which gate in response to the applied voltage when reconstituted into a planar lipid membrane, each VDAC isoform has a distinct physiological role. Among the three isoforms, VDAC2 is unique because of its embryonic lethality upon knockout. Using single-molecule electrophysiology, we investigated the biophysical properties that distinguish VDAC2 from VDAC1 and VDAC3. We found that, unlike the latter, VDAC2 exhibits dynamic switching between multiple high-conductance, anion-selective substates. Using -synuclein (Syn)—a known VDAC1 cytosolic regulator—we established that higher-conductance substates correlate with increased on-rates of Syn-VDAC2 interaction but shorter blockage times, thus maintaining an unchanged equilibrium constant across all substates. This suggests that Syn detects VDAC2’s structural variations before the final step in the binding reaction. We explored the dependence of VDAC2’s unique amino-terminal extension and cysteines on the substate behavior, finding that both structural elements modulate substate occurrence. The discovered conformational flexibility of VDAC2 may enable its interactions with diverse binding partners, explaining its critical physiological role via dynamic adaptation to cellular needs. Specifically, we propose that the appearance of distinct substates within the same channel and their different kinetics of interaction with Syn reveal a unique structural plasticity of VDAC2. This feature suggests a key to understanding the exceptional role of this multifaceted channel in the cell. It could tentatively explain the physiological significance of VDAC2: its ability to adapt to cell conditions and change the rates of interaction with its multiple cytosolic protein partners. III. Particle dynamics in biconical cavities: First-passage, direct-transit, and looping time distributions. Theoretical analysis in the framework of a continuous diffusion model. Earlier, we analyzed the effects of monotonically changing entropy potentials imposed by expanding or narrowing tubes on particle diffusion. This year, we examined particle dynamics in biconical cavities, wherein particle motion is influenced by either an entropy potential well, as in a cavity composed of first expanding and then narrowing cones, or an entropy potential barrier, as in a cavity made up of first narrowing and then expanding identical cones. Both types of cavities are relevant to multiple technological and biological problems, with examples of such structures found at both the micro- and nano-scales. We derive analytical expressions for the Laplace transforms of the distributions for the first-passage, direct-transit, and looping times in such structures. We find that not only the average values but also the distributions of the first-passage times in both cavities are identical. However, the direct-transit and looping time distributions are drastically different. In particular, the mean direct-transit time for the expanding–narrowing cavity (entropy potential well) approaches a constant value with the increasing depth of the entropy potential well. This corresponds to the increasing ratio of the cavity’s largest radius to the radius of its openings. In contrast, the mean direct-transit time goes to infinity in the case of the narrowing–expanding cavity with the increasing height of the entropy potential barrier. Importantly, in various single-molecule experiments, it has been shown that the first passage time distributions, not just their mean values, carry important information on the molecular intermediates governing the dynamics of complex chemical and biological systems. In the present study, we now demonstrate that distributions of looping and direct transit times can report on the drastic differences in the system geometry, even when the systems’ first passage time averages and distributions are identical.