I. Alpha-synuclein lipid-dependent membrane binding and translocation through beta-barrel channels Alpha-synuclein (alpha-syn), a neuronal water-soluble protein of 140-aminoacids long, attracts great attention due to its involvement in the etiology of Parkinson disease (PD), and some other neurodegenerative dementias. Pathological hallmarks of PD include death of dopamine-secreting neurons in substantia nigra and the presence of intracellular inclusions, or Lewy bodies, composed primarily of aggregated alpha-syn. Further evidence links gene multiplications and missense mutations in the alpha-syn gene to early-onset and familial PD. Still, the direct implication of alpha-syn in cellular toxicity and neurodegeneration has not been clearly identified;moreover, its native cellular functions also remain a matter of debate. Gauging the interactions of alpha-syn with membranes and its pathways between and within cells is important for understanding its pathogenesis. This year, to address these questions, we use a robust beta-barrel channel, alpha-Hemolysin, reconstituted into planar lipid bilayers. Transient, 95% blockage of the channel current by monomeric alpha-syn was observed when alpha-syn was added from the membrane side where the shorter (stem) part of the channel is exposed and the applied potential was lower on the side of alpha-syn addition. While the on-rate of alpha-syn binding to the channel strongly increased with the applied field, the off-rate displayed a turnover behavior. Statistical analysis suggests that at voltages >50 mV, a significant fraction of the alpha-syn molecules bound to the channel undergoes subsequent translocation. The observed on-rate varied by >100 times depending on the bilayer lipid composition. Removal of the last 25 amino acids from the highly negatively charged C-terminal of alpha-syn resulted in a significant decrease in the binding rates. Taken together, these results demonstrate that beta-barrel channels may serve as sensitive probes of alpha-syn interactions with membranes as well as model systems for studies of channel-assisted protein transport. These findings allow us to hypothesize that alpha-syn is also able to translocate through the human beta-barrel channel, the voltage-dependent anion channel of the outer mitochondrial membrane, and, therefore, target complexes of the mitochondrial respiratory chain in the inner membrane. This may reveal the elusive physiological and pathophysiological roles for monomeric alpha-syn, and thus reconcile and explain previous observations of the effects of this protein on mitochondrial bioenergetics. II. VDAC regulation by tubulin Mitochondrial dysfunction is involved in the pathogenesis of numerous metabolic diseases of human development. We have extended our studies on the voltage dependent anion channel (VDAC) of the outer mitochondrial membrane regulation by dimeric tubulin. Elucidating molecular interactions by which transport properties of this major channel of the outer mitochondrial membrane are modified by cytosolic proteins is important for understanding of mitochondrial functions that control cell survival and death. Using umbrella-sampling simulations of molecular dynamics, we established the thermodynamic and kinetic components governing ATP transport across the VDAC1 channel. We found that there are several low-affinity binding sites for ATP along the translocation pathway and that the main barrier for ATP transport is located around the center of the channel and is formed predominantly by residues in the N-terminus. The binding affinity of ATP to an open channel was found to be in the millimolar to micromolar range. However, we show that this weak binding increases the ATP translocation probability by about 10-fold compared with the VDAC pore in which attractive interactions were artificially removed. We have also applied methods of molecular dynamics simulations to our recent finding that free dimeric tubulin induces a highly efficient, reversible blockage of VDAC reconstituted into planar lipid membranes and thus controls VDAC permeability for ATP/ADP and other mitochondrial respiratory substrates. Using the Rosetta protein−protein docking algorithm, we established a tentative structure of the VDAC−tubulin complex. An extensive set of equilibrium and nonequilibrium (under applied electric field) molecular dynamics (MD) simulations was used to establish the conductance of the open and tubulin-blocked channel. It was found that the presence of the unstructured C-terminal tail of tubulin in the VDAC pore decreases its conductance by more than 40% and switches its selectivity from anionic to cationic. The subsequent one-dimensional potential of mean force computation for the VDAC-tubulin complex shows that the state renders ATP transport virtually impossible. A number of residues pivotal for tubulin binding to the channel were identified that help to clarify the molecular details of VDAC-tubulin interaction and to provide new insight into the mechanism of the control of mitochondria respiration by VDAC in health and disease. III. Physical theory of transport The so-called anomalous diffusion reported to take place at the surface of biological membranes and in the crowded environment of the cell has been attracting a lot of attention recently. This year we have concentrated on creating analytical tools which would allow one to discriminate between bona fide anomalous diffusion and transient behavior in micro-heterogeneous environments. Free diffusion is a universal description of unbiased motion in a macro-homogeneous media, which is widely used in the analysis of various problems in physics, chemistry, and biology. It is well known that diffusion in biological systems frequently occurs in heterogeneous environments, e.g., diffusion in the crowded cytoplasm, characterized by a wide range of scales. Therefore, it is not surprising that the data on the mean-square displacements are frequently described by sub-linear dependences on time, whereas for free diffusion the mean-square displacements should be proportional to the time of observation. Because the sub-linear dependence is a fingerprint of anomalous subdiffusion, the question naturally arises how to discriminate between anomalous subdiffusion and transient behavior to effective free diffusion regime. There are three most widely used methods to characterize diffusion. One is to fit the time dependence of the mean-square displacement to the function of time with the exponent as a free fitting parameter. Second is to integrate the dependence over time, find the area under the curve, and use it to obtain the exponent. Finally, third is to differentiate the mean-square displacement with respect to time and use the result for the exponent determination. We show that when diffusion is anomalous, all three methods lead to the same value of the exponent;however, in the case of transient behavior the three methods yield the time-dependent exponents which, importantly, are different functions of time. In a separate study we analyzed diffusion in a comb-like structure, formed a main cylindrical tube with identical periodic dead ends. We have developed a formalism which allows us to study the mean square displacement of the particle along the structure axis and demonstrated that the transient behavior occurs naturally in such a system, which, within certain intervals of observation times, can be interpreted as anomalous.
| Rostovtseva, Tatiana K; Gurnev, Philip A; Hoogerheide, David P et al. (2018) Sequence diversity of tubulin isotypes in regulation of the mitochondrial voltage-dependent anion channel. J Biol Chem 293:10949-10962 |
| Berezhkovskii, Alexander M; Bezrukov, Sergey M (2018) Stochastic Gating as a Novel Mechanism for Channel Selectivity. Biophys J 114:1026-1029 |
| Hoogerheide, David P; Gurnev, Philip A; Rostovtseva, Tatiana K et al. (2018) Real-Time Nanopore-Based Recognition of Protein Translocation Success. Biophys J 114:772-776 |
| Berezhkovskii, Alexander M; Bezrukov, Sergey M (2018) Effect of stochastic gating on the flux through a membrane channel: a steady-state approach. J Phys Condens Matter 30:254006 |
| Hoogerheide, David P; Gurnev, Philip A; Rostovtseva, Tatiana K et al. (2017) Mechanism of ?-synuclein translocation through a VDAC nanopore revealed by energy landscape modeling of escape time distributions. Nanoscale 9:183-192 |
| Berezhkovskii, Alexander M; Dagdug, Leonardo; Bezrukov, Sergey M (2017) Mean Direct-Transit and Looping Times as Functions of the Potential Shape. J Phys Chem B 121:5455-5460 |
| Hoogerheide, David P; Noskov, Sergei Y; Jacobs, Daniel et al. (2017) Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes. Proc Natl Acad Sci U S A 114:E3622-E3631 |
| Gurnev, Philip A; Stanley, Christopher B; Aksoyoglu, M Alphan et al. (2017) Poly(ethylene glycol)s in Semidilute Regime: Radius of Gyration in the Bulk and Partitioning into a Nanopore. Macromolecules 50:2477-2483 |
| Gurnev, Philip A; Roark, Torri C; Petrache, Horia I et al. (2017) Cation-Selective Channel Regulated by Anions According to Their Hofmeister Ranking. Angew Chem Int Ed Engl 56:3506-3509 |
| Berezhkovskii, Alexander M; Dagdug, Leonardo; Bezrukov, Sergey M (2017) Bulk-mediated surface transport in the presence of bias. J Chem Phys 147:014103 |
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