I. Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes Tubulin has emerged as a highly unexpected component of mitochondrial membranes involved in regulation of membrane permeability. The discovery of this functional role has reawakened interest in the nature of the tubulinmembrane interaction to answer a new question: How does tubulin, a cytosolic protein famous for its role in microtubule structure and dynamics, come to target mitochondrial membranes? This year, using a combination of five biophysical methodssurface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations, we have studied peripheral binding of tubulin to biomimetic membranes of different lipid compositions. We find that tubulin distinguishes between lamellar and nonlamellar lipids through a highly conserved amphipathic binding motif. Specifically, alpha-tubulin targets cell and organelle membranes by sensing lipid-packing defects, with broad consequences for both normal cellular function and disease. We show that tubulin belongs to the group of peripherally bound amphitropic proteins. These proteins are the subfamily of peripheral membrane proteins that interact directly with the lipid membrane rather than with intrinsic membrane proteins and are therefore strongly influenced by lipid composition. A number of diseases, such as atherosclerosis, type II diabetes, and lysosomal storage disorders, are associated with defects in maintaining the correct distribution of intracellular lipids. Although involvement of amphitropic proteins in various cell functions is soundly established, the mechanisms of their interaction with cellular membranes are only beginning to be understood because their characteristic reversible binding to the membranes creates obvious experimental difficulties in assessing binding conformations and kinetics. II. Partitioning of soft water-soluble polymers into beta-barrel channels in their functional states Understanding polymer partitioning into nanoscale cavities of different nature is important for many technological applications that include, but are not limited to, analytical chromatography, separation techniques, and purification methods. It is also critical in the qualitative interpretation and quantitative analysis of molecular interactions and biological regulation in the crowded cellular environment. This necessitates model studies with polymer solutions explored in both dilute and semidilute regimes. Recently, we studied polymer partitioning from semidilute solutions of poly(ethylene glycol) (PEG) mixtures into a number of membrane-spanning beta-barrel channels of different origin, and the results were rationalized within the earlier formulated polymers-pushing-polymers model of nanopore partitioning. This model is based on the assumptions that the larger component of the polymer mixture, being preferentially excluded from the cavity, pushes the smaller component into the cavity, thus representing forced polymer redistribution between the bulk and the channel. This year we studied polymer mixtures by using two different methods, small-angle neutron scattering and nanopore conductance fluctuation analysis, to quantify the larger polymer parameters in the bulk and the degree of its partitioning in the pore, respectively. We first show that the reduction of the PEG 3400 characteristic size with its increasing concentration in the bulk is statistically significant but small. We then demonstrate that partitioning of the larger polymer in the nanopore is negligible if its relative weight fraction is kept under 50%, in excellent agreement with the major assumptions of the model mentioned above. These findings are important for understanding and quantifying polymer behavior in the bulk and polymer partitioning into nanopores and different protein cavities in dilute and semidilute regimes mimicking those of the crowded conditions of the cell. III. Understanding ion specificity of the Hofmeister ranking Ion specificity and, in particular, the distinctive effects of anions in salt-induced protein precipitation have been known since the 1880s, when Franz Hofmeister established the ranking of anions in their ability to regulate egg yolk protein water solubility. Experimental and theoretical studies have given a detailed empirical picture of the phenomenon, but the nature of the ionic interactions with the surfaces leading to the Hofmeister effect is still under debate. The only consensus is that it cannot be explained by standard theories of electrolytes. For example, bromide is unique in that its salts were recognized as a drug to treat epilepsy a couple of dozen years before Hofmeisters studies and they are still in use to treat specific types of refractory seizures in children, but the mechanism of their action remains elusive. Though the Hofmeister ranking of salts has been a frequent target of biological studies including channel-facilitated membrane transport, this year we decided to take advantage of arguably the simplest ion transport model of modern biophysicsthe channel formed by a linear pentadecapeptide, gramicidin A. Counterintuitively, we found that conductance of this perfectly cation-selective channel increases about twofold in the Hofmeister anion series H2PO4

Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2017
Total Cost
Indirect Cost
Name
U.S. National Inst/Child Hlth/Human Dev
Department
Type
DUNS #
City
State
Country
Zip Code
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
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
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

Showing the most recent 10 out of 66 publications