title: Alpha-synuclein lipid-dependent membrane binding and translocation through the α-hemolysin channel.
abstract: Gauging the interactions of a natively unfolded Parkinson disease-related protein, alpha-synuclein (α-syn) with membranes and its pathways between and within cells is important for understanding its pathogenesis. Here, to address these questions, we use a robust β-barrel channel, α-hemolysin, reconstituted into planar lipid bilayers. Transient, ~95% blockage of the channel current by α-syn was observed when 1), α-syn was added from the membrane side where the shorter (stem) part of the channel is exposed; and 2), the applied potential was lower on the side of α-syn addition. While the on-rate of α-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 α-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 α-syn resulted in a significant decrease in the binding rates. Taken together, these results demonstrate that β-barrel channels may serve as sensitive probes of α-syn interactions with membranes as well as model systems for studies of channel-assisted protein transport.
authors: Physics Department, University of Massachusetts, Amherst, Massachusetts; Section on Molecular Transport, Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland. Electronic address: gurnev@physics.umass.edu.
authors: Philip A
authors: PA
authors: Gurnev
authors: Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
authors: Thai Leong
authors: TL
authors: Yap
authors: Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
authors: Candace M
authors: CM
authors: Pfefferkorn
authors: Section on Molecular Transport, Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.
authors: Tatiana K
authors: TK
authors: Rostovtseva
authors: Mathematical and Statistical Computing Laboratory, Division for Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland.
authors: Alexander M
authors: AM
authors: Berezhkovskii
authors: Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
authors: Jennifer C
authors: JC
authors: Lee
authors: Physics Department, University of Massachusetts, Amherst, Massachusetts.
authors: V Adrian
authors: VA
authors: Parsegian
authors: Section on Molecular Transport, Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.
authors: Sergey M
authors: SM
authors: Bezrukov
mesh_terms: D000595:Amino Acid Sequence; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051844:alpha-Synuclein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D008563:Membrane Lipids; D051844:alpha-Synuclein