Journal
BIOPHYSICAL JOURNAL
Volume 106, Issue 3, Pages 556-565Publisher
CELL PRESS
DOI: 10.1016/j.bpj.2013.12.028
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Funding
- Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development
- National Heart, Lung, and Blood Institute
- Center for Information Technology
- National Institutes of Health
- National Science Foundation EAGER [1249199]
- Div Of Chem, Bioeng, Env, & Transp Sys
- Directorate For Engineering [1249199] Funding Source: National Science Foundation
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Gauging the interactions of a natively unfolded Parkinson disease-related protein, alpha-synuclein (alpha-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 beta-barrel channel, alpha-hemolysin, reconstituted into planar lipid bilayers. Transient, similar to 95% blockage of the channel current by alpha-syn was observed when 1), alpha-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 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.
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