Facilitated Polypeptide Translocation through a Protein Pore

Abstract

Facilitated translocation of proteins through a transmembrane protein pore is a ubiquitous and fundamental process in biology. Protein translocation machineries possess various binding sites within the pore interior, but a clear mechanistic understanding of how the interaction of the polypeptides with the binding site alters the underlying kinetics is still missing. Here, we employed standard protein engineering and single-channel electrical recordings to obtain detailed kinetic information of polypeptide translocation through the staphylococcal α-hemolysin (αHL) transmembrane pore, a robust, tractable, and versatile β-barrel protein. Binding sites comprised of rings of negatively-charged aspartic acid residues, engineered at different positions within the β barrel, produced significant alterations in the functional features pf the protein pore, facilitating the transport of cationic polypeptides from one side of the membrane to the other. The translocation of polypeptides through the engineered protein pore was dependent on the position of the binding site, the length of the polypeptide as well as its hydrophobic index. Acknowledgements. This research was supported by grants from the National Science Foundation (DMR-0706517 and HRD-0703452) and the National Institutes of Health (R01 GM088403) as well as by the Syracuse Biomaterials Institute (SBI).