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Interactions of Peptides with a Protein Pore

Liviu Movileanu ^* , Jason P. Schmittschmitt , J. Martin Scholtz   and Hagan Bayley ^¶

^* Department of Physics, Syracuse University, College of Arts and Sciences, Syracuse, New York; Structural Biology, Biochemistry and Biophysics Program, Syracuse University, Syracuse, New York; Department of Medical Biochemistry & Genetics, The Texas A&M University System Health Science Center, College Station, Texas; Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, College Station, Texas; and ^¶ Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford, United Kingdom

Correspondence: Address reprint requests to L. Movileanu, Tel: 315-443-8078; Fax: 315-443-9103; E-mail: lmovilea{at}physics.syr.edu.

The partitioning of polypeptides into nanoscale transmembrane pores is of fundamental importance in biology. Examples include protein translocation in the endoplasmic reticulum and the passage of proteins through the nuclear pore complex. Here we examine the exchange of cationic -helical peptides between the bulk aqueous phase and the transmembrane ß-barrel of the -hemolysin (HL) protein pore at the single-molecule level. The experimental kinetic data suggest a two-barrier, single-well free energy profile for peptide transit through the HL pore. This free energy profile is strongly voltage- and peptide-length-dependent. We used the Woodhull-Eyring formalism to rationalize the values measured for the association and dissociation rate constants k_on and k_off, and to separate k_off into individual rate constants for exit through each of the openings of the protein pore. The rate constants k_on, and decreased with the length of the peptide. At high transmembrane potentials, the alanine-based peptides, which include bulky lysine side chains, bind more strongly (formation constants K_f tens of M^–1) than highly flexible polyethylene glycols (K_f M^–1) to the lumen of the HL protein pore. In contrast, at zero transmembrane potential, the peptides bind weakly to the lumen of the pore, and the affinity decreases with the peptide length, similar to the case of the polyethylene glycols. The binding is enhanced at increased transmembrane potentials, because the free energy contribution G = –FV/RT predominates with the peptides. We suggest that the HL protein may serve as a robust and versatile model for examining the interactions between positively charged signal peptides and a ß-barrel pore.

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