Interaction of peptides with a protein pore

¹ Syracuse University
² Texas A&M
³ Oxford University

^* To whom correspondence should be addressed. E-mail: lmovilea{at}physics.syr.edu.

Submitted on December 3, 2004
Revised on December 26, 2004
Accepted on 23 May 2005

	Abstract

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 -barrel of the -hemolysin (HL) protein pore at the single-molecule level. The experimental kinetic data suggest a two-barrier, single-well free energy diagram 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 kinetic rate constants k_on, k_off^cis and k_off^trans 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 poly(ethylene glycol)s (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 HL, and the binding decreases with the peptide length, similar to PEGs. The binding is enhanced at increased transmembrane potentials because 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 -barrel pore.

Key Words: Driving force, Folding, Free energy barrier, Transmembrane pore, alfa-helical peptide, beta-barrel

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