Catalyzing the translocation of polypeptides through attractive interactions.
Facilitated translocation of polypeptides through a protein pore is a ubiquitous and fundamental process in biology. Several translocation systems possess various well-defined binding sites within the pore lumen, 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 rational protein design and single-channel electrical recordings to obtain detailed kinetic signatures of polypeptide translocation through the staphylococcal alpha-hemolysin (alphaHL) transmembrane pore, a robust, tractable, and versatile beta-barrel protein. Acidic binding sites composed of rings of negatively charged aspartic acid residues, engineered at strategic positions within the beta barrel, produced dramatic changes in the functional properties of the alphaHL protein, facilitating the transport of cationic polypeptides from one side of the membrane to the other. When two electrostatic binding sites were introduced, at the entry and exit of the beta barrel, both the rate constants of association and dissociation increased substantially, diminishing the free energy barrier for translocation. By contrast, more hydrophobic polypeptides exhibited a considerable decrease in the rate constant of association to the pore lumen, having to overcome a greater energetic barrier because of the hydrophilic nature of the pore interior.
Wolfe
Aaron J
AJ
Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, USA.
Mohammad
Mohammad M
MM
Cheley
Stephen
S
Bayley
Hagan
H
Movileanu
Liviu
L
D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002412:Cations / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology; D013210:Staphylococcus / Q000737:chemistry / Q000502:physiology; D013997:Time Factors
D001427:Bacterial Toxins; D002412:Cations; D006460:Hemolysin Proteins; D010455:Peptides; C021091:staphylococcal alpha-toxin