TY - JOUR
T1 - Interactions of a Polypeptide with a Protein Nanopore under Crowding Conditions
AU - Larimi, Motahareh Ghahari
AU - Mayse, Lauren Ashley
AU - Movileanu, Liviu
N1 - Funding Information:
The authors would like to thank A. Thakur for his assistance during the very early stages of this project. This research project was supported by US National Institutes of Health grants GM088403 (to L.M.) and GM129429 (to L.M.) and the National Science Foundation grant REU DMR-1460784 (to L.A.M.).
Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/4/23
Y1 - 2019/4/23
N2 - Molecular crowding, a ubiquitous feature of the cellular environment, has significant implications in the kinetics and equilibrium of biopolymer interactions. In this study, a single charged polypeptide is exposed to competing forces that drive it into a transmembrane protein pore versus forces that pull it outside. Using single-molecule electrophysiology, we provide compelling experimental evidence that the kinetic details of the polypeptide-pore interactions are substantially affected by high concentrations of less-penetrating polyethylene glycols (PEGs). At a polymer concentration above a critical value, the presence of these neutral macromolecular crowders increases the rate constant of association but decreases the rate constant of dissociation, resulting in a stronger polypeptide-pore interaction. Moreover, a larger-molecular weight PEG exhibits a lower rate constant of association but a higher rate constant of dissociation than those values corresponding to a smaller-molecular weight PEG. These outcomes are in accord with a lower diffusion constant of the polypeptide and higher depletion-attraction forces between the polypeptide and transmembrane protein pore under crowding and confinement conditions.
AB - Molecular crowding, a ubiquitous feature of the cellular environment, has significant implications in the kinetics and equilibrium of biopolymer interactions. In this study, a single charged polypeptide is exposed to competing forces that drive it into a transmembrane protein pore versus forces that pull it outside. Using single-molecule electrophysiology, we provide compelling experimental evidence that the kinetic details of the polypeptide-pore interactions are substantially affected by high concentrations of less-penetrating polyethylene glycols (PEGs). At a polymer concentration above a critical value, the presence of these neutral macromolecular crowders increases the rate constant of association but decreases the rate constant of dissociation, resulting in a stronger polypeptide-pore interaction. Moreover, a larger-molecular weight PEG exhibits a lower rate constant of association but a higher rate constant of dissociation than those values corresponding to a smaller-molecular weight PEG. These outcomes are in accord with a lower diffusion constant of the polypeptide and higher depletion-attraction forces between the polypeptide and transmembrane protein pore under crowding and confinement conditions.
KW - free-energy landscape
KW - peptide-protein interactions
KW - polymer
KW - single-channel electrical recordings
KW - single-molecule kinetics
KW - α-hemolysin
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U2 - 10.1021/acsnano.9b00008
DO - 10.1021/acsnano.9b00008
M3 - Article
C2 - 30925041
AN - SCOPUS:85065343741
SN - 1936-0851
VL - 13
SP - 4469
EP - 4477
JO - ACS nano
JF - ACS nano
IS - 4
ER -