期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 110, 期 47, 页码 E4417-E4426出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1312739110
关键词
substrate binding kinetics; single-molecule sensor; Coulombic interaction; phosphorylation
资金
- National Institutes of Health
- Oxford Nanopore Technologies
- Biotechnology and Biological Sciences Research Council
- Structural Genomics Consortium [1097737]
- AbbVie
- Boehringer Ingelheim
- Canada Foundation for Innovation
- Canadian Institutes for Health Research
- Genome Canada
- GlaxoSmithKline
- Janssen, Lilly Canada
- Novartis Research Foundation
- Ontario Ministry of Economic Development and Innovation
- Pfizer
- Takeda
- Wellcome Trust
In stochastic sensing, the association and dissociation of analyte molecules is observed as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free determination of rate and equilibrium constants with respect to a specific binding site. We engineered sensors based on the staphylococcal a-hemolysin pore to allow the single-molecule detection and characterization of protein kinase-peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we observed unusually high association rate constants for the interaction of Pim kinases with their consensus substrate Pimtide (similar to 10(7) to 10(8) M-1.s(-1)), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.
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