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Anal Biochem. 2019 Jun 12. pii: S0003-2697(19)30180-0. doi: 10.1016/j.ab.2019.05.017. [Epub ahead of print]

MHC-I peptide binding activity assessed by exchange after cleavage of peptide covalently linked to β2-microglobulin.

Author information

1
Program in Immunology and Microbiology, University of Massachusetts Medical School, Worcester, MA, 01605, United States; Department of Pathology, University of Massachusetts Medical School, Worcester, MA, 01605, United States.
2
Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, GA, 30329, United States.
3
Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, GA, 30329, United States; Department of Microbiology and Immunology, Emory Vaccine Center at Yerkes, Emory University School of Medicine, Atlanta, GA, 30329, United States.
4
Program in Immunology and Microbiology, University of Massachusetts Medical School, Worcester, MA, 01605, United States; Department of Pathology, University of Massachusetts Medical School, Worcester, MA, 01605, United States; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, 01605, United States. Electronic address: lawrence.stern@umassmed.edu.

Abstract

A common approach to measuring binding constants involves combining receptor and ligand and measuring the distribution of bound and free states after equilibration. For class I major histocompatibility (MHC-I) proteins, which bind short peptides for presentation to T cells, this approach is precluded by instability of peptide-free protein. Here we develop a method wherein a weakly-binding peptide covalently attached to the N-terminus of the MHC-I β2m subunit is released from the peptide binding site after proteolytic cleavage of the linker. The resultant protein is able to bind added peptide. A direct binding assay and method for estimation of peptide binding constant (Kd) are described, in which fluorescence polarization is used to follow peptide binding. A competition binding assay and method for estimation of inhibitor binding constant (Ki) using the same principle also are also described. The method uses a cubic equation to relate observed binding to probe concentration, probe Kd, inhibitor concentration, and inhibitor Ki under general reaction conditions without assumptions relating to relative binding affinities or concentrations. We also delineate advantages of this approach compared to the Cheng-Prusoff and Munson-Rodbard approaches for estimation of Ki using competition binding data.

KEYWORDS:

Competition assay; Equilibrium binding constant; Fluorescence polarization; IC(50); Major histocompatibility protein; Peptide exchange

PMID:
31201791
DOI:
10.1016/j.ab.2019.05.017

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