Probing Membrane Protein Association Using Concentration-Dependent Number and Brightness

Overview

Journal Angew Chem Int Ed Engl

Specialty Chemistry

Date 2020 Dec 22

PMID 33351993

Citations 7

Authors

Michael D Paul

Randall Rainwater

Yi Zuo

Luo Gu

Kalina Hristova

Affiliations

Soon will be listed here.

Abstract

We introduce concentration-dependent number and brightness (cdN&B), a fluorescence fluctuation technique that can be implemented on a standard confocal microscope and can report on the thermodynamics of membrane protein association in the native plasma membrane. It uses transient transfection to enable measurements of oligomer size as a function of receptor concentration over a broad range, yielding the association constant. We discuss artifacts in cdN&B that are concentration-dependent and can distort the oligomerization curves, and we outline procedures that can correct for them. Using cdN&B, we characterize the association of neuropilin 1 (NRP1), a protein that plays a critical role in the development of the embryonic cardiovascular and nervous systems. We show that NRP1 associates into a tetramer in a concentration-dependent manner, and we quantify the strength of the association. This work demonstrates the utility of cdN&B as a powerful tool in biophysical chemistry.

Citing Articles

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Ligand bias underlies differential signaling of multiple FGFs via FGFR1.

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Utility of FRET in studies of membrane protein oligomerization: The concept of the effective dissociation constant.

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Regulation of the EphA2 receptor intracellular region by phosphomimetic negative charges in the kinase-SAM linker.

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