Premium
Probing Membrane Protein Association Using Concentration‐Dependent Number and Brightness
Author(s) -
Paul Michael D.,
Rainwater Randall,
Zuo Yi,
Gu Luo,
Hristova Kalina
Publication year - 2021
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202010049
Subject(s) - oligomer , tetramer , biophysical chemistry , biophysics , membrane , chemistry , confocal microscopy , fluorescence , confocal , brightness , biology , biochemistry , physics , microbiology and biotechnology , optics , organic chemistry , enzyme
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.