Probing Membrane Protein Association Using Concentration‐Dependent Number and Brightness

• Published in: Angewandte Chemie International Edition Vol. 60; no. 12; pp. 6503 - 6508
• Main Authors: Paul, Michael D., Rainwater, Randall, Zuo, Yi, Gu, Luo, Hristova, Kalina
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 15.03.2021
• Edition: International ed. in English
• Subjects: association constants; Brightness; Embryogenesis; Fluorescence; Fluorescence Resonance Energy Transfer; Humans; Membrane proteins; membrane receptors; Membranes; Neuropilin; Neuropilin-1 - analysis; neuropilin 1; number and brightness; Oligomerization; Proteins; thermodynamics; Transfection; thermodynamics; neuropilin 1; membrane receptors; association constants; number and brightness
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202010049

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. Concentration‐dependent number and brightness (cdN&B) is a fluorescence fluctuation technique that can be implemented on a standard confocal microscope and report on the thermodynamics of membrane protein association in the native plasma membrane. cdN&B uses transient transfection to enable measurements of oligomer size as a function of receptor concentration over a broad range, yielding the association constant.