Probing Membrane Protein Association Using Concentration-Dependent Number and Brightness.


Paul MD(1), Rainwater R(2), Zuo Y(2), Gu L(2), Hristova K(2)(1).
Author information:
(1)Program in Molecular Biophysics, Johns Hopkins University, Baltimore, MD, 21218, USA.
(2)Department of Materials Science and Engineering and Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, 21218, USA.


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.