Probing Membrane Protein Association Using Concentration-Dependent Number and Brightness

Michael D. Paul, Randall Rainwater, Yi Zuo, Luo Gu, Kalina Hristova

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
JournalAngewandte Chemie - International Edition
DOIs
StateAccepted/In press - 2020

Keywords

  • association constants
  • membrane receptors
  • neuropilin 1
  • number and brightness
  • thermodynamics

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)

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