Quantifying Membrane Protein Oligomerization with Fluorescence Cross-correlation Spectroscopy

Overview

Journal Methods

Specialty Biochemistry

Date 2018 Feb 16

PMID 29448037

Citations 19

Authors

Megan J Kaliszewski

Xiaojun Shi

Yixuan Hou

Ryan Lingerak

Soyeon Kim

Paul Mallory

Adam W Smith

Affiliations

Soon will be listed here.

Abstract

Fluorescence cross-correlation spectroscopy (FCCS) is an advanced fluorescence technique that can quantify protein-protein interactions in vivo. Due to the dynamic, heterogeneous nature of the membrane, special considerations must be made to interpret FCCS data accurately. In this study, we describe a method to quantify the oligomerization of membrane proteins tagged with two commonly used fluorescent probes, mCherry (mCH) and enhanced green (eGFP) fluorescent proteins. A mathematical model is described that relates the relative cross-correlation value (f) to the degree of oligomerization. This treatment accounts for mismatch in the confocal volumes, combinatoric effects of using two fluorescent probes, and the presence of non-fluorescent probes. Using this model, we calculate a ladder of f values which can be used to determine the oligomer state of membrane proteins from live-cell experimental data. Additionally, a probabilistic mathematical simulation is described to resolve the affinity of different dimeric and oligomeric protein controls.

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