Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Summary

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique.  

Abstract

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Introduction

Biological activities are ultimately carried out by proteins, most of which interact with others for proper biological functions. Using a computational approach, the total amount of protein-protein interactions in human is estimated to be ~650,000¹, and disruption of these interactions often leads to diseases². Due to their essential roles in controlling cellular and organismal processes, numerous methods have been developed to study protein-protein interactions, such as yeast-two-hybrid, bimolecular fluorescence complementation, split-luciferase complementation, and co-immunoprecipitation assay³.....

Protocol

1. Design the FRET assay.

1. Download the structure file of the Cul1•Cand1 complex from the Protein Data Bank (file 1U6G).
2. View the structure of the Cul1•Cand1 complex in PyMOL.
3. Use the Measurement function under the Wizard menu of PyMOL to estimate the distance between the first amino acid of Cand1 and the last amino acid of Cul1 (Figure 1).
4. Load the online spectra viewer (see Table of Materials) and view the excitation and emission spectra of 7-amino-4-methylcoumarin (AMC) and FlAsH simultaneously (Figure 2). Note that....

Results

To test the FRET between Cul1^AMC and ^FlAsHCand1, we first determined the emission intensity of 70 nM Cul1^AMC (the donor) and 70 nM ^FlAsHCand1 (the acceptor), respectively (Figure 3A-C, blue lines). In each analysis, only one emission peak was present, and the emission of ^FlAsHCand1 (the acceptor) was low. When 70 nM each of Cul1^AMC and ^FlAsHCand1 were mixed to genera.......

Discussion

FRET is a physical phenomenon that is of great interest for studying and understanding biological systems¹⁹. Here, we present a protocol for testing and using FRET to study the binding kinetics of two interacting proteins. When designing FRET, we considered three major factors: the spectral overlap between donor emission and acceptor excitation, the distance between the two fluorophores, and the dipole orientation of the fluorophores²⁸. To choose the fluorophores for FRET, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anion exchange chromatography column	GE Healthcare	17505301	HiTrap Q FF anion exchange chromatography column
Benchtop refrigerated centrifuge	Eppendorf	2231000511
BL21 (DE3) Competent Cells	ThermoFisher Scientific	C600003
Calcium Chloride	Fisher Scientific	C78-500
Cation exchange chromatography column	GE Healthcare	17505401	HiTrap SP Sepharose FF
Desalting Column	GE Healthcare	17085101
Floor model centrifuge (high speed)	Beckman Coulter	J2-MC
Floor model centrifuge (low speed)	Beckman Coulter	J6-MI
Fluorescence SpectraViewer	ThermoFisher Scientific		https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html
FluoroMax fluorimeter	HORIBA	FluoroMax-3
FPLC	GE Healthcare	29018224
GGGGAMC peptide	New England Peptide	custom synthesis
Glutathione beads	GE Healthcare	17075605
Glycerol	Fisher Scientific	G33-500
HEPES	Fisher Scientific	BP310-100
Isopropyl-β-D-thiogalactoside (IPTG)	Fisher Scientific	15-529-019
LB Broth	Fisher Scientific	BP1426-500
Ni-NTA agarose	Qiagen	30210
Ovalbumin	MilliporeSigma	A2512
pGEX-4T-2 vector	GE Healthcare	28954550
Protease inhibitor cocktail	MilliporeSigma	4693132001
Reduced glutathione	Fisher Scientific	BP25211
Refrigerated shaker	Eppendorf	M1282-0004
Rosetta Competent Cells	MilliporeSigma	70953-3
Size exclusion chromatography column	GE Healthcare	28990944	Superdex 200 10/300 GL column
Sodium Chloride (NaCl)	Fisher Scientific	S271-500
Stopped-flow fluorimeter	Hi-Tech Scientific	SF-61 DX2
TCEP·HCl	Fisher Scientific	PI20490
Thrombin	MilliporeSigma	T4648
Tris Base	Fisher Scientific	BP152-500
Ultrafiltration membrane	MilliporeSigma	UFC903008	Amicon Ultra-15 Centrifugal Filter Units, Ultra-15, 30,000 NMWL