Förster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

Summary

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

Abstract

Förster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

Introduction

For proteins, elucidation of dynamics along with 3-dimensional (3-D) structural knowledge leads to an enhanced understanding of structure-function relationships of biomolecular systems. Structural methods, such as X-ray crystallography and cryogenic electron microscopy, capture a static structure and often require the determination of multiple structures to elucidate aspects of biomolecule binding and dynamics¹. This article discusses a solution-based method for mapping global structural elements, such as binding sites or binding interactions, that are potentially more transient and less easily captured by static methods. Strong candidate syste....

Protocol

1. Selection of labeling sites

1. Identify at least three potential labeling sites to triangulate the putative binding site on the existing protein structures. In this case, SecA, SecYEG, and preprotein attached to SecA through genetic fusion were identified².
   1. Choose labeling sites within 25-75 Å of the putative binding site and in relatively static regions of the protein, the distance will determine the specific FRET dye pair to be used³⁶. Locate the labeling sites in protein regions that are relatively distinct from each other, so the sites describe the vertices of a triangle ....

Results

This study focused on determining the location of the preprotein binding site on SecA prior to insertion of the preprotein into the SecYEG channel. To map the binding site, FRET experiments were performed between different regions of the preprotein and three distinct locations on the SecA and SecYEG proteins (Figure 1A-D). From the distances obtained and three-dimensional structures of SecA, SecYEG, and the preprotein, the location of the preprotein binding .......

Discussion

Through the use of the FRET mapping methodology, we identified the signal sequence binding site on the SecA protein. Importantly, the presence of a 3-D crystal structure of the complex greatly facilitated our study. The strength of this mapping methodology lies in the ability to use an existing structure to identify locations for labeling. This methodology cannot be used to determine a 3-D structure; however, determination of structural elements⁵⁶, refinement of an existing structure

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
490 nm LED laser	Horiba	1684-LED
Alexa Fluor 647 C2 Maleimide//DIBO Alkyne	Life Technologies	A20347
Agar	Difco	DF0812
Alexa Fluor 488 C5 Maleimide/DIBO Alkyne	Life Technologies	A10254
Alexa Fluor 488 DIBO Alkyne	Life Technologies	S10904
Alexa Fluor 647 DIBO Alkyne	Life Technologies	S10906
Amicon Ultra­4 Centrifugal filter (50kDa MWCO)	Sigma	UFC805008
Dodecylmaltoside (DDM)	Anatrace	D310
E. coli alkaline phosphatase signal peptide SP22	Biomolecules Midwest	N/A	Synthesized custom item
extended signal peptide SP41	Biomolecules Midwest	N/A	Synthesized custom item
FluorEssence	Horiba	version 2.4	spectral acquisition program for Fluoromax4 spectrofluorometer
Fluoromax 4 spectrofluorometer	Horiba	N/A
GlobalsWE	Laboratory for Fluorescence Dynamics, University of California, Irvine		spectral analysis program for time-resolved decays
H­4­Azido­Phe­OH	BACHEM	4020250.0001
LB (Miller) Broth	Fisher Scientific	BP9723
Ludox HS-40 colloidal silica (40 wt.% suspension in H2O)	Sigma-Aldrich	420816	dilution is needed to make a proper scattering solution
PTI Felix GX	Horiba	version 4.1.0.4096	spectral acquisition program for PTI Time Master Instrument
PTI Time Master Instrument	Horiba	NA
Pymol Molecular Graphics Program	Schrodinger	version 2.4
Water bath	Thermo Scientific	NESLAB RTE 10