标题：Site-specific fluorescence double-labeling of proteins and analysis of structural changes in solution by Fluorescence Resonance Energy Transfer (FRET)
摘要：Graphical abstractDisplay OmittedHighlights•Insertion of a Cys4 sequence and single Cys motif allowedsite-specific labeling with FlAsH and Alexa568of a protein.•FRET is a well-known technique useful for analysis of structural changes of proteins labeled with donor and accepter fluorophores.•Inter-fluorophore distances and proximity ratios of the peakintensities are used to understand structural changes.AbstractFluorescence Resonance Energy Transfer (FRET) is a well-known methodology for detection and quantitation of structural changes of proteins in solution. FRET requires site-specific protein labeling with two fluorophores, one of which functions as an energy donor and the other one as an energy acceptor. However, the site-specific labeling of protein is often complex and difficult, particularly when inserting two fluorophores in specific sites. We have examined several protein labeling approaches with a varying degree of success. Described here is a dual labeling strategy that worked reproducibly in a number of protein targets and we believe will be applicable to a variety of proteins, which have few or no native cysteine (Cys) residues. We have successfully double-labeled DnaA protein ofBacillus anthracis, which lacks intrinsic Cys residues. A cysteine residue was inserted at the N-terminus byin vitromutagenesis and a Cys-Cys-Phe-Gly-Cys-Cys (CCPGCC) sequence at the C-terminus by PCR. This protein was labeled site-specifically with a fluorescein derivative, FlAsH, at the CCPGCC sequence followed by Alexa568maleimide at the N-terminus Cys residue. Structural changes of the protein with nucleotide, DNA and an inhibitor protein binding were determined by FRET analysis of the double-labeled protein. This comprehensive novel methodology for site-specific protein labeling with different fluorophores is applicable for understanding differentin vitroproteomic structural studies. Here, we describe a verified technique used for FRET spectral analysis and quantitative evaluation of structural changes using fluorophore labeled DnaA protein constructs as an example.
