Design consideration and probes for fluorescence resonance energy transferstudies

Spectroscopic properties of two newly synthesized water-soluble thiol-react ive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca ) and N-iodoacetyl-beta-(2-naphthyl)alanine (I-Nal), were characterized usi ng single cysteine mutants of Escherichia coli adenylate kinase. Together w ith two known water-soluble thiol-reactive dyes (Lucifer yellow iodoacetami de and 5-iodoacetamidosalicylic acid) and as well, tryptophan residues (eit her native or inserted into a protein by site directed mutagenesis), these probes can be arranged pairwise in a molecular tool set for studies of stru ctural transitions in proteins by means of fluorescence resonance energy-tr ansfer (FRET) experiments. A set of seven donor/acceptor pairs which allow determination of intramolecular distances and their distributions over the range 10-40 Angstrom in labeled protein derivatives is described. The charg ed groups present in the probes facilitate the conjugation reaction and imp rove postlabeling purification. General considerations for design of charge d probes and site-directed labeling for applications of FRET methods in stu dies of protein structure and dynamics are presented.