INTRINSIC FLUORESCENCE STUDIES OF THE CHAPERONIN GROEL CONTAINING SINGLE TYR -] TRP REPLACEMENTS REVEAL LIGAND-INDUCED CONFORMATIONAL-CHANGES

Two mutants of GroEL containing the single tyrosine to tryptophan repl acement of either residue 203 or 360 in the apical domain have been pu rified, characterized, and used for fluorescence studies, Both mutants can facilitate the in vitro refolding of rhodanese in an ATP- and Gro ES-dependent manner, producing yields of recoverable activity comparab le to the wild-type chaperonin. Y203W shows some increased hydrophobic exposure and easier urea-induced disassembly compared with wild-type or Y360W, although the unfolding of all the species was similar at hig h concentrations of urea. Intrinsic fluorescence studies of the two mu tants reveal that nucleotide binding (ADP or AMP-PNP (adenosine 5'-(be ta,gamma-imino)triphosphate)) induces conformational changes in the te tradecamer that are independent presence of the co chaperonin, GroES. The K-1/2 for this transition is approximately 5 mu M for both mutants , Energy transfer experiments show that the tryptophan fluorescence of the Y360W mutant is partially quenched (similar to 50%) upon binding of the fluorescent, hydrophobic probe 4,4'-bis(1-anilino-8-naphthalene sulfonic acid), while the fluorescence of the Y203W mutant is signific antly quenched (similar to 75%). These results are discussed in relati on to the molecular mechanism for GroEL function.