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 replacement of either residue 203 or 360 in the apical domain have been purified, characterized, and used for fluorescence studies. Both mutants can facilitate the in vitro refolding of rhodanese in an ATP- and GroES-dependent manner, producing yields of recoverable activity comparable 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 high concentrations of urea. Intrinsic fluorescence studies of the two mutants reveal that nucleotide binding (ADP or AMP-PNP (adenosine 5'-(beta,gamma-imino)triphosphate)) induces conformational changes in the tetradecamer that are independent of the presence of the co-chaperonin, GroES. The K1/2 for this transition is approximately 5 microM for both mutants. Energy transfer experiments show that the tryptophan fluorescence of the Y360W mutant is partially quenched ( approximately 50%) upon binding of the fluorescent, hydrophobic probe 4,4'-bis(1-anilino-8-naphthalenesulfonic acid), while the fluorescence of the Y203W mutant is significantly quenched ( approximately 75%). These results are discussed in relation to the molecular mechanism for GroEL function.
Gibbons
D L
DL
Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78284-7760, USA. horowitz@bioc02.uthscsa.edu
Hixson
J D
JD
Hay
N
N
Lund
P
P
Gorovits
B M
BM
Ybarra
J
J
Horowitz
P M
PM
D000244:Adenosine Diphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D014443:Tyrosine
D018834:Chaperonin 60; D000266:Adenylyl Imidodiphosphate; D014443:Tyrosine; D000244:Adenosine Diphosphate; D014364:Tryptophan; D008274:Magnesium; D011188:Potassium