Detection and characterization of intermediates in the folding of large proteins by the use of genetically inserted tryptophan probes.
L-Lactate dehydrogenase from Bacillus stearothermophilus was rebuilt by using site-directed mutagenesis to produce an enzymically active, tryptophan-less enzyme by replacing all the wild-type tryptophans (80, 150, and 203) by tyrosines. Nine single tryptophan-containing active enzymes were constructed from this enzyme by genetically replacing one of the tyrosines 36, 85, 147, 190, 203, 237, 248, 279, or 285 by tryptophan. The equilibrium and the time-resolved tryptophan fluorescence intensity and anisotropy were used to report unfolding events in guanidine hydrochloride (GHCl) monitored from these nine defined positions. Three structural transitions, half complete at 0.55, 1.7, and 2.8 M GHCl, were identified and defined four folding intermediates, I (native), II (expanded monomer 1), III (expanded monomer 2), and IV (random coil), stable at 0, 1, 2.2, and 4 M GHCl, respectively. Intermediate II is a globular monomer. All the probed alpha-helices and most of the beta-structure was intact. There was an increase in the rate but not the extent of the mobilities of six of the probed tryptophan side chains, indicating loss of tertiary structure. Circular dichroism (CD) showed all the secondary structure to be intact. Intermediate III is monomeric and still globular, but the tryptophan anisotropy indicated an increase mobility at positions 36, 85, 190, 203, 279, and 285. Helix alpha-B is further disrupted but helices alpha-1F, alpha-2G, and alpha 3G were still rigid. CD showed half the secondary structure to be still intact. Intermediate IV is a random coil in which all tryptophans have complete rotational freedom and the helix CD signal is lost.(ABSTRACT TRUNCATED AT 250 WORDS)
Smith
C J
CJ
Molecular Recognition Centre, University of Bristol School of Medical Sciences, University Walk, U.K.
Clarke
A R
AR
Chia
W N
WN
Irons
L I
LI
Atkinson
T
T
Holbrook
J J
JJ
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009710:Nucleotide Mapping; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry
D006146:Guanidines; D015335:Molecular Probes; D014364:Tryptophan; D007770:L-Lactate Dehydrogenase; D019791:Guanidine