Probing intradomain and interdomain conformational changes during equilibrium unfolding of phosphoglycerate kinase: fluorescence and circular dichroism study of tryptophan mutants.
Phosphoglycerate kinase is a monomeric protein composed of two globular domains of the alpha/beta type. Extensive domain-domain interactions involve three segments of the polypeptide chain that are distant from one another in the primary sequence: the N-terminus, the C-terminus, and a centrally located alpha-helix. In order to monitor spectroscopically the conformational changes that occur in the individual domains and at the interdomain interface during the unfolding process, we have constructed a series of single-tryptophan mutants. In addition to two previously described mutants, each with single tryptophans in the C-terminal domain (W308 and W333) [Szpikowska, B. K., Beechem, J. M., Sherman, M. A., & Mas, M. T. (1994) Biochemistry 33, 2217-2225], four new single-tryptophan mutants have been constructed: two with tryptophans located in the interdomain region (W194 and W399) and two with tryptophans in the N-terminal domain (W48 and W122). The equilibrium unfolding transitions induced by guanidine hydrochloride were monitored using far-UV CD, near-UV CD, steady-state, and time-resolved fluorescence. These studies reveal two unfolding transitions and suggest a sequential unfolding process for the mutants described in this paper. During the first transition (Cm approximately 0.5 M) the interdomain region and C-terminal domain unfold; the N-terminal domain remains relatively compact but lacks much of the tertiary structure that characterizes the native state. A hyperfluorescent intermediate is detected during this transition by tryptophan probes placed within the N-terminal domain. Complete unfolding of the N-terminal domain occurs during the second transition (Cm approximately 0.9 M).
Sherman
M A
MA
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Beechem
J M
JM
Mas
M T
MT
D002942:Circular Dichroism; D005454:Fluorescence Polarization; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014364:Tryptophan / Q000737:chemistry; D015003:Yeasts / Q000201:enzymology*
D006146:Guanidines; D014364:Tryptophan; D010735:Phosphoglycerate Kinase; D019791:Guanidine