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). Study holds ProTherm entries: 5347, 5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358, 5359, 5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370, 5371, 5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379 Extra Details: alpha-helix; conformational changes; single-tryptophan mutants;,interdomain region
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:29 p.m.
|Number of data points||125|
|Proteins||Phosphoglycerate kinase ; Phosphoglycerate kinase|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: ddG_H2O ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence SLSSKLSVQDLDLKDKRVFIRVDFNVPLDGKKITSNQRIVAALPTIKYVLEHHPRYVVLASHLGRPNGERNEKYSLAPVAKELQSLLGKDVTFLNDCVGPEVEAAVKASAPGSVILLENLRYHIEEEGSRKVDGQKVKASKEDVQKFRHELSSLADVYINDAFGTAHRAHSSMVGFDLPQRAAGFLLEKELKYFGKALENPTRPFLAILGGAKVADKIQLIDNLLDKVDSIIIGGGMAFTFKKVLENTEIGDSIFDKAVGPEIAKLMEKAKAKGVEVVLPVDFIIADAFSASANTKTVTDKEGIPAGWQGLDNGPESRKLFAATVAKATVILWNGPPGVFEFEKFAAGTKALLDEVVKSSAAGNTVIIGGGDTATVAKKYGVTDKISHVSTGGGASLELLEGKELPGVAFLSEKK|