The unfolding-refolding transition of phosphoglycerate kinase followed by steady-state fluorescence has clearly shown the existence of a hyperfluorescent form [Missiakas et al. (1990) Biochemistry 29, 8683-8689]. In order to determine the contribution of each of the two tryptophans to the fluorescence properties of the enzyme in the equilibrium transition and to characterize the hyperfluorescent form, two single tryptophan mutants in which tryptophans 308 and 333 were replaced by a tyrosine and a phenylalanine, respectively, were constructed. Neither the catalytic nor the physicochemical properties of the enzyme are significantly altered by these mutations. The unfolding-refolding transitions were studied using circular dichroism and tryptophan fluorescence emission. Both tryptophans contribute to the hyperfluorescence observed in the first transition. For guanidine hydrochloride concentrations higher than 0.9 M, it clearly appears that the second transition results from a further unfolding. It is accompanied by a decrease in fluorescence intensity and a 5 mm red shift of the maximum emission wavelength. When the unfolding is induced by urea, the end of the transition corresponds to the hyperfluorescent state. Further addition of guanidine hydrochloride induces complete unfolding. These results suggest the presence of residual microstructures around tryptophan 308 and tryptophan 333 in the hyperfluorescent state. The characterization of these clusters and their contribution as starting structures in the folding process are now under investigation. Study holds ProTherm entries: 5034, 5035 Extra Details: additive : EDTA(0.5 mM), unfolding-refolding transition; tryptophans;,physicochemical properties; hyperfluorescent state
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:28 p.m.
|Number of data points||6|
|Proteins||Phosphoglycerate kinase ; Phosphoglycerate kinase|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence SLSSKLSVQDLDLKDKRVFIRVDFNVPLDGKKITSNQRIVAALPTIKYVLEHHPRYVVLASHLGRPNGERNEKYSLAPVAKELQSLLGKDVTFLNDCVGPEVEAAVKASAPGSVILLENLRYHIEEEGSRKVDGQKVKASKEDVQKFRHELSSLADVYINDAFGTAHRAHSSMVGFDLPQRAAGFLLEKELKYFGKALENPTRPFLAILGGAKVADKIQLIDNLLDKVDSIIIGGGMAFTFKKVLENTEIGDSIFDKAVGPEIAKLMEKAKAKGVEVVLPVDFIIADAFSASANTKTVTDKEGIPAGWQGLDNGPESRKLFAATVAKATVILWNGPPGVFEFEKFAAGTKALLDEVVKSSAAGNTVIIGGGDTATVAKKYGVTDKISHVSTGGGASLELLEGKELPGVAFLSEKK|