Cold denaturation of yeast phosphoglycerate kinase (yPGK) was investigated by a combination of far UV circular dichroism (CD), steady-state and time-resolved fluorescence, and small angle X-ray scattering. It was shown that cold denaturation of yPGK cannot be accounted for by a simple two-state process and that an intermediate state can be stabilized under mild denaturing conditions. Comparison between far UV CD and fluorescence shows that in this state the protein displays a fluorescence signal corresponding mainly to exposed tryptophans, whereas its CD signal is only partially modified. Comparison with spectroscopic data obtained from a mutant missing the last 12 amino-acids (yPGK delta404) suggests that lowering the temperature mainly results in a destabilization of hydrophobic interactions between the two domains. Small angle X-ray scattering measurements give further information about this stabilized intermediate. At 4 degrees C and in the presence of 0.45 M Gdn-HCl, the main species corresponds to a protein as compact as native yPGK, whereas a significant proportion of ellipticity has been lost. Although various techniques have shown the existence of residual structures in denatured proteins, this is one example of a compact denatured state devoid of its main content in alpha helices. Study holds ProTherm entries: 7039, 7040, 7041, 7042, 7043, 7044 Extra Details: additive : EDTA(0.5 mM), phosphoglycerate kinase; cold denaturation;,hydrophobic interactions; SAXS
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:33 p.m.
|Number of data points||18|
|Proteins||Phosphoglycerate kinase ; Phosphoglycerate kinase|
|Assays/Quantities/Protocols||Experimental Assay: Cm temp:4.0 C ; Experimental Assay: m temp:4.0 C ; Experimental Assay: dG_H2O temp:4.0 C ; Experimental Assay: Cm temp:22.0 C ; Experimental Assay: m temp:22.0 C ; Experimental Assay: dG_H2O temp:22.0 C ; Experimental Assay: Cm temp:4.0 C ; Experimental Assay: m temp:4.0 C ; Experimental Assay: dG_H2O temp:4.0 C ; Experimental Assay: Cm temp:22.0 C ; Experimental Assay: m temp:22.0 C ; Experimental Assay: dG_H2O temp:22.0 C|
|Libraries||Mutations for sequence SLSSKLSVQDLDLKDKRVFIRVDFNVPLDGKKITSNQRIVAALPTIKYVLEHHPRYVVLASHLGRPNGERNEKYSLAPVAKELQSLLGKDVTFLNDCVGPEVEAAVKASAPGSVILLENLRYHIEEEGSRKVDGQKVKASKEDVQKFRHELSSLADVYINDAFGTAHRAHSSMVGFDLPQRAAGFLLEKELKYFGKALENPTRPFLAILGGAKVADKIQLIDNLLDKVDSIIIGGGMAFTFKKVLENTEIGDSIFDKAVGPEIAKLMEKAKAKGVEVVLPVDFIIADAFSASANTKTVTDKEGIPAGWQGLDNGPESRKLFAATVAKATVILWNGPPGVFEFEKFAAGTKALLDEVVKSSAAGNTVIIGGGDTATVAKKYGVTDKISHVSTGGGASLELLEGKELPGVAFLSEKK|