Sugars and polyols are used to stabilize proteins. The degree of stabilization conferred on a model protein by sucrose was calculated in terms of the free energy of folding. Phosphoglycerate kinase (PGK) was denatured by guanidine hydrochloride (GuHCl) in different sucrose concentrations. The linear extrapolation method [1,2] was used to calculate the free energy of folding in the absence of denaturant. Although sucrose increased the concentration of GuHCl required to unfold the protein, the free energy of folding in water was unchanged. In order to probe the nature of the stabilizing effect of sucrose, an FT-Raman spectroscopic study of denaturant-polyol systems was undertaken. Investigations of interactions between GuHCl, urea or formamide and polyhydric compounds, revealed no evidence for hydrogen bonding or dipole-dipole associations. Polyhydric compounds caused minor changes in denaturant spectra although the converse was not observed. The structure of deuterated water changed on addition of denaturants. For non-ionic denaturants, addition of polyhydric solutes countered this change in water structure. Thus polyhydric compounds oppose the effect of denaturants on water structure. The observed increase in GuHCl concentration required to unfold PGK in the presence of sucrose may be attributed to this property of sucrose. Study holds ProTherm entries: 9340 Extra Details: dithiothreitol(2 mM) was added in the experiment
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:37 p.m.
|Number of data points||1|
|Proteins||Phosphoglycerate kinase ; Phosphoglycerate kinase|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence MNKKTIRDVDVRGKRVFCRVDFNVPMEQGAITDDTRIRAALPTIRYLIEHGAKVILASHLGRPKGKVVEELRLDAVAKRLGELLERPVAKTNEAVGDEVKAAVDRLNEGDVLLLENVRFYPGEEKNDPELAKAFAELADLYVNDAFGAAHRAHASTEGIAHYLPAVAGFLMEKELEVLGKALSNPDRPFTAIIGGAKVKDKIGVIDNLLEKVDNLIIGGGLAYTFVKALGHDVGKSLLEEDKIELAKSFMEKAKEKGVRFYMPVDVVVADRFANDANTKVVPIDAIPADWSALDIGPKTRELYRDVIRESKLVVWNGPMGVFEMDAFAHGTKAIAEALAEALDTYSVIGGGDSAAAVEKFGLADKMDHISTGGGASLEFMEGKQLPGVVALEDK|