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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 31, Number 10
BKCSDE 31(10)
October 20, 2010

Title	Folding Mechanism of WT* Ubiquitin Variant Studied by Stopped-flow Fluorescence Spectroscopy
Author	Soon Ho Park
Keywords	Protein folding kinetics, Folding intermediate, Native interaction
Abstract	The folding kinetics of WT* ubiquitin variant with valine to alanine mutation at sequence position 26 (HubWA) was studied by stopped-flow fluorescence spectroscopy. While unfolding kinetics showed a single exponential phase, refolding reaction showed three exponential phases. The semi-logarithmic plot of urea concentration vs. rate constant for the first phase showed v-shape pattern while the second phase showed v-shape with roll-over effect at low urea concentration. The rate constant and the amplitude of the third phase were constant throughout the urea concentrations, suggesting that this phase represents parallel process due to the configurational isomerization. Interestingly, the first and second phases appeared to be coupled since the amplitude of the second phase increased at the expense of the amplitude of the first phase in increasing urea concentrations. This observation together with the roll-over effect in the second folding phase indicates the presence of intermediate state during the folding reaction of HubWA. Quantitative analysis of Hub- WA folding kinetics indicated that this intermediate state is on the folding pathway. Folding kinetics measurement of a mutant HubWA with hydrophobic core residue mutation, Val to Ala at residue position 17, suggested that the intermediate state has significant amount of native interactions, supporting the interpretation that the intermediate is on the folding pathway. It is considered that HubWA is a useful model protein to study the contribution of residues to protein folding process using folding kinetics measurements in conjunction with protein engineering.
Page	2877 - 2883
Full Text	PDF

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