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Protein Sci. 2012 Jun;21(6):819-27. doi: 10.1002/pro.2065. Epub 2012 Apr 23.

A "Link-Psi" strategy using crosslinking indicates that the folding transition state of ubiquitin is not very malleable.

Author information

1
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.

Abstract

Using a combined crosslinking-ψ analysis strategy, we examine whether the structural content of the transition state of ubiquitin can be altered. A synthetic dichloroacetone crosslink is first introduced across two β strands. Whether the structural content in the transition state ensemble has shifted towards the region containing the crosslink is probed by remeasuring the ψ value at another region (ψ identifies the degree to which an inserted bi-Histidine metal ion binding site is formed in the transition state). For sites around the periphery of the obligate transition state nucleus, we find that the resulting changes in ψ values are near or at our detection limit, thereby indicating that the structural content of the transition state has not measurably changed upon crosslinking. This work demonstrates the utility of the simultaneous application of crosslinking and ψ-analysis for examining potential transition state heterogeneity in globular proteins.

PMID:
22528473
PMCID:
PMC3403417
DOI:
10.1002/pro.2065
[Indexed for MEDLINE]
Free PMC Article

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