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Proc Natl Acad Sci U S A. 2015 May 26;112(21):6631-6. doi: 10.1073/pnas.1418673112. Epub 2015 May 11.

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions.

Author information

1
Interdepartmental Program in Biomolecular Science and Engineering and.
2
Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637; and.
3
Departments of Physics and.
4
Los Alamos National Laboratory, Los Alamos Neutron Scattering Center, Los Alamos, NM 87545.
5
Interdepartmental Program in Biomolecular Science and Engineering and Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106; kwp@chem.ucsb.edu.

Abstract

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.

KEYWORDS:

Flory scaling; SAXS; protein folding; statistical coil; two state

PMID:
25964362
PMCID:
PMC4450392
DOI:
10.1073/pnas.1418673112
[Indexed for MEDLINE]
Free PMC Article

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