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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.

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


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.


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

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