Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 112, Issue 21, Pages 6631-6636Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1418673112
Keywords
protein folding; SAXS; two state; statistical coil; Flory scaling
Categories
Funding
- Department of Energy [DE-AC52-06NA25396]
- NIH [GM055694, GM093263, GM097463]
- Whitaker Fellowship
- National Science Foundation [DMR-0960331]
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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 (similar to 6 M) to low (similar to 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.
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