Details

Autor(en) / Beteiligte

• Watkins, Herschel M.
• Simon, Anna J.
• Sosnick, Tobin R.
• Lipman, Everett A.
• Hjelm, Rex P.
• Plaxco, Kevin W.

Titel

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

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2015-05, Vol.112 (21), p.6631-6636

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2015

Quelle

MEDLINE

Beschreibungen/Notizen

• 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. Significance The relationship between proteins unfolded under physiological conditions and those unfolded by chemical denaturation remains controversial. Specifically, although FRET studies suggest that unfolded proteins invariably contract with falling denaturant levels, scattering studies argue that they do not. Here, we explore the origins of this discrepancy using PEG as a negative control. Scattering indicates that, as expected, the polymer’s dimensions are denaturant-independent. The dye-labeled polymer, nevertheless, exhibits denaturant-dependent changes in measured transfer efficiency similar to those seen for unfolded proteins. This similarity raises questions regarding the interpretation of such changes as being indicative of hydrophobic or hydrogen bond-driven collapse.