Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions
| Autor: | Kevin W. Plaxco, Anna J. Simon, Herschel M. Watkins, Tobin R. Sosnick, Everett A. Lipman, Rex P. Hjelm |
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| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular Small Angle Protein Denaturation Protein Folding Protein Conformation Molecular Conformation Flory scaling Polyethylene Glycols two state Scattering chemistry.chemical_compound Protein structure X-Ray Diffraction Models PEG ratio Scattering Small Angle Fluorescence Resonance Energy Transfer Guanidine Coloring Agents Multidisciplinary Chemistry Small-angle X-ray scattering Proteins Molecular SAXS Biological Sciences statistical coil Random coil Denaturation midpoint Crystallography Förster resonance energy transfer biological sciences Biophysics Protein folding Hydrophobic and Hydrophilic Interactions |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America, vol 112, iss 21 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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