title: Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions.
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.
authors.affiliation: Interdepartmental Program in Biomolecular Science and Engineering and.
authors.forename: Herschel M
authors.initials: HM
authors.lastname: Watkins
authors.affiliation: Interdepartmental Program in Biomolecular Science and Engineering and.
authors.forename: Anna J
authors.initials: AJ
authors.lastname: Simon
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637; and.
authors.forename: Tobin R
authors.initials: TR
authors.lastname: Sosnick
authors.affiliation: Departments of Physics and.
authors.forename: Everett A
authors.initials: EA
authors.lastname: Lipman
authors.affiliation: Los Alamos National Laboratory, Los Alamos Neutron Scattering Center, Los Alamos, NM 87545.
authors.forename: Rex P
authors.initials: RP
authors.lastname: Hjelm
authors.affiliation: Interdepartmental Program in Biomolecular Science and Engineering and Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106; kwp@chem.ucsb.edu.
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Plaxco
mesh_terms: D004396:Coloring Agents; D031541:Fluorescence Resonance Energy Transfer; D019791:Guanidine; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008968:Molecular Conformation; D011092:Polyethylene Glycols / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D004396:Coloring Agents; D011506:Proteins; D011092:Polyethylene Glycols; D019791:Guanidine
keywords: Flory scaling; SAXS; protein folding; statistical coil; two state