title: Site-specific dimensions across a highly denatured protein; a single molecule study.
abstract: Do highly denatured proteins adopt random coil configurations? Here, we address this question by measuring residue-to-residue separations across the denatured FynSH3 domain. Using single-molecule Forster resonance energy transfer techniques, we have collected transfer efficiency probability distributions for dye-labeled, denatured protein. Applying maximum likelihood analysis to the interpretation of these distributions, we have determined the through-space distance between five residue pairs in the protein's guanidine hydrochloride-unfolded and trifluoroethanol-unfolded states. We find that, while the dimensions of the guanidine hydrochloride -unfolded molecule generally coincide with the dimensions predicted for a random coil ensemble, potentially statistically significant deviations from random coil behavior are also evident. These small, site-specific deviations may provide a means of reconciling earlier, scattering-based evidence for the random coil nature of the unfolded state with more site-specific spectroscopic evidence suggesting residual structure. We have also studied the unfolded ensemble populated in 50% trifluoroethanol, a denaturant that induces a highly helical unfolded state. We find that the size and shape of the unfolded ensemble under these conditions is effectively indistinguishable from that populated in guanidinium hydrochloride solutions, suggesting that the gross structure of the denatured state is, perhaps surprisingly, independent of the chemistry of the cosolvent.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA.
authors.forename: Evan R
authors.initials: ER
authors.lastname: McCarney
authors.forename: James H
authors.initials: JH
authors.lastname: Werner
authors.forename: Summer L
authors.initials: SL
authors.lastname: Bernstein
authors.forename: Ingo
authors.initials: I
authors.lastname: Ruczinski
authors.forename: Dmitrii E
authors.initials: DE
authors.lastname: Makarov
authors.forename: Peter M
authors.initials: PM
authors.lastname: Goodwin
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Plaxco
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D018909:src Homology Domains
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D011506:Proteins; D011994:Recombinant Proteins