Small-angle X-ray scattering and single-molecule FRET spectroscopy produce highly divergent views of the low-denaturant unfolded state.
The results of more than a dozen single-molecule Förster resonance energy transfer (smFRET) experiments suggest that chemically unfolded polypeptides invariably collapse from an expanded random coil to more compact dimensions as the denaturant concentration is reduced. In sharp contrast, small-angle X-ray scattering (SAXS) studies suggest that, at least for single-domain proteins at non-zero denaturant concentrations, such compaction may be rare. Here, we explore this discrepancy by studying protein L, a protein previously studied by SAXS (at 5 °C), which suggested fixed unfolded-state dimensions from 1.4 to 5 M guanidine hydrochloride (GuHCl), and by smFRET (at 25 °C), which suggested that, in contrast, the chain contracts by 15-30% over this same denaturant range. Repeating the earlier SAXS study under the same conditions employed in the smFRET studies, we observe little, if any, evidence that the unfolded state of protein L contracts as the concentration of GuHCl is reduced. For example, scattering profiles (and thus the shape and dimensions) collected within ∼4 ms after dilution to as low as 0.67 M GuHCl are effectively indistinguishable from those observed at equilibrium at higher denaturant. Our results thus argue that the disagreement between SAXS and smFRET is statistically significant and that the experimental evidence in favor of obligate polypeptide collapse at low denaturant cannot be considered conclusive yet.
Department of Physics, University of Chicago, Chicago, IL 60637, USA.
Tae Yeon
TY
Yoo
Steve P
SP
Meisburger
James
J
Hinshaw
Lois
L
Pollack
Gilad
G
Haran
Tobin R
TR
Sosnick
Kevin
K
Plaxco
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D019791:Guanidine / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D053838:Scattering, Small Angle; D014508:Urea / Q000737:chemistry / Q000378:metabolism; D014965:X-Rays
D011506:Proteins; D014508:Urea; D019791:Guanidine