Urea-induced unfolding of the immunity protein Im9 monitored by spFRET.
We have studied the urea-induced unfolding of the E colicin immunity protein Im9 using diffusion single-pair fluorescence resonance energy transfer. Detailed examination of the proximity ratio of the native and denatured molecules over a wide range of urea concentrations suggests that the conformational properties of both species are denaturant-dependent. Whereas native molecules become gradually more expanded as urea concentration increases, denatured molecules show a dramatic dependence of the relationship between proximity ratio and denaturant concentration, consistent with substantial compaction of the denatured ensemble at low denaturant concentrations. Analysis of the widths of the proximity ratio distributions for each state suggests that whereas the native state ensemble is relatively narrow and homogeneous, the denatured state may possess heterogeneity in mildly denaturing conditions.
Tezuka-Kawakami
Tomoko
T
Institute of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
Gell
Chris
C
Brockwell
David J
DJ
Radford
Sheena E
SE
Smith
D Alastair
DA
D001426:Bacterial Proteins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D011489:Protein Denaturation; D017510:Protein Folding; D014508:Urea / Q000032:analysis* / Q000737:chemistry*
D001426:Bacterial Proteins; D014508:Urea