Single-molecule studies of the Im7 folding landscape.
Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted I(eqm)) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the I(eqm) variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na(2)SO(4) in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding.
Pugh
Sara D
SD
Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.
Gell
Christopher
C
Smith
D Alastair
DA
Radford
Sheena E
SE
Brockwell
David J
DJ
D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014508:Urea / Q000494:pharmacology
D002352:Carrier Proteins; C509253:E colicin-binding immunity protein Im7, E coli; D029968:Escherichia coli Proteins; D014508:Urea