title: Conformational dynamics is more important than helical propensity for the folding of the all α-helical protein Im7.
abstract: Im7 folds via an on-pathway intermediate that contains three of the four native α-helices. The missing helix, helix III, is the shortest and its failure to be formed until late in the pathway is related to frustration in the structure. Im7H3M3, a 94-residue variant of the 87-residue Im7 in which helix III is the longest of the four native helices, also folds via an intermediate. To investigate the structural basis for this we calculated the frustration in the structure of Im7H3M3 and used NMR to investigate its dynamics. We found that the native state of Im7H3M3 is highly frustrated and in equilibrium with an intermediate state that lacks helix III, similar to Im7. Model-free analysis identified residues with chemical exchange contributions to their relaxation that aligned with the residues predicted to have highly frustrated interactions, also like Im7. Finally, we determined properties of urea-denatured Im7H3M3 and identified four clusters of interacting residues that corresponded to the α-helices of the native protein. In Im7 the cluster sizes were related to the lengths of the α-helices with cluster III being the smallest but in Im7H3M3 cluster III was also the smallest, despite this region forming the longest helix in the native state. These results suggest that the conformational properties of the urea-denatured states promote formation of a three-helix intermediate in which the residues that form helix III remain non-helical. Thus it appears that features of the native structure are formed early in folding linked to collapse of the unfolded state.
authors.affiliation: Centre for Structural and Molecular Biochemistry, School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, United Kingdom.
authors.forename: Angelo Miguel
authors.initials: AM
authors.lastname: Figueiredo
authors.forename: Sara B-M
authors.initials: SB
authors.lastname: Whittaker
authors.forename: Stuart E
authors.initials: SE
authors.lastname: Knowling
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Moore
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; C509253:E colicin-binding immunity protein Im7, E coli; D029968:Escherichia coli Proteins; D014508:Urea
keywords: Im7; NMR; frustration; protein folding