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Conformational dynamics is more important than helical propensity for the folding of the all ?-helical protein Im7.

ABSTRACT: Im7 folds via an on-pathway intermediate that contains three of the four native ?-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 ?-helices of the native protein. In Im7 the cluster sizes were related to the lengths of the ?-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.

SUBMITTER: Figueiredo AM

PROVIDER: S-EPMC3843627 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Conformational dynamics is more important than helical propensity for the folding of the all α-helical protein Im7.

Figueiredo Angelo Miguel AM Whittaker Sara B-M SB Knowling Stuart E SE Radford Sheena E SE Moore Geoffrey R GR

Protein science : a publication of the Protein Society 20131019 12

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 ...[more]

PMID: 24123274

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Project description:Previous work shows that the transiently populated, on-pathway intermediate in Im7 folding contains three of the four native alpha-helices docked around a core stabilised by native and non-native interactions. To determine the structure and dynamic properties of this species in more detail, we have used protein engineering to trap the intermediate at equilibrium and analysed the resulting proteins using NMR spectroscopy and small angle X-ray scattering. Four variants were created. In L53AI54A, two hydrophobic residues within helix III are truncated, preventing helix III from docking stably onto the developing hydrophobic core. In two other variants, the six residues encompassing the native helix III were replaced with three (H3G3) or six (H3G6) glycine residues. In the fourth variant, YY, two native tyrosine residues (Tyr55 and Tyr56) were re-introduced into H3G6 to examine their role in determining the properties of the intermediate ensemble. All four variants show variable peak intensities and broad peak widths, consistent with these proteins being conformationally dynamic. Chemical shift analyses demonstrated that L53AI54A and YY contain native-like secondary structure in helices I and IV, while helix II is partly formed and helix III is absent. Lack of NOEs and rapid NH exchange for L53AI54A, combined with detailed analysis of the backbone dynamics, indicated that the hydrophobic core of this variant is not uniquely structured, but fluctuates on the NMR timescale. The results demonstrate that though much of the native-like secondary structure of Im7 is present in the variants, their hydrophobic cores remain relatively fluid. The comparison of H3G3/H3G6 and L53AI54A/YY suggests that Tyr55 and/or Tyr56 interact with the three-helix core, leading other residues in this region of the protein to dock with the core as folding progresses. In this respect, the three-helix bundle acts as a template for formation of helix III and the creation of the native fold.

Dataset Information

Conformational dynamics is more important than helical propensity for the folding of the all ?-helical protein Im7.

Publications

Conformational dynamics is more important than helical propensity for the folding of the all α-helical protein Im7.

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