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Interrupted Pressure-Jump NMR Experiments Reveal Resonances of On-Pathway Protein Folding Intermediate.

ABSTRACT: Previous pressure-jump NMR experiments on a pressure-sensitized double mutant of ubiquitin showed evidence that its folding occurs via two parallel, comparably efficient pathways: a single barrier and a two-barrier pathway. An interrupted folding NMR experiment is introduced, where for a brief period the pressure is dropped to atmospheric conditions (1 bar), followed by a jump back to high pressure for signal detection. Conventional, forward sampling of the indirect dimension during the low-pressure period correlates the 15N or 13C' chemical shifts of the unfolded protein at 1 bar to the 1H frequencies of both the unfolded and folded proteins at high pressure. Remarkably, sampling the data of the same experiment in the reverse direction yields the frequencies of proteins present at the end of the low-pressure interval, which include unfolded, intermediate, and folded species. Although the folding intermediate 15N shifts differ strongly from natively folded protein, its 13C' chemical shifts, which are more sensitive probes for secondary structure, closely match those of the folded protein and indicate that the folding intermediate must have a structure that is quite similar to the native state.

SUBMITTER: Charlier C

PROVIDER: S-EPMC6698896 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Interrupted Pressure-Jump NMR Experiments Reveal Resonances of On-Pathway Protein Folding Intermediate.

Charlier Cyril C Courtney Joseph M JM Anfinrud Philip P Bax Ad A

The journal of physical chemistry. B 20181010 49

Previous pressure-jump NMR experiments on a pressure-sensitized double mutant of ubiquitin showed evidence that its folding occurs via two parallel, comparably efficient pathways: a single barrier and a two-barrier pathway. An interrupted folding NMR experiment is introduced, where for a brief period the pressure is dropped to atmospheric conditions (1 bar), followed by a jump back to high pressure for signal detection. Conventional, forward sampling of the indirect dimension during the low-pres ...[more]

PMID: 30256104

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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

Interrupted Pressure-Jump NMR Experiments Reveal Resonances of On-Pathway Protein Folding Intermediate.

Publications

Interrupted Pressure-Jump NMR Experiments Reveal Resonances of On-Pathway Protein Folding Intermediate.

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