High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape

Fossat, Martin J.; Dao, Thuy P.; Jenkins, Kelly A; Dellarole, Mariano; Yang, Yinshan; McCallum, Scott A.; Garcı́a, Angel E.; Barrick, Doug; Roumestand, Christian; Royer, Catherine A.

doi:10.1016/j.bpj.2016.08.027

Cited by 35 publications

(53 citation statements)

References 33 publications

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“…Furthermore, alternative states may be elicited by pressure perturbation and studied by NMR spectroscopy (14,15). Relative populations of excited states are shifted under pressure as a result of associated volume differences, which includes the loss of cavities or packing defects in the protein interior (16), cavity hydration (17)(18)(19), local and global unfolding (20)(21)(22), and structure relaxation from side-chain rearrangement (23). As internal cavities make a major contribution to the total volume change upon protein structural transition, hydrostatic pressure emerges as a key variable to the study of protein excited states (16).…”

mentioning

confidence: 99%

How internal cavities destabilize a protein

Xue

Wakamoto

Kejlberg

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified “invisible” folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å−3.

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confidence: 99%

How internal cavities destabilize a protein

Xue

Wakamoto

Kejlberg

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…To visualize more precisely which regions of the protein become disordered at intermediate pressures, we constructed fractional contact maps (22) for I27 single-module. We define the probability of contact for any pair of residues, P i,j , as the geometric mean of the fractional probability that each of the two residues is in the folded state at a given pressure (P ij = [P i x P j ] 1/2 ) (26). The pressure dependence of the contact maps showed that the region first affected by an increase of pressure is the short parallel ß-sheet (Strands A’G).…”

Section: Resultsmentioning

confidence: 99%

“…Native contact maps were obtained by using software CMView (36) with a threshold of 9 Å around the Cα of each residue, using the best structure among the 20 refined ones. Using the geometric mean, rather than the joint probability as previously done (22,26), ensures the correct unfolding profile in the case of two-state unfolding.…”

Section: Methodsmentioning

confidence: 99%

“…NMR spectroscopy has emerged as a particularly powerful tool to obtain high-resolution structural information about protein folding events because an abundance of site-specific probes can be studied simultaneously in a multidimensional NMR spectrum. During the last past years, we have shown that the combination of high pressure and NMR constitutes a powerful tool that can yield unprecedented details on protein unfolding landscape (22-26; for a recent review 27). Indeed, the atomic resolution offered by multidimensional NMR experiments provides an intrinsic multi-probe approach to assess the degree of protein folding cooperativity, which is otherwise difficult to characterize using techniques such as circular dichroism or fluorescence.…”

Section: Introductionmentioning

confidence: 99%

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Monitoring Unfolding of Titin I27 single- and bi-Domain with High-Pressure NMR Spectroscopy

Roumestand

Herrada

Heusden

et al. 2018

Preprint

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A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the folding energy landscape. Simulations, when corroborated by experimental data yielding global information on the folding process, can provide this level of insight. Molecular Dynamics (MD) has been associated often to force spectroscopy experiments to decipher the unfolding mechanism of titin Ig-like single- or multi-domain, the giant multi-modular protein from sarcomere, yielding information on the sequential events during titin unfolding under stretching. Here, we used high-pressure NMR to monitor the unfolding of titin I27 Ig-like single-domain and tandem. Since this method brings residue-specific information on the folding process, it can provide quasi-atomic details on this process, without the help of MD simulations. Globally, the results of our high-pressure analysis are in agreement with previous results obtained by the association of experimental measurements and MD simulation and/or protein engineering, although the intermediate folding state caused by the early detachment of the AB ß-sheet, often reported in previous works based on MD or force spectroscopy, cannot be detected. On the other hand, the A’G parallel ß-sheet of the ß-sandwich has been confirmed as the Achilles heel of the 3D scaffold: its disruption yields complete unfolding, with very similar characteristics (free energy, unfolding volume, kinetics constant rates) for the two constructs.

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“…see Ref. 136). In addition, conformational distributions from folding simulations may be used to predict time-resolved experimental results.…”

Section: Harnessing the Predictive Power Of Folding Simulationsmentioning

confidence: 95%

Successes and challenges in simulating the folding of large proteins

Gershenson

Gosavi

Faccioli

et al. 2020

Journal of Biological Chemistry

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Computational simulations of protein folding can be used to interpret experimental folding results, to design new folding experiments, and to test the effects of mutations and small molecules on folding. However, whereas major experimental and computational progress has been made in understanding how small proteins fold, research on larger, multidomain proteins, which comprise the majority of proteins, is less advanced. Specifically, large proteins often fold via long-lived partially folded intermediates, whose structures, potentially toxic oligomerization, and interactions with cellular chaperones remain poorly understood. Molecular dynamics based folding simulations that rely on knowledge of the native structure can provide critical, detailed information on folding free energy landscapes, intermediates, and pathways. Further, increases in computational power and methodological advances have made folding simulations of large proteins practical and valuable. Here, using serpins that inhibit proteases as an example, we review native-centric methods for simulating the folding of large proteins. These synergistic approaches range from Gō and related structurebased models that can predict the effects of the native structure on folding to all-atom-based methods that include side-chain chemistry and can predict how disease-associated mutations may impact folding. The application of these computational approaches to serpins and other large proteins highlights the successes and limitations of current computational methods and underscores how computational results can be used to inform experiments. These powerful simulation approaches in combination with experiments can provide unique insights into how large proteins fold and misfold, expanding our ability to predict and manipulate protein folding.

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High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape

Cited by 35 publications

References 33 publications

How internal cavities destabilize a protein

How internal cavities destabilize a protein

Monitoring Unfolding of Titin I27 single- and bi-Domain with High-Pressure NMR Spectroscopy

Successes and challenges in simulating the folding of large proteins

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