Effect of Internal Cavities on Folding Rates and Routes Revealed by Real-Time Pressure-Jump NMR Spectroscopy

Roche, Julien; Dellarole, Mariano; José, A.; Norberto, Douglas R.; Garcı́a, Angel E.; García-Moreno, Bertrand; Roumestand, Christian; Royer, Catherine A.

doi:10.1021/ja406682e

Cited by 69 publications

(110 citation statements)

References 53 publications

(124 reference statements)

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“…SI Appendix, Table S1, summarizes the unanimous ΔV u behavior upon increasing urea concentrations for three different protein models, including skeletal troponin C in the apo and holo states, MpNep2, and an SH2 domain. Additionally, similar behavior has been observed for different mutants of staphylococcal nuclease (SNAse) with increasing concentrations of guanidinium chloride (Gdm-Cl) (52,53).…”

Section: Measuring Urea Effects Based On Nmr Peak Intensities and Saxssupporting

confidence: 59%

A hypothesis to reconcile the physical and chemical unfolding of proteins

Oliveira

Silva

2015

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

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High pressure (HP) or urea is commonly used to disturb folding species. Pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the protein-solvent system. However, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though proteinurea interactions are considered to be crucial. Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by HP. In studying MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. At subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by NMR intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to pressure denaturation (lower p 1/2 and larger ΔV u ); thus, urea and HP share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. These observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes.protein folding | hydrostatic pressure | urea | NMR | SAXS

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Section: Measuring Urea Effects Based On Nmr Peak Intensities and Saxssupporting

confidence: 59%

A hypothesis to reconcile the physical and chemical unfolding of proteins

Oliveira

Silva

2015

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

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“…Internal hydration can also be used to kinetically stabilize specific regions of the protein. However, it must be pointed out that the creation of cavities and their hydrated or dry states can result in a complex thermodynamic and kinetic response of the proteins, as pointed out in the context of pressure induced unfolding 19,88 . Moreover, these changes however must be effective in a broad range of temperatures.…”

Section: Resultsmentioning

confidence: 99%

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

et al. 2014

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In this work we address the question whether the enhanced stability of thermophilic proteins has a direct connection with internal hydration. Our model systems are two homologues G-domains of different stability: the mesophilic G domain of the Elongation-Factor thermal unstable protein from E. coli and the hyperthermophilic G domain of the EF-α protein from S. solfataricus. Using molecular dynamics simulation at the microsecond time-scale we show that both proteins host water molecules in internal cavities and that these molecules exchange with the external solution in the nanosecond time scale. The hydration free energy of these sites evaluated via extensive calculations is found to be favourable for both systems, with the hyperthermophilic protein offering a slightly more favourable environment to host water molecules. We estimate that under ambient conditions, the free energy gain due to internal hydration is about 1.3 kcal/mol in favor of the hyperthermophilic variant. However, we also find that at the high working temperature of the hyperthermophile, the cavities are rather dehydrated, meaning that at extreme conditions other molecular factors secure the stability of the protein. Interestingly, we detect a clear correlation between the hydration of internal cavities and the protein conformational landscape. The emerging picture is that internal hydration is an effective observable to probe the conformational landscape of proteins. In the specific context of our investigation, the analysis confirms that the hyperthermophilic G-domain is characterized by multiple states and it has a more flexible structure than its mesophilic homologue.

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“…In future, it would be very interesting to compare simulation results with experimental data for other model peptides such as β-hairpins, should such data become available. Experimental kinetics results for the pressure-dependence of helix 15 and protein folding 34,35 kinetics are also a rich source of information for future detailed comparison with molecular simulation. …”

Section: Discussionmentioning

confidence: 99%

Role of solvation in pressure-induced helix stabilization

Best

Miller

Mittal

2014

The Journal of Chemical Physics

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In contrast to the well-known destabilization of globular proteins by high pressure, recent work has shown that pressure stabilizes the formation of isolated α-helices. However, all simulations to date have obtained a qualitatively opposite result within the experimental pressure range. We show that using a protein force field (Amber03w) parametrized in conjunction with an accurate water model (TIP4P/2005) recovers the correct pressure-dependence and an overall stability diagram for helix formation similar to that from experiment; on the other hand, we confirm that using TIP3P water results in a very weak pressure destabilization of helices. By carefully analyzing the contributing factors, we show that this is not merely a consequence of different peptide conformations sampled using TIP3P. Rather, there is a critical role for the solvent itself in determining the dependence of total system volume (peptide and solvent) on helix content. Helical peptide structures exclude a smaller volume to water, relative to non-helical structures with both the water models, but the total system volume for helical conformations is higher than non-helical conformations with TIP3P water at low to intermediate pressures, in contrast to TIP4P/2005 water. Our results further emphasize the importance of using an accurate water model to study protein folding under conditions away from standard temperature and pressure.

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Effect of Internal Cavities on Folding Rates and Routes Revealed by Real-Time Pressure-Jump NMR Spectroscopy

Cited by 69 publications

References 53 publications

A hypothesis to reconcile the physical and chemical unfolding of proteins

A hypothesis to reconcile the physical and chemical unfolding of proteins

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

Role of solvation in pressure-induced helix stabilization

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