Thermodynamics of the Trp‐cage miniprotein unfolding in urea

Wafer, Lucas N.; Streicher, Werner; Makhatadze, George I.

doi:10.1002/prot.22681

Cited by 37 publications

(57 citation statements)

References 38 publications

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“…The m-value for Trp-cage in presence of TMAO is much smaller in magnitude than the m-value in presence of urea, which we previously estimated to be 1.36 ± 0.08 kJ/(mol M). 58 …”

Section: Resultsmentioning

confidence: 99%

Molecular Mechanism for the Preferential Exclusion of TMAO from Protein Surfaces

et al. 2012

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Trimethylamine N-oxide (TMAO) is a naturally occurring protecting osmolyte that stabilizes the folded state of proteins and also counteracts the destabilizing effect of urea on protein stability. Experimentally, it has been inferred that TMAO is preferentially excluded from the vicinity of protein surfaces. Here, we combine computer modeling and experimental measurements to gain an understanding of the mechanism of the protecting effect of TMAO on proteins. We have developed an all-atom molecular model for TMAO that captures the exclusion of TMAO from model compounds and protein surfaces, as a consequence of incorporating realistic TMAO-water interactions through osmotic pressure measurements. Osmotic pressure measurements also suggest no significant attraction between urea and TMAO molecules in solution. To obtain an accurate potential for molecular simulations of protein stability in TMAO solutions, we have explored different ways of parameterizing the protein/osmolyte and osmolyte/osmolyte interactions by scaling charges and the strength of Lennard-Jones interactions and carried out equilibrium folding experiments of Trp-cage miniprotein in presence of TMAO to guide the parameterization. Our calculations suggest a general principle for preferential interaction behavior of cosolvents with protein surfaces - preferentially excluded osmolytes have repulsive self-interaction given by osmotic coefficient φ > 1, while denaturants, in addition to having attractive interactions with the proteins, have favorable self-interaction given by osmotic coefficient φ < 1, to enable preferential accumulation in the vicinity of proteins.

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“…The m-value for Trp-cage in presence of TMAO is much smaller in magnitude than the m-value in presence of urea, which we previously estimated to be 1.36 ± 0.08 kJ/(mol M). 58 …”

Section: Resultsmentioning

confidence: 99%

Molecular Mechanism for the Preferential Exclusion of TMAO from Protein Surfaces

et al. 2012

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“…60 It is an ultrafast folder 61 and has been studied with available experimental denaturation data. 62 …”

Section: Resultsmentioning

confidence: 99%

Empirical Optimization of Interactions between Proteins and Chemical Denaturants in Molecular Simulations

Zheng

Borgia

et al. 2015

J. Chem. Theory Comput.

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Chemical denaturants are the most commonly used perturbation applied to study protein stability and folding kinetics, as well as the properties of unfolded polypeptides. We build on recent work balancing the interactions of proteins and water, and accurate models for the solution properties of urea and guanidinium chloride, to develop a combined force field which is able to capture the strength of interactions between proteins and denaturants. We use solubility data for a model tetraglycine peptide in each denaturant to tune the protein-denaturant interaction by a novel simulation methodology. We validate the results against data for more complex sequences: single molecule Förster resonance energy transfer data for a 34-residue fragment of the globular protein CspTm, and photoinduced electron transfer quenching data for the disordered peptides C(AGQ)nW in denaturant solution, as well as the chemical denaturation of the mini-protein Trp cage. The combined force field model should aid our understanding of denaturation mechanisms and the interpretation of experiment.

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“…Recent reports for TC5b have reported comparable Tm values: 43.7 ± 1.8 °C from a combined analysis of calorimetry and CD data (54) and 41 ± 2 °C from an analysis of CD melts over a range of urea denaturant concentrations (51). Recent computational estimates (55) of the thermodynamic stability of TC5b, Tm = 48 °C (55) are also approaching the experimental values rather than yielding fold stabilities far in excess of that observed.…”

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confidence: 89%

Optimal Salt Bridge for Trp-Cage Stabilization

et al. 2011

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Gai and co-workers (Bunagan, M. R. (2006) J. Phys. Chem. B 110, 3759-3763) reported computational design studies suggesting that a D9E mutation would stabilize the Trp-cage. Experimental studies for this mutation were reported in 2008 (Hudaky, P. (2008) Biochemistry 47, 1007-1016); the authors suggested that [D9E]-TC5b presented a more compact, and melting resistant structure due to the “optimal distance between the two sides of the molecule”. Nonetheless, the authors reported essentially the same CD melting temperature, 38±0.3 °C, for TC5b and its [D9E] mutant. In this study, a more stable Trp-cage, DAYAQ WLKDG GPSSG RPPPS, was examined by NMR and CD with the following mutations: [D9E], [D9R,R16E], [R16Orn], [D9E,R16Orn], [R16K], and [D9E,R16K]. Of these, the [D9E]-mutant displayed the smallest acidification induced change in the apparent Tm. In analogy to the prior study, the CD melts of TC10b and its [D9E] mutant were, however, very similar; all of the other mutations were significantly fold destabilizing by all measures. A detailed analysis indicates that the original D9/R16 salt bridge is optimal with regard to fold cooperativity and fold stabilization. Evidence for salt-bridging is also provided for a swapped pair, the [D9R,R16E]-mutant. Model systems reveal that an ionized aspartate at the C-terminus of a helix significantly decreases intrinsic helicity, a requirement for Trp-cage fold stability. The CD evidence which was cited as supporting increased fold stability for the [D9E]-TC5b at higher temperatures appears to be a reflection of increased helix stability in both the folded and unfolded state rather than a more favorable salt bridge. The present study also provides evidence for other Trp-cage stabilizing roles of the R16 sidechain.

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Thermodynamics of the Trp‐cage miniprotein unfolding in urea

Cited by 37 publications

References 38 publications

Molecular Mechanism for the Preferential Exclusion of TMAO from Protein Surfaces

Molecular Mechanism for the Preferential Exclusion of TMAO from Protein Surfaces

Empirical Optimization of Interactions between Proteins and Chemical Denaturants in Molecular Simulations

Optimal Salt Bridge for Trp-Cage Stabilization

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