[7] Mutatational analysis of protein folding mechanisms

Jennings, Patricia A.; Saalau-Bethell, S.; Finn, Bryan E.; Chen, Xiaowu; Matthews, C. Robert

doi:10.1016/0076-6879(91)02009-x

Cited by 25 publications

(16 citation statements)

References 18 publications

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“…A quantitative relationship between protein folding kinetics and the thermodynamic stability of the native state can be obtained with linear free energy relationships [70,[108][109][110]. In the past these relations have been applied to the interpretation of data from site-directed mutagenesis experiments [111][112][113][114]. They are also the mainstay of the analysis of many other biochemical reactions [115][116][117].…”

Section: Energy Landscape Analysis Of Folding Simulationsmentioning

confidence: 99%

Funnels, pathways, and the energy landscape of protein folding: A synthesis

et al. 1995

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The understanding, and even the description of protein folding is impeded by the complexity of the process. Much of this complexity can be described and understood by taking a statistical approach to the energetics of protein conformation, that is, to the energy landscape. The statistical energy landscape approach explains when and why unique behaviors, such as specific folding pathways, occur in some proteins and more generally explains the distinction between folding processes common to all sequences and those peculiar to individual sequences. This approach also gives new, quantitative insights into the interpretation of experiments and simulations of protein folding thermodynamics and kinetics. Specifically, the picture provides simple explanations for folding as a two‐state first‐order phase transition, for the origin of metastable collapsed unfolded states and for the curved Arrhenius plots observed in both laboratory experiments and discrete lattice simulations. The relation of these quantitative ideas to folding pathways, to uniexponential vs. multiexponential behavior in protein folding experiments and to the effect of mutations on folding is also discussed. The success of energy landscape ideas in protein structure prediction is also described. The use of the energy landscape approach for analyzing data is illustrated with a quantitative analysis of some recent simulations, and a qualitative analysis of experiments on the folding of three proteins. The work unifies several previously proposed ideas concerning the mechanism protein folding and delimits the regions of validity of these ideas under different thermodynamic conditions. © 1995 Wiley‐Liss, Inc.

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Section: Energy Landscape Analysis Of Folding Simulationsmentioning

confidence: 99%

Funnels, pathways, and the energy landscape of protein folding: A synthesis

et al. 1995

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“…It was proposed that the accuracy of f f -values can be improved by measuring the effect of two mutations at each probed position. 38 However, if all possible groups of three variants in our reference data sets are used to calculate the corresponding f f -values by a global fit of the rate and equilibrium constants, the calculated f f -values still converge to the true values only if the two variants with the largest difference in stability have lDDG 0 l . 7 kJ/mol (data not shown).…”

Section: Origin Of Unusual F F -Values In Protein Foldingmentioning

confidence: 99%

Origin of Unusual φ-values in Protein Folding: Evidence Against Specific Nucleation Sites

Sánchez

Kiefhaber

2003

Journal of Molecular Biology

146

167

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“…3A) marks the resolution limit for rates measured in our force assay. The dependence of the combined rate (λ 1 ) on force yields a chevronlike plot well known from bulk kinetic protein-folding experiments (38) (Fig. 3A) (HP35stab: red circles, HP35WT: blue circles in Fig.…”

Section: Significancementioning

confidence: 99%

Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy

Žoldák

Stigler

Pelz

et al. 2013

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

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In this study we expand the accessible dynamic range of singlemolecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 μs thus approaching the timescales accessible for all atom molecular dynamics simulations. P rotein folding is a spontaneous process where a linear polypeptide chain acquires a functional and highly complex 3D structure. Conformational folding can occur on timescales ranging from hours down to microseconds (1, 2). The study of small fast-folding proteins has provided key information for understanding fundamental aspects of protein-folding mechanisms. Recently, it has even become possible to simulate folding trajectories of small proteins in full atomic detail using molecular dynamics simulations up to the millisecond time range (3, 4). However, direct time-resolved experimental measurements of such ultrafast processes have largely been limited to a few ensemble methods like temperature-jump, continuous-flow experiments, and triplet-lifetime measurements.The C-terminal subdomain of the actin-binding protein villin (villin headpiece, HP35) has been used as a model system in a number of both experimental and simulation studies of protein folding at the speed limit (5-19). The folding kinetics of HP35 has been studied extensively mostly by T-jump and by tripletlifetime experiments (6-8, 11, 12, 16). Folding of the wild-type HP35 at 30°C occurs at 348 × 10 3 s −1 , and unfolding at 7.4 × 10 3 s −1 , which in combination yields a free energy of folding of ∼4 k B T (6). Before crossing the major folding barrier, HP35 undergoes a rapid local rearrangement on the nanosecond timescale, which results in stable contacts of tertiary structure. Triplettriplet-energy transfer (TTET) measurements have reported a native-state heterogeneity and an additional high-energy intermediate on the native-state side of the major unfolding barrier (16). A detailed structural picture of the locking-unlocking reaction of the native states as observed in TTET data were given by recent all-atom molecular dynamics simulations (5). Along this line, a variety of native conformations was found consiste...

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[7] Mutatational analysis of protein folding mechanisms

Cited by 25 publications

References 18 publications

Funnels, pathways, and the energy landscape of protein folding: A synthesis

Funnels, pathways, and the energy landscape of protein folding: A synthesis

Origin of Unusual φ-values in Protein Folding: Evidence Against Specific Nucleation Sites

Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy

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