Elimination of the C-cap in ubiquitin—structure, dynamics and thermodynamic consequences

Ermolenko, Dmitri N.; Dangi, Bindi; Gvritishvili, Anzor; Gronenborn, Angela M.; Makhatadze, George I.

doi:10.1016/j.bpc.2006.03.017

Cited by 8 publications

(7 citation statements)

References 66 publications

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“…More recently, residues around Gly53 have been implicated in a conformational switch that involves a concerted change at residues 51–54, , in excellent agreement with the structures of minor state 3. Structures similar to minor state 1 have been observed in experiments, both at high pressure and in a mutant in which the helix C-cap has been removed, and recent solid-state NMR data also suggested structural heterogeneity in the same region as observed in minor state 1. Finally, we note that reversible transitions to minor states 1 and 3 were also observed in a recently published simulation of ubiquitin performed with a slightly different force field …”

Section: Resultssupporting

confidence: 57%

Picosecond to Millisecond Structural Dynamics in Human Ubiquitin

et al. 2016

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Human ubiquitin has been extensively characterized using a variety of experimental and computational methods and has become an important model for studying protein dynamics. Nevertheless, it has proven difficult to characterize the microsecond time scale dynamics of this protein with atomistic resolution. Here we use an unbiased computer simulation to describe the structural dynamics of ubiquitin on the picosecond to millisecond time scale. In the simulation, ubiquitin interconverts between a small number of distinct states on the microsecond to millisecond time scale. We find that the conformations visited by free ubiquitin in solution are very similar to those found various crystal structures of ubiquitin in complex with other proteins, a finding in line with previous experimental studies. We also observe weak but statistically significant correlated motions throughout the protein, including long-range concerted movement across the entire β sheet, consistent with recent experimental observations. We expect that the detailed atomistic description of ubiquitin dynamics provided by this unbiased simulation may be useful in interpreting current and future experiments on this protein.

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Section: Resultssupporting

confidence: 57%

Picosecond to Millisecond Structural Dynamics in Human Ubiquitin

et al. 2016

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“…In the remaining 10% of the structures, the α helix is partially frayed at its C terminus; similar unfolding of the C-terminal end of the helix has been observed in experiment both at high pressure (76) and in a mutant in which the helix C-cap has been removed (77), suggesting that ubiquitin may undergo such motion, although the force field appears to overestimate its population. We conclude that the force field provides a reasonable description of the folded state of ubiquitin, although it seems to slightly underestimate the stability of the α helix in the context of the folded state.…”

Section: Resultsmentioning

confidence: 82%

Atomic-level description of ubiquitin folding

Piana

Lindorff‐Larsen

Shaw

2013

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

300

350

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Equilibrium molecular dynamics simulations, in which proteins spontaneously and repeatedly fold and unfold, have recently been used to help elucidate the mechanistic principles that underlie the folding of fast-folding proteins. The extent to which the conclusions drawn from the analysis of such proteins, which fold on the microsecond timescale, apply to the millisecond or slower folding of naturally occurring proteins is, however, unclear. As a first attempt to address this outstanding issue, we examine here the folding of ubiquitin, a 76-residue-long protein found in all eukaryotes that is known experimentally to fold on a millisecond timescale. Ubiquitin folding has been the subject of many experimental studies, but its slow folding rate has made it difficult to observe and characterize the folding process through all-atom molecular dynamics simulations. Here we determine the mechanism, thermodynamics, and kinetics of ubiquitin folding through equilibrium atomistic simulations. The picture emerging from the simulations is in agreement with a view of ubiquitin folding suggested from previous experiments. Our findings related to the folding of ubiquitin are also consistent, for the most part, with the folding principles derived from the simulation of fast-folding proteins, suggesting that these principles may be applicable to a wider range of proteins. CHARMM22* | energy landscape | enthalpy | phi-value analysis | prefactor U nderstanding the principles that govern protein folding, the self-assembly process that leads from an unstructured polypeptide chain to a fully functional protein, has been one of the major challenges in the area of physical biochemistry over the last 50 years. Naturally occurring proteins typically fold on timescales ranging from milliseconds to minutes, but as our understanding of the principles of protein folding has improved, a number of fastfolding proteins that fold on the microsecond timescale have been engineered and characterized (1-12). The design of such fastfolding proteins was in part intended to narrow the gap between the timescales of protein folding and the timescale accessible to physics-based atomistic molecular dynamics (MD) simulations, thus enabling fruitful combinations of experiments and simulations to study the mechanisms of folding (13-18). We recently studied 12 fast-folding proteins using equilibrium MD simulations, for example, with the aim of elucidating general principles underlying the folding of these proteins (19). The folding of these proteins was found to be a relatively sequential process that follows a few paths, in which the order of formation of native structure is correlated with relative structural stability in the unfolded state. The extent to which these observations pertained to fast-folding proteins only or to protein folding more generally, however, was unclear. Here we address this question by applying the same methodology and physics-based force field used in our previous investigation of fast-folding proteins to the study of ubiquitin, a nat...

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“…Such substitutions can sometimes lead to dramatic structural changes (33). These changes in the structure of the native-state protein could, in turn, alter stability in a way that could compromise experimental proofs of enhancing protein stability solely by optimization of charge-charge interactions on the protein surface.…”

Section: Resultsmentioning

confidence: 99%

Rational stabilization of enzymes by computational redesign of surface charge–charge interactions

Gribenko

Patel

Liu

et al. 2009

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

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208

177

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Here, we report the application of a computational approach that allows the rational design of enzymes with enhanced thermostability while retaining full enzymatic activity. The approach is based on the optimization of the energy of charge-charge interactions on the protein surface. We experimentally tested the validity of the approach on 2 human enzymes, acylphosphatase (AcPh) and Cdc42 GTPase, that differ in size (98 vs. 198-aa residues, respectively) and tertiary structure. We show that the designed proteins are significantly more stable than the corresponding WT proteins. The increase in stability is not accompanied by significant changes in structure, oligomerization state, or, most importantly, activity of the designed AcPh or Cdc42. This success of the design methodology suggests that it can be universally applied to other enzymes, on its own or in combination with the other strategies based on redesign of the interactions in the protein core.Until man duplicates a blade of grass, nature will laugh at his so-called scientific knowledge.Thomas Edison computational design ͉ protein engineering ͉ protein stability R ational engineering of proteins to enhance stability and yet retain their enzymatic activity is well motivated (1). One motivation is the practical significance of expanding the use of enzymes in many areas of the modern world, including protein therapeutics, enzymes for food industry, diagnostics, and other areas of industrial biotechnology. Another motivation is validation of the existing scientific knowledge. In this case, predictions made by the existing models for protein stability are subjected to thorough experiments, testing their applicability to protein design. In this paper, we present the results of rational design of enzymes with enhanced stability and unchanged enzymatic activity. This approach has 2 major differences from previously described successful protein design methods (2-5): (i) it concentrates only on the residues on the protein surface, and (ii) it optimizes just one type of interactions, namely, charge-charge interactions on the protein surface (6-15).One of the most important aspects of engineering proteins with enhanced stability, retaining the enzymatic activity, is often forgotten. However, for all of these design efforts to be practically useful, it is important that the engineered proteins retain their biological and enzymatic activity. This issue is particularly important when enhanced protein stability is achieved by redesigning the charge-charge interactions on the protein surface. Such redesign can lead to several potentially detrimental effects on the activity: (i) it can affect the electrostatic potential in the active center, thus reducing or even abolishing the activity; (ii) it can affect substrate/product binding and again reduce or abolish the enzymatic activity; or (iii) it can have effects on the kinetics of substrate binding and, thus, lower the activity via reduced rates of electrostatic steering (3,(16)(17)(18)(19)(20)(21)(22).To this end, it is imp...

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Elimination of the C-cap in ubiquitin—structure, dynamics and thermodynamic consequences

Cited by 8 publications

References 66 publications

Picosecond to Millisecond Structural Dynamics in Human Ubiquitin

Picosecond to Millisecond Structural Dynamics in Human Ubiquitin

Atomic-level description of ubiquitin folding

Rational stabilization of enzymes by computational redesign of surface charge–charge interactions

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