Recognition Dynamics Up to Microseconds Revealed from an RDC-Derived Ubiquitin Ensemble in Solution

Lange, Oliver; Lakomek, Nils‐Alexander; Farès, Christophe; Schröder, Gunnar F.; Walter, Korvin F. A.; Becker, Stefan; Meiler, Jens; Grubmüller, Helmut; Griesinger, Christian; Groot, Bert L. de

doi:10.1126/science.1157092

Cited by 989 publications

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“…As shown in many works, 0.5 to 1 μs-long MD simulations of ubiquitin can reproduce NMR order parameters and residual dipolar couplings very well, indicating good agreement between simulations and experimental observables. 16,69,72,90 Also, such simulations reproduce local and large-scale conformational fluctuations observed in X-rays structures. The latter are easily visualized by projecting the MD trajectory on a two-dimensional plane built from available X-rays structures as shown in a number of recent works.…”

Section: ■ Resultsmentioning

confidence: 75%

“…Logarithmic probability distributions were built by projecting the trajectories on a reference frame, binning them into a grid and counting the number of frames inside each cell of the grid (N i ) to compute −k B T ln(N i /N 0 ), where N 0 corresponds to the most populated bin thus setting the offset. The method is basically the same that Lange et al introduced, 72 plus the calculation of logarithmic densities that Long et al 90 employed to visualize the projected trajectories.…”

Section: ■ Introductionmentioning

confidence: 99%

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Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics

et al. 2014

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We present herein a thorough description of the effects of high glucose concentrations on the diffusion, hydration and internal dynamics of ubiquitin, as predicted from extensive molecular dynamics simulations on several systems described at fully atomistic level. We observe that the protein acts as a seed that speeds up the natural propensity of glucose to cluster at high concentration; the sugar molecules thus aggregate around the protein trapping it inside a dynamic cage. This process extensively dehydrates the protein surface, restricts the motions of the remaining water molecules, and drags the large-scale, collective motions of protein atoms slowing down the rate of exploration of the conformational space despite only a slight dampening of fast, local dynamics. We discuss how these effects could be relevant to the function of sugars as preservation agents in biological materials, and how crowding by small sticky molecules could modulate proteins across different reaction coordinates inside the cellular cytosol. ■ INTRODUCTIONSugars play roles as agents for the preservation of biological material in nature and in biotechnological manufacture, 1−3 most importantly through their capacity to stabilize proteins against cold and hot denaturation, both in solution and in the solid state. 4−7 Other potential effects of sugars on protein properties have been less explored, but changes in activity, dynamics, and regulation can be expected by analogy to the effects known to be caused by high concentrations of other molecules. Whereas most hydrophilic molecules have the capacity to agglomerate and disrupt water structure, sugars and polyols generally have exceptionally large solubilities, which allows them to strongly dehydrate other molecules and to cluster at very high concentrations forming glassy states. 8−11 Because of these special properties, questions about the effects of sugars on protein properties are intimately related to those revolving around the effects of viscosity, molecular crowding, encapsulation and even vitrification on proteins, all meeting at the crossroads between chemistry, biology, medicine, and applications in the food and pharmacological industries. Within all these closely related fields, the effects of high concentrations of small hydrophilic molecules on protein stability and translational and rotational diffusion have been explored, but studies of their effects on protein hydration and on internal protein dynamics are scarce. 4−7,12−16 More specifically to sugars, reports on the structure and dynamics of sugar-only solutions abound, 8,17−23 but works dealing with proteins in sugar solutions are mostly focused on the stability of the proteins and do not pay much attention to other effects related to hydration, diffusion, and internal mobility. 4−7 We recently reported in a preliminary work that concentrated glucose solutions can perturb protein dynamics by restricting exploration of the conformational space, through a mechanism that presumably involves protein−sugar interac...

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Section: ■ Resultsmentioning

confidence: 75%

Section: ■ Introductionmentioning

confidence: 99%

Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics

et al. 2014

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“…Analysis of the DER ensemble provided new biophysical insights, suggesting that while the protein's core is tightly packed, it nevertheless exhibits appreciable fluid-like character. Another ubiquitin ensemble that was constructed by refinement against RDCs and is sensitive to motions on a ps-to-ms time scale is described in some detail in Section 4 (Lange et al 2008). Ensemble approaches have also been used to visualize transient encounter complexes (Bashir et al 2010;Tang et al 2006), quantify enzyme dynamics , describe the solution structures of intrinsically disordered proteins (Jensen et al 2013(Jensen et al , 2014Silvestre-Ryan et al 2013), and probe correlated motions in proteins (Bouvignies et al 2005;Bryn Fenwick et al 2014;Clore & Schwieters, 2004a;Fenwick et al 2011).…”

Section: Ensemble Approaches For Analyzing Protein Dynamicsmentioning

confidence: 99%

Protein dynamics and function from solution state NMR spectroscopy

Kovermann

Rogne

Wolf‐Watz

2016

Quart. Rev. Biophys.

148

122

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Abstract. It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.

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“…Protein-protein interactions and ligand recognition have been shown to occur on a µs time scale. 1 In membrane proteins, the presence of slow motion has been a hurdle for X-ray crystallography, as in the case of the 2 -adrenergic G-protein coupled receptor (GPCR). 2 Also for NMR spectroscopy, intermediate (ns-µs) motion tends to be an obstacle.…”

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confidence: 99%

Assignment of Dynamic Regions in Biological Solids Enabled by Spin-State Selective NMR Experiments

2010

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Biological function largely depends on protein dynamics. Protein-protein interactions and ligand recognition have been shown to occur on a µs time scale. 1 In membrane proteins, the presence of slow motion has been a hurdle for X-ray crystallography, as in the case of the 2 -adrenergic G-protein coupled receptor (GPCR). 2 Also for NMR spectroscopy, intermediate (ns-µs) motion tends to be an obstacle. In many amyloidogenic peptides and proteins, large parts of the primary sequence are often obscured and do not yield detectable resonances in solid-state NMR spectra, presumably due to dynamics. 3 In the voltage gated membrane protein VDAC, 4,5 large segments of the constricting N-terminal R-helix, which seems important for the gating process, could not be assigned due to dynamics. 5 Ironically, those parts of a protein undergoing slow motion are particularly interesting for the understanding of its biology. We and others have demonstrated that sparsely protonated proteins can be successfully employed in Magic Angle Spinning (MAS) solid-state NMR for proton detection, 6 INEPT based resonance assignment strategies, 7 and mapping of solvent accessibility. 8 This approach allowed us to show for the first time that mutual cancellation of dipole-dipole and chemical shift anisotropy (CSA) relaxation pathways 9,10 can give rise to differential transverse relaxation times T 2 also in the solid state. While in the solution state, the effect is due to isotropic tumbling of the molecule, differential relaxation in the solid-state was shown to be due to internal mobility. 11,12 We show here that TROSY 10 type scalar coupling based triple resonance experiments in combination with perdeuteration are beneficial for the detection and assignment of those residues in the protein which undergo intermediate (ns-µs) dynamics. Standard (non-spin-state selective) triple resonance experiments, in contrast, perform extremely poorly in these cases. 7 Conventional MAS solidstate NMR experiments that are based on dipolar transfers are shown not to detect these flexible residues at all. Figures 1A and B depict the first 1 H/ 15 N planes of tripleresonance out and back HNCO experiments, in which 1 H magnetization is transferred to C′ and back. For these experiments, a sample of the chicken R-spectrin SH3 is employed which is 100% perdeuterated at nonexchangeable sites and partially back-exchanged with protons at labile sites. 7 Paramagnetic Relaxation Enhancement (PRE) using Cu-edta was employed for accelerated data acquisition. 13 While the spectrum in Figure 1A was recorded using a standard HNCO pulse scheme, 14 the spectrum in Figure 1B was recorded with spin-state selection, 15 using the pulse schemes shown in Supplementary Figure 1E and F, respectively. Even for the temperature which yields the best signal-to-noise ratio, residues located in mobile parts of the protein (labeled in black) have severely reduced intensities in comparison to residues that are situated in immobile -sheet regions (labeled in gray). For those dynamic residues, sel...

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Recognition Dynamics Up to Microseconds Revealed from an RDC-Derived Ubiquitin Ensemble in Solution

Cited by 989 publications

References 45 publications

Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics

Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics

Protein dynamics and function from solution state NMR spectroscopy

Assignment of Dynamic Regions in Biological Solids Enabled by Spin-State Selective NMR Experiments

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