Time Scales of Slow Motions in Ubiquitin Explored by Heteronuclear Double Resonance

Salvi, Nicola; Ulzega, Simone; Ferrage, Fabien; Bodenhausen, Geoffrey

doi:10.1021/ja210238g

Cited by 33 publications

(69 citation statements)

References 53 publications

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“…In addition, a previous study using mutagenesis and extreme pH values suggested that rotation of this peptide bond may explain the microsecond motion observed in two nearby residues (19). Microsecond motions in this region have also been observed with heteronuclear doubleresonance (20) and solid-state RD (21) experiments. Furthermore, in the 100-ns simulations used for modeling the RD fit MD mode, peptide flipping was the structural feature with the slowest time scale, with flips occurring in 21 of 170 independent simulations (Fig.…”

Section: Resultsmentioning

confidence: 73%

Allosteric switch regulates protein–protein binding through collective motion

Smith

Ban

Pratihar

et al. 2016

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

105

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Many biological processes depend on allosteric communication between different parts of a protein, but the role of internal protein motion in propagating signals through the structure remains largely unknown. Through an experimental and computational analysis of the ground state dynamics in ubiquitin, we identify a collective global motion that is specifically linked to a conformational switch distant from the binding interface. This allosteric coupling is also present in crystal structures and is found to facilitate multispecificity, particularly binding to the ubiquitin-specific protease (USP) family of deubiquitinases. The collective motion that enables this allosteric communication does not affect binding through localized changes but, instead, depends on expansion and contraction of the entire protein domain. The characterization of these collective motions represents a promising avenue for finding and manipulating allosteric networks.allostery | protein dynamics | concerted motion | relaxation dispersion | nuclear magnetic resonance I ntermolecular interactions are one of the key mechanisms by which proteins mediate their biological functions. For many proteins, these interactions are enhanced or suppressed by allosteric networks that couple distant regions together (1). The mechanisms by which these networks function are just starting to be understood (2-4), and many of the important details have yet to be uncovered. In particular, the role of intrinsic protein motion and kinetics remains particularly poorly characterized. A number of structural ensembles representing ubiquitin motion have been recently proposed (5-9). Additionally, it has been suggested that through motion at the binding interface, its free state visits the same conformations found in complex with its many binding partners (5, 10). However, it remains an unanswered question if the dynamics that enable this multispecificity are only clustered around the canonical binding interface or whether this motion is allosterically coupled to the rest of the protein, especially given the presence of motion at distal sites (11). ResultsTo answer this question and to provide a detailed structural picture of the underlying mechanism, we applied recently developed high-power relaxation dispersion (RD) experiments (12, 13) to both the backbone amide proton ( 1 H N ) and nitrogen ( 15 N) nuclei of ubiquitin. This survey yielded a nearly twofold increase in the number of nuclei where RD had been previously observed (11)(12)(13)(14) (from 17 to 31; Fig. 1A and Fig. S1). When fit individually, the full set of backbone and side-chain nuclei shows a consistent time scale of motion [exchange lifetime (τ ex ) = 55 μs; Fig. 1B]. Furthermore, the nuclei showing exchange are spread throughout the structure (Fig. 1C). Put together, these data suggest that the motions are not independent but share a common molecular mechanism.To determine whether the RD data could be modeled using a single collective motion, we developed a computational method to take a set of molecu...

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Section: Resultsmentioning

confidence: 73%

Allosteric switch regulates protein–protein binding through collective motion

Smith

Ban

Pratihar

et al. 2016

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

105

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“…The picture emerging from these data shows that there is an exchange process in a well-defined region, enclosing the loop of residues D52-T55 (which forms a so-called type-II β-turn structure in microcrystals), as well as the neighboring helix. The exchange process is thought to correspond to a flip of peptide plane D52/G53, and some rearrangement of hydrogen bonds and side chain orientations, as discussed before [44,190,191]. The fitted exchange rate is about 3000 s −1 , and the relative populations are approximately 90:10.…”

Section: Selected Examples Of Recent Applications: How Do Protein Motmentioning

confidence: 83%

Studying dynamics by magic-angle spinning solid-state NMR spectroscopy: Principles and applications to biomolecules

Schanda

Ernst

2016

Progress in Nuclear Magnetic Resonance Spectroscopy

198

295

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Magic-angle spinning solid-state NMR spectroscopy is an important technique to study molecular structure, dynamics and interactions, and is rapidly gaining importance in biomolecular sciences. Here we provide an overview of experimental approaches to study molecular dynamics by MAS solid-state NMR, with an emphasis on the underlying theoretical concepts and differences of MAS solid-state NMR compared to solution-state NMR. The theoretical foundations of nuclear spin relaxation are revisited, focusing on the particularities of spin relaxation in solid samples under magic-angle spinning. We discuss the range of validity of Redfield theory, as well as the inherent multi-exponential behavior of relaxation in solids. Experimental challenges for measuring relaxation parameters in MAS solid-state NMR and a few recently proposed relaxation approaches are discussed, which provide information about time scales and amplitudes of motions ranging from picoseconds to milliseconds. We also discuss the theoretical basis and experimental measurements of anisotropic interactions (chemical-shift anisotropies, dipolar and quadrupolar couplings), which give direct information about the amplitude of motions. The potential of combining relaxation data with such measurements of dynamically-averaged anisotropic interactions is discussed. Although the focus of this review is on the theoretical foundations of dynamics studies rather than their application, we close by discussing a small number of recent dynamics studies, where the dynamic properties of proteins in crystals are compared to those in solution.

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“…Using a 15 N-labeled ubiquitin sample at 277 K, we measured relaxation dispersion for residues Ile13, Thr55, and Val70, which have been previously reported to show dispersion [1,17,18]. We used an on-resonance R 1q experiment [15] in which the spin-lock field strength was varied from 1 to 6 kHz allowing for residues that undergo chemical exchange to be modulated solely by the utilized spin-lock field strength (Fig.…”

Section: Validation For Relaxation Dispersion Experimentsmentioning

confidence: 99%

Exceeding the limit of dynamics studies on biomolecules using high spin-lock field strengths with a cryogenically cooled probehead

Ban

Gossert

Giller

et al. 2012

Journal of Magnetic Resonance

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Internal motions in the microsecond timescale have been proposed to play an active part in a protein's biological function. Nuclear magnetic resonance (NMR) relaxation dispersion is a robust method sensitive to this timescale with atomic resolution. However, due to technical limitations, the observation of motions faster than ∼40 μs for ¹⁵N nuclei was not possible. We show that with a cryogenically cooled NMR probehead, a high spin-lock field strength can be generated that is able to detect motions as fast as 25 μs. We apply this high spin-lock field strength in an NMR experiment used for characterizing dynamical processes. An on-resonance rotating-frame transverse relaxation experiment was implemented that allows for the detection of a 25 μs process from a dispersion curve, and transverse relaxation rates were compared at low and high spin-lock field strengths showing that at high field strengths contributions from chemical exchange with lifetimes up to 25 μs can be removed. Due to the increase in sensitivity towards fast motion, relaxation dispersion for a residue that undergoes smaller chemical shift variations due to dynamics was identified. This technique reduces the previously inaccessible window between the correlation time and the relaxation dispersion window that covers four orders of magnitude by a factor of 2.

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Time Scales of Slow Motions in Ubiquitin Explored by Heteronuclear Double Resonance

Cited by 33 publications

References 53 publications

Allosteric switch regulates protein–protein binding through collective motion

Allosteric switch regulates protein–protein binding through collective motion

Studying dynamics by magic-angle spinning solid-state NMR spectroscopy: Principles and applications to biomolecules

Exceeding the limit of dynamics studies on biomolecules using high spin-lock field strengths with a cryogenically cooled probehead

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