Measurement of Site-Specific <sup>13</sup>C Spin−Lattice Relaxation in a Crystalline Protein

Lewandowski, Józef; Sein, Julien; Sass, Hans Jürgen; Grzesiek, Stephan; Blackledge, Martin; Emsley, Lyndon

doi:10.1021/ja102744b

Cited by 83 publications

(195 citation statements)

References 26 publications

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“…S5). We probed polyQ side-chain motion via 15 N R 1 , 15 N R 1ρ , 13 C R 1 , and 13 C R 1ρ relaxation measurements, in part enabled by ultrafast MAS (36,37) (Fig. 3 G-I and SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

Hoop

Lin

Kar

et al. 2016

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

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Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.solid-state NMR | Huntington's disease | amyloid disease | protein aggregation | amyloid

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Section: Resultsmentioning

confidence: 99%

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

Hoop

Lin

Kar

et al. 2016

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

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158

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“…For example, for protonated alanine spinning below 20 kHz MAS, the 13 C relaxation rate constants of carbonyl, C α and methyl-13 C are essentially the same (about 1.5 to 2.5 s −1 ) [102]. When increasing the MAS frequency to 60 kHz the respective rates differ between each other by almost a factor 50, showing that spin diffusion is strongly reduced (although the rates still may not necessarily reflect the actual relaxation rate constants).…”

Section: Longitudinal Relaxation Parameters In Solid-state Nmrmentioning

confidence: 98%

“…When increasing the MAS frequency to 60 kHz the respective rates differ between each other by almost a factor 50, showing that spin diffusion is strongly reduced (although the rates still may not necessarily reflect the actual relaxation rate constants). It was proposed that ~60 kHz MAS frequency may suffice to provide site-specific R 1 relaxation rate constants [102]. However, a recent study challenged this view, and showed that for 13 C α sites fast MAS alone is insufficient, and even deuteration and >50 kHz MAS (in a fully 13 Clabeled protein) is insufficient to suppress the effect of spin diffusion on 13 C α R 1 relaxationrate constants.…”

Section: Longitudinal Relaxation Parameters In Solid-state Nmrmentioning

confidence: 99%

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

Schanda

Ernst

2016

Progress in Nuclear Magnetic Resonance Spectroscopy

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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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“…1 103 resonances were resolved in the CP-HSQC, indicating that the CP-HSQC experiment is an efficient detection block for 15 N or 13 C relaxation measurements. Thus, the 15 N-1 H CP-HSQC dipolar correlation module was combined with a 15 N inversion-recovery block (30,31), 15 N spin-lock (32,33), or additional 13 C-15 N specific transfers (34) and 13 C inversion-recovery (35) into a new set of extremely sensitive and resolved experiments. Pulse schemes are shown in Fig.…”

mentioning

confidence: 99%

“…This is particularly relevant in the site-specific measurement of the 15 N R 1ρ and 13 CO R 1 , which are both particularly susceptible to residual effects. It has been shown that these parameters can be accurately measured under MAS by using MAS frequencies >40 kHz and a spin-lock nutation frequency >10 kHz (31,33,35).…”

mentioning

confidence: 99%

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

Knight

Pell

Bertini

et al. 2012

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

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We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with 1 H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of 15 N and 13 C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu þ (diamagnetic) or Cu 2þ (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to 1 H-1 H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.paramagnetism | nuclear relaxation rates | copper | microcrystal S tructure determination of proteins plays a central role in understanding key events in biology. Although the structure of many proteins can be obtained from single-crystal X-ray diffraction, or by solution-state nuclear magnetic resonance (NMR) spectroscopy, there is nevertheless a range of important substrates for which structures cannot be determined today. These include immobile systems lacking long-range order such as protein aggregates, large complexes and membrane-bound systems.Solid-state NMR has the unique potential to study, with atomic resolution, systems of this nature, and spectacular progress has been made in this area over the last decade (1). There are today a small handful of structures obtained by solid-state NMR, from microcrystalline samples to fibrils and membrane-associated systems (2). Additionally, solid-state NMR is uniquely sensitive to site-specific protein dynamics over a broad range of timescales, and a number of demonstration studies have recently appeared for model proteins (3).The use of perdeuterated proteins has very recently opened the way to highly sensitive proton-detected solid-state experiments (4). Despite early proof-of-principle papers (5), this approach only became popular with the realization that amide sites must be only partially reprotonated (typically 10-30% back exchange) (6-8) to yield well-resolved 1 H spectra. This represented a significant compromise in sensitivity to gain resolution, and effectively made the determination of internuclear distances impractical, with few exceptions (9-11). We have recently shown how this problem can be completely overcome by using 100% reprotonation of exchangeable sites, without loss of resolution, if perdeuteration is combined wit...

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Measurement of Site-Specific ¹³C Spin−Lattice Relaxation in a Crystalline Protein

Cited by 83 publications

References 26 publications

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

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

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

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Measurement of Site-Specific 13C Spin−Lattice Relaxation in a Crystalline Protein

Cited by 83 publications

References 26 publications

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

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

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

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Measurement of Site-Specific ¹³C Spin−Lattice Relaxation in a Crystalline Protein