Multidimensional 2H-Based NMR Methods for Resonance Assignment, Structure Determination, and The Study of Protein Dynamics

Gardner, Kevin H.; Kay, Lewis E.

doi:10.1007/0-306-47083-7_2

Cited by 4 publications

(4 citation statements)

References 161 publications

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“…Obtaining an enhancement in signal-to-noise ratio of the same magnitude as the carbonyl groups in the hyperpolarized spectrum, but by conventional NMR, would require us to average 5 × 10 5 transients. The resonances from aromatic amino acid side chains show stronger attenuation, which is consistent with their shorter relaxation time due to lower levels of deuteration . It is also to be noted that the enhancements obtained from proteins hyperpolarized in native and denaturing conditions were similar over the entire spectrum.…”

Section: Resultssupporting

confidence: 60%

Solution NMR of Polypeptides Hyperpolarized by Dynamic Nuclear Polarization

et al. 2011

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Hyperpolarization of nuclear spins through techniques such as dynamic nuclear polarization (DNP) can greatly increase the signal-to-noise ratio in NMR measurements, thus eliminating the need for signal averaging. This enables the study of many dynamic processes which would otherwise not be amenable to study by NMR spectroscopy. A report of solid- to liquid-state DNP of a short peptide, bacitracin A, as well as of a full-length protein, L23, is presented here. The polypeptides are hyperpolarized at low temperature and dissolved for NMR signal acquisition in the liquid state in mixtures of organic solvent and water. Signal enhancements of 300-2000 are obtained in partially deuterated polypeptide when hyperpolarized on (13)C and of 30-180 when hyperpolarized on (1)H. A simulated spectrum is used to identify different resonances in the hyperpolarized (13)C spectra, and the relation between observed signal enhancement for various groups in the protein and relaxation parameters measured from the hyperpolarized samples is discussed. Thus far, solid- to liquid-state DNP has been used in conjunction with small molecules. The results presented here, however, demonstrate the feasibility of hyperpolarizing larger proteins, with potential applications toward the study of protein folding or macromolecular interactions.

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Section: Resultssupporting

confidence: 60%

Solution NMR of Polypeptides Hyperpolarized by Dynamic Nuclear Polarization

et al. 2011

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“…Another option is the side chain dynamics of solvent-exposed, polar, and charged groups. NMR studies of side chain dynamics indicate a wide range of time scales from picoseconds to nanoseconds. , While the presence of such processes in the MHz/GHz region is undoubted, it is difficult to assess the magnitude of the resulting dipole moment fluctuations. There are, however, hints from MD simulations that such processes indeed play a role in dielectric relaxation. − In particular, Simonson and Perahia 10b observed dipolar fluctuations of polar groups of cytochrome c to contribute to the dielectric spectrum at 100−800 ps.…”

Section: Spectral Assignmentmentioning

confidence: 99%

What Can Really Be Learned from Dielectric Spectroscopy of Protein Solutions? A Case Study of Ribonuclease A

2004

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We report on a dielectric relaxation study of aqueous solutions of ribonuclease A at 298.15 K as a function of protein concentration between 0.5 and 6 wt % in the MHz/GHz frequency range. The spectra can be decomposed into five modes of Debye type diffusive behavior. In agreement with the standard interpretation, we assign the two dominant modes at low and high frequency (β-relaxation and γ-relaxation, respectively) to protein tumbling and bulk water relaxation. We observe three further modes (δ1−δ3) between β- and γ-relaxation, in contrast to a bimodal δ-dispersion frequently reported. We attribute the high frequency part (δ3) near 40 ps to hydration water reorientation, which, in the notion of other authors, corresponds to “loosely bound water”. We argue that the existence of “tightly bound” water, often deduced from the low frequency part in the nanosecond regime (δ1), is inconsistent with a highly mobile hydration layer observed by NMR techniques and molecular dynamics (MD) simulations. On the same grounds, we reject hydration water−bulk water exchange as a mechanism for δ-dispersion. In accordance with MD simulations, we assume that protein−water cross-correlations drive the nanosecond (δ1) process. We also discuss the role of intraprotein motions, which may contribute near 500 MHz (δ2). We discuss the meaning of the hydrodynamic radius and of the hydration numbers in light of the high mobility of hydration waters. We show that because of protein−protein interactions, the effective dipole moment of the protein decreases with increasing protein concentration.

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“…Finally, cells are transferred to a medium with 100% D 2 O for induction of protein synthesis. This method results in deuteration levels in proteins that are >99% when d 7 -glucose is used as the carbon source . This method is optimized for both 2 H, 15 N and 2 H, 15 N, 13 C labeling.…”

Section: Sample Preparation (Labeling Strategies)mentioning

confidence: 99%

“…This method results in deuteration levels in proteins that are >99% when d 7 -glucose is used as the carbon source. 119 This method is optimized for both 2 H, 15 N and 2 H, 15 N, 13 C labeling.…”

Section: Perdeuterationmentioning

confidence: 99%

Solid-State NMR: Methods for Biological Solids

et al. 2022

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In the last two decades, solid-state nuclear magnetic resonance (ssNMR) spectroscopy has transformed from a spectroscopic technique investigating small molecules and industrial polymers to a potent tool decrypting structure and underlying dynamics of complex biological systems, such as membrane proteins, fibrils, and assemblies, in near-physiological environments and temperatures. This transformation can be ascribed to improvements in hardware design, sample preparation, pulsed methods, isotope labeling strategies, resolution, and sensitivity. The fundamental engagement between nuclear spins and radio-frequency pulses in the presence of a strong static magnetic field is identical between solution and ssNMR, but the experimental procedures vastly differ because of the absence of molecular tumbling in solids. This review discusses routinely employed state-of-the-art static and MAS pulsed NMR methods relevant for biological samples with rotational correlation times exceeding 100’s of nanoseconds. Recent developments in signal filtering approaches, proton methodologies, and multiple acquisition techniques to boost sensitivity and speed up data acquisition at fast MAS are also discussed. Several examples of protein structures (globular, membrane, fibrils, and assemblies) solved with ssNMR spectroscopy have been considered. We also discuss integrated approaches to structurally characterize challenging biological systems and some newly emanating subdisciplines in ssNMR spectroscopy.

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Multidimensional 2H-Based NMR Methods for Resonance Assignment, Structure Determination, and The Study of Protein Dynamics

Cited by 4 publications

References 161 publications

Solution NMR of Polypeptides Hyperpolarized by Dynamic Nuclear Polarization

Solution NMR of Polypeptides Hyperpolarized by Dynamic Nuclear Polarization

What Can Really Be Learned from Dielectric Spectroscopy of Protein Solutions? A Case Study of Ribonuclease A

Solid-State NMR: Methods for Biological Solids

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