High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles

Fricke, Pascal; Mance, Deni; Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Baldus, Marc; Lange, Adam

doi:10.1007/s10858-016-0044-y

Cited by 56 publications

(65 citation statements)

References 41 publications

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“…To tackle this problem, judicious sample choices and preparation, as evidenced by the good linewidths observed in highly rigid fibrils [57] or carefully prepared tubular assemblies of the HIV-capsid protein CA [172], will be key to successful DNP implementation. The introduction of multidimensional pulse sequences tailored for DNP samples [15] can also overcome resolution challenges.…”

Section: Future Perspectivesmentioning

confidence: 99%

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

Rogawski

McDermott

2017

Archives of Biochemistry and Biophysics

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Magic angle spinning solid state NMR studies of biological macromolecules [1–3] have enabled exciting studies of membrane proteins [4,5], amyloid fibrils [6], viruses, and large macromolecular assemblies [7]. Dynamic nuclear polarization (DNP) provides a means to enhance detection sensitivity for NMR, particularly for solid state NMR, with many recent biological applications and considerable contemporary efforts towards elaboration and optimization of the DNP experiment. This review explores precedents and innovations in biological DNP experiments, especially highlighting novel chemical biology approaches to introduce the radicals that serve as a source of polarization in DNP experiments.

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Section: Future Perspectivesmentioning

confidence: 99%

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

Rogawski

McDermott

2017

Archives of Biochemistry and Biophysics

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“…49–52 Examples of these systems include bR in its native purple membrane and PI3-SH3. In both of these cases, the signal minimum due to the dynamics of −CH 3 groups occurs at a lower temperature (95 K) and appears to be localized as opposed to the case of APG.…”

Section: Introductionmentioning

confidence: 99%

Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR

Markhasin

Can

et al. 2017

J. Phys. Chem. B

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In DNP MAS NMR experiments at ~80–110 K, the structurally important −13CH3 and −15NH3+ signals in MAS spectra of biological samples disappear due to the interference of the molecular motions with the 1H decoupling. Here we investigate the effect of these dynamic processes on the NMR line shapes and signal intensities in several typical systems: (1) microcrystalline APG, (2) membrane protein bR, (3) amyloid fibrils PI3-SH3, (4) monomeric alanine-CD3, and (5) the protonated and deuterated dipeptide N-Ac-VL over 78–300 K. In APG, the three-site hopping of the Ala-Cβ peak disappears completely at 112 K, concomitant with the attenuation of CP signals from other 13C’s and 15N’s. Similarly, the 15N signal from Ala-NH3+ disappears at ~ 173 K, concurrent with the attenuation in CP experiments of other 15N’s as well as 13C’s. In bR and PI3-SH3, the methyl groups are attenuated at ~95 K, while all other 13C’s remain unaffected. However, both systems exhibit substantial losses of intensity at ~243 K. Finally, with spectra of Ala and N-Ac-VL, we show that it is possible to extract site specific dynamic data from the temperature dependence of the intensity losses. Furthermore, 2H labeling can assist with recovering the spectral intensity. Thus, our study provides insight into the dynamic behavior of biological systems over a wide range of temperatures, and serves as a guide to optimizing the sensitivity and resolution of structural data in low temperature DNP MAS NMR spectra.

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“…A thorough understanding of the DNP transfer mechanisms in conjugated systems will be essential for the selective signal enhancement of proteins of various sizes and shapes and to prevent polarization “leakage” into the cellular background. We are also excited at the prospect of combining our targeted DNP approach with other developments in the field, such as higher magnetic fields, time domain DNP experiments and optical approaches for the comprehensive description of protein structures in cells.…”

Section: Figurementioning

confidence: 99%

Targetable Tetrazine‐Based Dynamic Nuclear Polarization Agents for Biological Systems

2020

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Dynamic nuclear polarization (DNP) has shown great promise as a tool to enhance the nuclear magnetic resonance signals of proteins in the cellular environment. As sensitivity increases, the ability to select and efficiently polarize a specific macromolecule over the cellular background has become desirable. Herein, we address this need and present a tetrazine‐based DNP agent that can be targeted selectively to proteins containing the unnatural amino acid (UAA) norbornene‐lysine. This UAA can be introduced efficiently into the cellular milieu by genetic means. Our approach is bio‐orthogonal and easily adaptable to any protein of interest. We illustrate the scope of our methodology and investigate the DNP transfer mechanisms in several biological systems. Our results shed light on the complex polarization‐transfer pathways in targeted DNP and ultimately pave the way to selective DNP‐enhanced NMR spectroscopy in both bacterial and mammalian cells.

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High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles

Cited by 56 publications

References 41 publications

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR

Targetable Tetrazine‐Based Dynamic Nuclear Polarization Agents for Biological Systems

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