Dynamic nuclear polarization on a hybridized hammerhead ribozyme: An explorative study of RNA folding and direct DNP with a paramagnetic metal ion cofactor

Daube, Diane; Vogel, Marc; Suess, Beatrix; Corzilius, Björn

doi:10.1016/j.ssnmr.2019.04.005

Cited by 21 publications

(29 citation statements)

References 68 publications

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“…Making use of spin labels in the context of MAS-DNP was thus a natural next step in the development of the technique, and several groups in the last years have investigated the benefits of tagging their biomolecular system with the PA required for hyperpolarization. 57,58 The motivation and objectives of the different approaches were diverse, going from optimization of the sample preparation for best sensitivity 59,60 and biocompatibility, [61][62][63] investigation of the bleaching/quenching effect of the paramagnetic tag [64][65][66] under DNP conditions for its potential use as a structural probe, [67][68][69] selection of a particular protein in a complex environment, 62,63,[70][71][72][73][74][75] to the detection of protein-protein interactions, 76 and the development of highresolution localized techniques through selective DNP. 77…”

Section: Targeted Dnp Versus Conventional Dnpmentioning

confidence: 99%

“…2a). 65,69 Extension of this approach for diamagnetic biomolecules was obtained via chelator ligands compatible with site-directed spin-labeling of cysteines, like DOTA-M or 4MMDPA (see Chemical structure of maleimide derivatized Gd-DOTA and Gd-4MMDPA spin labels, which can react with the thiol group of exposed cysteines, forming either a carbon-sulfur or a disulfide bond, respectively. 68 The metal ion used as PA is highlighted with a red shadowed circle.…”

Section: Endogenous Metal Dnp and Site-directed Spin Labeling Of Metamentioning

confidence: 99%

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Targeted DNP for biomolecular solid-state NMR

et al. 2021

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Section: Targeted Dnp Versus Conventional Dnpmentioning

confidence: 99%

Section: Endogenous Metal Dnp and Site-directed Spin Labeling Of Metamentioning

confidence: 99%

Targeted DNP for biomolecular solid-state NMR

et al. 2021

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“…B. die metabolische Bildgebung in Echtzeit, [10] hochauflçsende NMR und Festkçrper-NMR erheblich vorangebracht. [11,12] Hierbei ist die Hyperpolarisation von Aminosäuren (ASs) und Peptiden besonders interessant, wenn auch noch nicht vollständig erforscht. Als Bausteine von Proteinen spielen ASs eine wesentliche Rolle in der Stoffwechselregulation.…”

Section: Introductionunclassified

Parawasserstoff‐induzierte Polarisation von Aminosäuren

et al. 2021

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Die Kernspinresonanz (NMR) hat sich zu einer universellen Methode für biochemische und biomedizinische Studien, einschließlich Metabolomik, Proteomik und Magnetresonanztomographie (MRT), entwickelt. Durch die Vervielfachung des NMR‐Signals ausgewählter Moleküle hat die Kernspin‐Hyperpolarisation das Anwendungsspektrum von NMR und MRT bedeutsam erweitert (z. B. hyperpolarisierte Festkörper‐NMR und metabolische Bildgebung in vivo). Parawasserstoff (pH2) erlaubt es, Moleküle schnell und kostengünstig zu hyperpolarisieren. In diesem Gebiet wurden in den letzten zehn Jahren umfangreiche Fortschritte erzielt, einschließlich der Synthese neuer Tracer, Katalysatoren und Methoden für die Polarisationsübertragung. Das Portfolio an hyperpolarisierten Molekülen umfasst nunmehr Aminosäuren, die für viele Anwendungen von großem Interesse sind. In diesem Beitrag beleuchten wir die aktuelle Literatur zur Hyperpolarisation von Aminosäuren und Peptiden.

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“…However, Corzilius and coworkers have introduced recently the integration of 1 H- 13 C heteronuclear Overhauser effect (hNOE), which has been used for a long time in solution-state NMR to DNP enhanced ssNMR. 207,208 Based on Solomon equation in steady-state solution 134 the mechanism of hNOE can be described as a cross relaxation (CR) process as follows:…”

Section: Introductionmentioning

confidence: 99%

“…The CR polarization transfer pathway results in a negative enhancement, which is distinguishable from the enhanced 13 C signal, derived from conventionally used cross polarization pathway and becomes even more efficient in the presence of paramagnetic nuclei such as Gd 3+ . 207 The use of dynamics-derived CR in the DNP hyperpolarization transfer pathway, called "specific cross-relaxation enhancement by active motions under DNP" (SCREAM-DNP) 15 , has been demonstrated in several biological systems, including Interleukin-1 β 207 , RNA aptamer 15 , ribozyme 208 , and GPR 176 . Aladin et al applied active methyl groups bearing tetracycline as a ligand to probe its binding and affinity to the RNA aptamer.…”

Section: Introductionmentioning

confidence: 99%

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR

Jakdetchai¹

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KR2 is a light-driven sodium ion pump found in marine flavobacterium Krokinobacter Eikastus. The protein belongs to the microbial rhodopsin family, which is characterized by seven transmembrane helices and a retinal cofactor covalently bound to a conserved lysine residue through a Schiff base linkage. Specific features of KR2 and other sodium pumping rhodopsins are the NDQ motif, the N-terminal helix capping the protein at the extracellular side, and the sodium ion bound at the protomer interface in the pentameric structure. The ability to pump sodium ions was a surprising discovery since the positive charge at the Schiff base was long thought to hinder the transport of non-proton cations and the Grotthuss mechanism could not be applied to explain the Na+ transport. The photocycle of KR2 revealed by flashed photolysis and ultrafast femtosecond absorption spectroscopy consists of consecutive intermediates, named K, L, M, and O. Here, DNP-enhanced ssNMR was used to analyze various aspects of these intermediate states. The K/L-state can be generated and trapped by in-situ illumination inside the magnet at 110 K. The trapping of L-state together with the K-state at this temperature is unexpected as this usually leads to the trapping of only K-state in bacteriorhodopsin (BR), proteorhodopsin (PR), and channelrhodopsin 2 (ChR2). This observation suggests a lower energy barrier between K- and L-state in KR2. For the O-state, the intermediate was generated by illuminating outside the magnet, followed by rapid freezing in liquid nitrogen and transfer to the magnet. Based on these procedures, the retinal conformation, and the electrostatic environment at the Schiff base in KR2 dark, K-, L- and O-intermediates were probed using 13C-labeled retinals bound to 15N-labeled KR2 by both 1D and 2D magic angle spinning (MAS) NMR experiments. The obtained data show an all-trans retinal conformation with the distortion of 150° at H-C14-C15-H in the dark state whereas the retinal has a 13-cis, 15-anti conformation in the K- and L-state after light activation. Differences between K- and L-intermediates were observed. The retinal chemical shifts of the K-state show a large deviation from the model compound behavior between the middle and end part of the polyene chain. In the L-state, these differences are much less pronounced. These observations indicate that the light energy stored in the K-state dissipates into the protein in the subsequent photointermediate states. Furthermore, an additional shielding observed for C14 in L-state indicates the slight rotation toward a more compact 13-cis, 15-syn conformation. The distortion of the H-C14-C15-H angle in the L-state (136°) is larger than in the dark state. This twist of the retinal in the L-state would play an important role in lowering the pKa of the Schiff base, which is a prerequisite for the proton transfer from the Schiff base to the proton acceptor (D116). The electrostatic environments at the Schiff base in K- and L-states cause a de-shielding of the 15N nitrogen compared to the dark state. This indicates a stepwise stronger interaction with the counterion as the Schiff base proton moves away from the Schiff base and comes closer to the D116 in the transition from K- to L-state and approaches the proton transfer step during the M-state formation. In the O-state, the retinal was found to be in the all-trans conformation but differed to the dark state in the C13, C20, and Schiff base nitrogen chemical shifts. The largest effect (9 ppm) was observed for the Schiff base nitrogen, which could be explained by the effect of the positive charge of bound Na+ near the Schiff base in the O-state, coordinated by N112 and D116 as observed in the O-state crystal structure in the pentameric form. The structural change at the opsin followed the retinal isomerization and the energy transfer from the chromophore to the surrounding were also investigated in this thesis using various amino acids labeling schemes. Moreover, 1H-13C hNOE in combination with CE-DNP was applied to probe the dynamics of retinylidene methyl groups and 23Na MAS NMR was employed to detect the bound sodium ion at the protomer interface in KR2 dark state.

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Dynamic nuclear polarization on a hybridized hammerhead ribozyme: An explorative study of RNA folding and direct DNP with a paramagnetic metal ion cofactor

Cited by 21 publications

References 68 publications

Targeted DNP for biomolecular solid-state NMR

Targeted DNP for biomolecular solid-state NMR

Parawasserstoff‐induzierte Polarisation von Aminosäuren

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR

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