Structure of a Protein–RNA Complex by Solid‐State NMR Spectroscopy

Ahmed, Mumdooh; Marchanka, Alexander; Carlomagno, Teresa

doi:10.1002/ange.201915465

Cited by 7 publications

(15 citation statements)

References 40 publications

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“…The two tags were chosen in order to compare and contrast their applicability for protein solid-state NMR studies. The diamagnetic reference for the PROXYL-M-tagged protein was obtained by reducing PROXYL-M with ascorbic acid after tagging of DnaB [22]. In case of the DOTA-M tag, the paramagnetic protein sample was prepared by using Gd 3+ , whereas the diamagnetic reference state was obtained by using Lu 3+ .…”

Section: Resultsmentioning

confidence: 99%

“…The spin labeling rection with PROXYL-M was performed at 4 °C and pH 7.5 for 18 h using a protein concentration of 110 µM and a 30-fold excess of spin label (per cysteine). The reduction of the PROXYL-M tag was performed in a new buffer (10 mM sodium phosphate, 150 mM NaCl, 30 mM ascorbic acid, 10% glycerine, pH 7.5) [22]. Finally, the solution was desalted with PD-10 column.…”

Section: Tagging Of Dnab With the Reduced Form Of Proxyl-mmentioning

confidence: 99%

“…For instance, the diamagnetic Mg 2+ cofactor in ATPdriven proteins can be exchanged with paramagnetic metal ions [20,21]. Additionally, diamagnetic proteins and nucleic acids [22] can be modified at specific positions with covalently bound paramagnetic tags [12,23], e.g. by using tags that react with the thiol groups of free cysteines which can be site-specifically introduced into proteins by mutagenesis [24][25][26][27].…”

Section: Introductionmentioning

confidence: 99%

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Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

Zehnder

Cadalbert

Yulikov

et al. 2021

Preprint

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Labeling of biomolecules with a paramagnetic probe for nuclear magnetic resonance (NMR) spectroscopy enables determining long-range distance restraints, which are otherwise not accessible by classically used dipolar coupling-based NMR approaches. Distance restraints derived from paramagnetic relaxation enhancements (PREs) can facilitate the structure determination of large proteins and protein complexes. We herein present the site-directed labeling of the large oligomeric bacterial DnaB helicase from Helicobacter pylori with cysteine-reactive maleimide tags carrying either a nitroxide radical or a lanthanide ion. The success of the labeling reaction was followed by quantitative continuous-wave electron paramagnetic resonance (EPR) experiments performed on the nitroxide-labeled protein. PREs were extracted site-specifically from 2D and 3D solid-state NMR spectra. A good agreement with predicted PRE values, derived by computational modeling of nitroxide and Gd3+ tags in the low-resolution DnaB crystal structure, was found. Comparison of experimental PREs and model-predicted spin label-nucleus distances indicated that the size of the "blind sphere" around the paramagnetic center, in which NMR resonances are not detected, is slightly larger for Gd3+ (~14 Å) than for nitroxide (~11 Å) in 13C-detected 2D spectra of DnaB. We also present Gd3+-Gd3+ dipolar electron-electron resonance EPR experiments on DnaB supporting the conclusion that DnaB was present as a hexameric assembly.

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Section: Resultsmentioning

confidence: 99%

Section: Tagging Of Dnab With the Reduced Form Of Proxyl-mmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

Zehnder

Cadalbert

Yulikov

et al. 2021

Preprint

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“… A) Depiction of the L7Ae‐wt+RNA complex (ssNMR structure PDB ID: 6TPH) [29b] . Color code: gray: L7Ae protein (residues I93 and V95 in dark gray, with their methyl carbons in purple and pink, respectively), light brown: RNA backbone, red: A, green: G, blue: C, yellow: U.…”

Section: Introductionmentioning

confidence: 99%

Specific Signal Enhancement on an RNA‐Protein Interface by Dynamic Nuclear Polarization

Aladin

Sreemantula

Biedenbänder

et al. 2023

Chemistry A European J

Self Cite

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Sensitivity and specificity are both crucial for the efficient solid-state NMR structure determination of large biomolecules. We present an approach that features both advantages by site-specific enhancement of NMR spectroscopic signals from the protein-RNA binding site within a ribonucleoprotein (RNP) by dynamic nuclear polarization (DNP). This approach uses modern biochemical techniques for sparse isotope labeling and exploits the molecular dynamics of 13 C-labeled methyl groups exclusively present in the protein. These dynamics drive heteronuclear cross relaxation and thus allow specific hyperpolarization transfer across the biomolecular complex's interface. For the example of the L7Ae protein in complex with a 26mer guide RNA minimal construct from the box C/D complex in archaea, we demonstrate that a single methyl-nucleotide contact is responsible for most of the polarization transfer to the RNA, and that this specific transfer can be used to boost both NMR spectral sensitivity and specificity by DNP.

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“…Structures of protein-RNA complexes are also prevalent, and were determined using various techniques such as cryogenic electron microscopy (CryoEM) (7), X-ray (8) and solution NMR (9). Solid state NMR has also provided structures of isolated RNA molecules or in complex with proteins (10, 11).…”

Section: Introductionmentioning

confidence: 99%

Characterizing Hydrogen Bonds in Intact RNA from MS2 Bacteriophage Using Solid State Magic Angle Spinning NMR

Meir

Goldbourt

2021

Preprint

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Ribonucleic acid (RNA) is a polymer with pivotal functions in many biological processes. RNA structure determination is thus a vital step towards understanding its function. The secondary structure of RNA is stabilized by hydrogen bonds formed between nucleotide base pairs and it defines the positions and shapes of functional stem-loops, internal loops, bulges, and other functional and structural elements. In this work we present a methodology for studying large intact RNA molecules using homonuclear 15N solid state nuclear magnetic resonance (NMR) spectroscopy. We show that Proton Driven Spin Diffusion (PDSD) experiments with long mixing times, up to 16s, improved by the incorporation of 1H Radiofrequency Dipolar Recoupling (RFDR) pulses, reveal key hydrogen-bond contacts. In the full-length RNA isolated from MS2 phage, we observed strong and dominant contributions of G-C Watson-Crick base pairs, and beyond these common interactions, we observe a significant contribution of the G-U wobble base pairs. Using the improved technique facilitates characterization of hydrogen-bond types in intact large-scale RNA using solid-state NMR. It can be highly useful to guide secondary structure prediction techniques, and possibly to refine higher resolution structure determination methods.

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Structure of a Protein–RNA Complex by Solid‐State NMR Spectroscopy

Cited by 7 publications

References 40 publications

Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

Specific Signal Enhancement on an RNA‐Protein Interface by Dynamic Nuclear Polarization

Characterizing Hydrogen Bonds in Intact RNA from MS2 Bacteriophage Using Solid State Magic Angle Spinning NMR

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