Carbon and Nitrogen Chemical Shifts of Solid State Enzymes

McDermott, Ann E.; Gu, Zhen

doi:10.1002/9780470034590.emrstm0053

Cited by 7 publications

(6 citation statements)

References 79 publications

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“…The chemical shift table generated by Sparky reported standard deviations of mostly 0.05-0.1 ppm for the 13 C shifts across the protein and <0.2 ppm for the 15 N shifts. 13 C and 15 N chemical shifts were referenced externally to the adamantane CH2 peak at 40.48 ppm 83 and the NH4Cl line at 39.27 ppm, 84 respectively.…”

Section: Methodsmentioning

confidence: 99%

Filamentous Phage Studied by Magic-Angle Spinning NMR: Resonance Assignment and Secondary Structure of the Coat Protein in Pf1

et al. 2007

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Assignments are presented for resonances in the magic-angle spinning solid-state NMR spectra of the major coat protein subunit of the filamentous bacteriophage Pf1. NMR spectra were collected on uniformly 13C and 15N isotopically enriched, polyethylene glycol precipitated samples of fully infectious and hydrated phage. Site-specific assignments were achieved for 231 of the 251 labeled atoms (92%) of the 46-residue-long coat protein, including 136 of the 138 backbone atoms, by means of two- and three-dimensional 15N and 13C correlation experiments. A single chemical shift was observed for the vast majority of atoms, suggesting a single conformation for the 7300 subunits in the 36 MDa virion in its high-temperature form. On the other hand, multiple chemical shifts were observed for the Calpha, Cbeta, and Cgamma atoms of T5 in the helix terminus and the Calpha and Cbeta atoms of M42 in the DNA interaction domain. The chemical shifts of the backbone atoms indicate that the coat protein conformation involves a 40-residue continuous alpha-helix extending from residue 6 to the C-terminus.

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Section: Methodsmentioning

confidence: 99%

Filamentous Phage Studied by Magic-Angle Spinning NMR: Resonance Assignment and Secondary Structure of the Coat Protein in Pf1

et al. 2007

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“…[183]. With a 15 NH 3 resonance at 38.9 ppm, this is in line with (liquid) CH 3 NO 2 or other secondary references, such as ammonium cloride [195].…”

Section: Shift Referencingsupporting

confidence: 56%

Structural characterization of membrane proteins by solid-state NMR spectroscopy

Seidel¹

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In this work, nuclear magnetic resonance spectroscopy was employed to characterize the three-dimensional atomic structure of proteins and other biomolecules in the solid phase.A method for obtaining sequential resonance assignments from ( 13 C, 13 C) correlation spectroscopy on a uniformly labeled protein under magic angle spinning was developed. This facilitated a largely complete assignment of a nano-crystalline form of the 76 residue protein ubiquitin. Comparison to results from other sample preparations indicates that chemical shifts variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. The use of measuring proton-proton distances via rare-spin encoding was described on l-tyrosine-ethylester (TEE), and on ubiquitin. On the example of TEE, it was shown that internuclear distance measurements can informatively be complemented by techniques that probe molecular motion. On ubiquitin, the calculation of a structural model from rare-spin encoded proton-proton distances was assessed using a homology model. Finally, Ca 2+ -ATPase-bound phospholamban (PLN) was investigated in a functional, non-crystalline lipid environment. Chemical shifts in all domains of AFA-PLN in the complex were assigned. The results were combined with other biophysical and biochemical data to calculate a three-dimensional model of the complex, which provides a structural basis for understanding the molecular interactions. These experimental studies were complemented by a statistical analysis of chemical shift predictions for proteins in the solid state with semi-empirical algorithms that are trained on solution-state NMR and X-ray diffraction data. The findings validate for the first time the applicability of such computer programs for solid-state NMR data, and highlight possible areas of improvement. vii viii Kurzzusammenfassung In dieser Arbeit wurde die kernmagnetische Resonanzspektroskopie zur Bestimmung der atomaren Raumstruktur von Proteinen und anderen Biomolekülen in fester Phase angewandt. Eine neue Methode zur sequenzspezifischen Resonanzzuordnung durch ( 13 C, 13 C) Korrelationsspektroskopie wurde entwickelt. Unter Verwendung dieser wurde eine nahezu vollständige Zuordnung der Resonanzen einer nano-kristallinen Form des 76-Reste Proteins Ubiquitin erreicht. Ein Vergleich mit Resultaten anderer Probenpräparationen weist auf einen Zusammenhang der Variation chemischer Verschiebungen mit dem Grad der molekularen Mobilität hin. Der Nutzen indirekt gemessener Proton-Proton Abstände wurde anhand von l-Tyrosin-Ethylester (TEE) und Ubiquitin beschrieben. Am Beispiel von TEE wurde gezeigt, dass die Verwendung internuklearer Abstände durch Techniken zur Messung molekulare Bewegungen sinnvoll ergänzt werden kann. Im Falle von Ubiquitin wurden Strukturrechnungen auf Grundlage der indirekt gemessenen Proton-Proton Abstände durchgeführt, wobei der Analyse eine Homologie zu bestehenden Modellen zu Grunde gelegt wurde. Abschließend wurde an Ca 2+ -ATPase gebundenes Phospholamban (PLN) in einer ...

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“…KBr was used as an external temperature sensor, 43 and the actual temperature of the sample (corrected for rotational heating) was maintained at 4 °C using the Bruker BCU temperature controller, which was stable to ±0.1°. 13 C and 15 N chemical shifts were referenced with respect to the external standards adamantane (CH 2 group referenced to DSS at 40.76 ppm 44 ) and NH 4 Cl (39.27 ppm 45 ), respectively. Typical 90°pulse lengths were 2.8 μs ( 1 H), 4.0 μs ( 13 C), and 4.8 μs ( 15 N), and the contact time of 1 H− 15 N/ 13 C cross-polarization (CP) was 2.2/1.8 ms. 1 H− 15 N/ 13 C CP employed a 95−105% linear amplitude ramp on the 1 H channel with the center Hartmann−Hahn matched to the first spinning sideband.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Toward Closing the Gap: Quantum Mechanical Calculations and Experimentally Measured Chemical Shifts of a Microcrystalline Lectin

et al. 2016

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NMR chemical shifts are exquisitely sensitive probes for conformation and dynamics in molecules and supramolecular assemblies. While isotropic chemical shifts are easily measured with high accuracy and precision in conventional NMR experiments, they remain challenging to calculate quantum mechanically, particularly in inherently dynamic biological systems. Using a model benchmark protein, the 133-residue agglutinin from Oscillatoria agardhii (OAA), which has been extensively characterized by us previously, we have explored the integration of X-ray crystallography, solution NMR, MAS NMR, and quantum mechanics/molecular mechanics (QM/MM) calculations for analysis of 13Cα and 15N isotropic chemical shifts. The influence of local interactions, quaternary contacts, and dynamics on the accuracy of calculated chemical shifts is analyzed. Our approach is broadly applicable and expected to be beneficial in chemical shift analysis and chemical-shift-based structure refinement for proteins and protein assemblies.

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Carbon and Nitrogen Chemical Shifts of Solid State Enzymes

Cited by 7 publications

References 79 publications

Filamentous Phage Studied by Magic-Angle Spinning NMR: Resonance Assignment and Secondary Structure of the Coat Protein in Pf1

Filamentous Phage Studied by Magic-Angle Spinning NMR: Resonance Assignment and Secondary Structure of the Coat Protein in Pf1

Structural characterization of membrane proteins by solid-state NMR spectroscopy

Toward Closing the Gap: Quantum Mechanical Calculations and Experimentally Measured Chemical Shifts of a Microcrystalline Lectin

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