NMR Structural Studies of Membrane Proteins

Opella, Stanley J.; McDonnell, Patricia A.

doi:10.1007/978-1-349-12749-8_6

Cited by 76 publications

(113 citation statements)

References 73 publications

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“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] Particularly, use of protein micro-/nano-crystals [17] has significantly improved resolution in high-resolution SSNMR of dilute spins such as 13 C and 15 N, permitting signal assignment and structural determination of various uniformly 13 C and/or 15 N-labeled proteins by SSNMR. [18][19][20][21][22][23][24][25] However, restricted sensitivity in 13 C and 15 N SSNMR has been still one of the major limiting factors in SSNMR analysis of proteins.…”

Section: Introductionmentioning

confidence: 99%

Sensitivity enhancement in 13C solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing 1H T1 relaxation

Wickramasinghe

Kotecha

Samoson

et al. 2007

Journal of Magnetic Resonance

118

111

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We discuss a simple approach to enhance sensitivity for 13 C high-resolution solid-state NMR for proteins in microcrystals by reducing 1 H T 1 relaxation times with paramagnetic relaxation reagents. It was shown that 1 H T 1 values can be reduced from 0.4-0.8 s to 60-70 ms for ubiquitin and lysozyme in D 2 O in the presence of 10 mM Cu(II)Na 2 EDTA without substantial degradation of the resolution in 13 C CPMAS spectra. Faster signal accumulation using the shorter 1 H T 1 attained by paramagnetic doping provided sensitivity enhancements of 1.4-2.9 for these proteins, reducing the experimental time for a given signal-to-noise ratio by a factor of 2.0-8.4. This approach presented here is likely to be applicable to various other proteins in order to enhance sensitivity in 13 C high-resolution solidstate NMR spectroscopy.

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Section: Introductionmentioning

confidence: 99%

Sensitivity enhancement in 13C solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing 1H T1 relaxation

Wickramasinghe

Kotecha

Samoson

et al. 2007

Journal of Magnetic Resonance

118

111

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“…The difficulty in obtaining high resolution three dimensional structures of the hydrophobic TM domains of integral membrane proteins (see section 1.1.3) cm be attributed in part to the insolubility of these domains (Garavito et al. 1996;Opella 1997 However, the extreme hydrophobicity of these segments does create obstacles to their characterization.…”

Section: Resultsmentioning

confidence: 99%

“…A detailed understanding of the mechanisms of normal and abnormal function of integral membrane pmteins is important for the treatment of diseases and conditions in which abnormally functioning integral membrane proteins are implicated. In order to achieve such a degree of understanding, it is necessary to determine the three-dimensional structure of integral membrane proteins to high (atomic) resolution (Opella, 1997).…”

Section: --------------------------------------47mentioning

confidence: 99%

“…The hydrophobicity of these domains and their resultant insolubility in aqueous solution provides serious challenges to the structural biologist (Opella, 1997). The standard techniques of atornic resolution protein structure determination, X-ray crystallography and nuclear magnetic resonance ('NMR') spectroscopy, have been optimized for the study of water-soluble proteins.…”

Section: Figure 22: Molecular Weights Of the Reactants Products And mentioning

confidence: 99%

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Solubilization of Hydrophobic Peptides by Reversible Cysteine PEGylation

Pomroy

Deber

1998

Biochemical and Biophysical Research Communications

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“…In order to obtain site-specific resolution, these involve either uniaxial orientation of the sample with respect to the static magnetic field [1] or magic angle spinning (MAS) [2]. In the latter case, anisotropic dipolar and chemical shift interactions which encode structural parameters are modulated by the sample spinning and must be re-introduced to yield useful information.…”

Section: Introductionmentioning

confidence: 99%

Dynamic nuclear polarization at 9 T using a novel 250 GHz gyrotron microwave source

Bajaj

Farrar

Hornstein

et al. 2011

Journal of Magnetic Resonance

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In this communication, we report enhancements of nuclear spin polarization by dynamic nuclear polarization (DNP) in static and spinning solids at a magnetic field strength of 9T (250 GHz for g = 2 electrons, 380 MHz for 1 H). In these experiments, 1 H enhancements of up to 170 ± 50 have been observed in 1-13 C-glycine dispersed in a 60:40 glycerol/water matrix at temperatures of 20 K; in addition, we have observed significant enhancements in 15 N spectra of unoriented pf1-bacteriophage. Finally, enhancements of ~17 have been obtained in two-dimensional 13 C-13 C chemical shift correlation spectra of the amino acid U-13 C, 15 N-proline during magic angle spinning (MAS), demonstrating the stability of the DNP experiment for sustained acquisition and for quantitative experiments incorporating dipolar recoupling. In all cases, we have exploited the thermal mixing DNP mechanism with the nitroxide radical 4-amino-TEMPO as the paramagnetic dopant. These are the highest frequency DNP experiments performed to date and indicate that significant signal enhancements can be realized using the thermal mixing mechanism even at elevated magnetic fields. In large measure, this is due to the high microwave power output of the 250 GHz gyrotron oscillator used in these experiments.

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NMR Structural Studies of Membrane Proteins

Cited by 76 publications

References 73 publications

Sensitivity enhancement in 13C solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing 1H T1 relaxation

Sensitivity enhancement in 13C solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing 1H T1 relaxation

Solubilization of Hydrophobic Peptides by Reversible Cysteine PEGylation

Dynamic nuclear polarization at 9 T using a novel 250 GHz gyrotron microwave source

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