The Guardian presents: Nuclear Magnetic Resonance Resource Lab
UCSD's Guardian captures Director of UCSD's Nuclear Magnetic Resonance Resource Lab Stanley J. Opella as he explains the research conducted in the campus' facility dubbed "the Bubble."
To view the narrated slideshow, click here.
Published January 15th, 2008
Abstract:
PISEMO, a separated local field experiment that can be performed with either direct 15N (or 13C) detection or indirect 1H detection, is demonstrated on a single crystal of a model peptide. The 1H signals modulated by 1H–15N heteronuclear dipole–dipole couplings are observed stroboscopically in the windows of the multiple-pulse sequence used to attenuate 1H–1H homonuclear dipole–dipole couplings. 1H-detection yields spectra with about 2.5 times the signal to noise ratio observed with 15N-detection under equivalent conditions. Resolution in both the 15N chemical shift and 1H–15N heteronuclear dipole–dipole coupling dimensions is similar to that observed with PISEMA, however, since only on-resonance pulses are utilized, the bandwidth is better.
Published October 10th, 2007
Abstract:
The construction and performance of a scroll coil double-resonance probe for solid-state NMR on stationary samples is described. The advantages of the scroll coil at the high resonance frequencies of 1H and 31P include: high efficiency, minimal perturbations of tuning by a wide range of samples, minimal RF sample heating of high dielectric samples of biopolymers in aqueous solution, and excellent RF homogeneity. The incorporation of a cable tie cinch for mechanical stability of the scroll coil is described. Experimental results obtained on a Hunter Killer Peptide 1 (HKP1) interacting with phospholipid bilayers of varying lipid composition demonstrate the capabilities of this probe on lossy aqueous samples.
Published August 6, 2007
Abstract:
Solid-state NMR spectra with single-site resolution of CXCR1, a G protein-coupled receptor (GPCR), were obtained in magnetically aligned phospholipid bicelles. These results demonstrate that GPCRs in phospholipid bilayers are suitable samples for structure determination by solid-state NMR. The spectra also enable studies of drug-receptor interactions.
Published February 8, 2006
Calculating Protein Structures Directly from Anisotropic Spin Interaction Constraints (p 283-293)
Yegor Smurnyy, Stanley J. Opella
Abstract:
Calculating protein structures directly from anisotropic spin interaction constraintsProtein structure determination by solid-state NMR of aligned samples relies on the fundamental characteristics of the anisotropic nuclear spin interactions present in isotopically labeled proteins. Progress in the implementation of algorithms that calculate protein structures from the orientational constraints in the chemical shift and heteronuclear dipolar coupling interactions is described using both simulated and experimental data. Copyright 2006 John Wiley & Sons, Ltd. Published Online: 14 Feb 2006
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This paper was published at ScienceDirect in the Journal of Magnetic Resonance:
In order to investigate the compensation mechanism of a trans-membrane helix in response to hydrophobic mismatch, the tilt and rotation angles of the trans-membrane helix of Vpu aligned in lipid bilayers of various thickness were determined using orientation-dependent frequencies obtained from solid-state NMR experiments of aligned samples. A tilt angle of 18° was observed in 18:1-O-PC/DOPG (9:1) lipid bilayers, which have a hydrophobic thickness that approximately matches the hydrophobic length of the trans-membrane helix of Vpu. Upon decreasing the hydrophobic thickness of lipid bilayers, no significant change in rotation angle was observed. However, the tilt angle increased systematically with increasing positive mismatch to 27° in 14:0-O-PC/DMPG (9:1), 35° in 12:0-O-PC/DLPG (9:1), and 51° in 10:0 PC/10:0 PG (9:1) lipid bilayers, indicating that the change in tilt angle of the trans-membrane helix is a principal compensation mechanism for hydrophobic mismatch. In addition, the distinctive kink in the middle of the helix observed in 18:1 bilayers disappears in thinner bilayers. Although the opposite of what might be expected, this finding suggests that a helix kink may also be a part of the hydrophobic matching mechanism for trans-membrane helices.
This paper was published at ScienceDirect in the Journal of Molecular Biology:
Volume 350 Issue 2 Page 605 -July 2005, Pages 310-318
doi:10.1016/j.jmb.2005.05.004
Abstract: Peptides have been instrumental in the development of solid-state nuclear magnetic resonance (NMR) spectroscopy, and their roles in the development of solid-state NMR of aligned samples is reviewed. In particular, the roles of synthetic peptides in the development of triple-resonance methods are described. Recent developments of pulse sequences and NMR probes for triple-resonance NMR of aligned samples are presented.
This paper was published in The Journal of Peptide Research:
Volume 65 Issue 6 Page 605 - June 2005
doi:10.1111/j.1399-3011.2005.00262.x
The filamentous bacteriophage Pf1 undergoes a reversible temperature-dependent transition that is also influenced by salt concentrations. This structural responsiveness may be a manifestation of the important biological property of flexibility, which is necessary for long, thin filamentous assemblies as a protection against shear forces. To investigate structural changes in the major coat protein, one- and two-dimensional solid-state NMR spectra of concentrated solutions of Pf1 bacteriophage were acquired, and the structure of the coat protein determined at 0°C was compared with the structure previously determined at 30°C. Despite dramatic differences in the NMR spectra, the overall change in the coat protein structure is small. Changes in the orientation of the C-terminal helical segment and the conformation of the first five residues at the N-terminus are apparent. These results are consistent with prior studies by X-ray fiber diffraction and other biophysical methods.
This paper was published in Protein Science Journal on March 1, 2005,
NMR methods can be used to determine the structures of membrane proteins. Lipids can be chosen so that protein-containing micelles, bicelles, or bilayers are available as samples. All three types of samples can be aligned weakly or strongly, depending on their rotational correlation time. Solution NMR methods can be used with weakly aligned micelle and small bicelle samples. Solid-state NMR methods can be used with mechanically aligned bilayer and magnetically aligned bicelle samples.
This paper was published in Methods of Enzymology, Volume 394, Pages 350-382
The three-dimensional backbone structure of a membrane protein with two transmembrane helices in micelles was determined using solution NMR methods that rely on the measurement of backbone 1H-15N residual dipolar couplings (RDCs) from samples of two different constructs that align differently in stressed polyacrylamide gels. Dipolar wave fitting to the 1H-15N RDCs determines the helical boundaries based on periodicity and was utilized in the generation of supplemental dihedral restraints for the helical segments. The 1H-15N RDCs and supplemental dihedral restraints enable the determination of the structure of the helix-loop-helix core domain of the mercury transport membrane protein MerF with a backbone RMSD of 0.58 Å. Moreover, the fold of this polypeptide demonstrates that the two vicinal pairs of cysteine residues, shown to be involved in the transport of Hg(II) across the membrane, are exposed to the cytoplasm. This finding differs from earlier structural and mechanistic models that were based primarily on the somewhat atypical hydropathy plot for MerF and related transport proteins.
This paper has been published in American Chemical Society
Posted: Feb 22, 2005
High-resolution solid-state NMR spectra can be obtained from uniformly 15N-labeled membrane proteins in magnetically aligned bicelles. Fast uniaxial diffusion about the axis of the bilayer normal results in single-line spectra that contain the orientation information necessary for protein structure determination.
The paper has been published in the American Chemical Society.
