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News and Updates Archive

Posted: Nov 8, 2004

Weak alignment of membrane proteins in stressed polyacrylamide gels

David H. Jones and Stanley J. Opella
Abstract:

Residual dipolar couplings are important as angular constraints for the structure determination of membrane proteins in micelles. Strained polyacrylamide gels are one of the few available mechanisms available for inducing the requisite weak alignment for these samples. However, their use is frequently limited by the ability to incorporate proteins and buffer solutions into the gel matrix. The implementation of several methods of incorporating membrane proteins into gels are described. Conditions for copolymerizing the protein in the absence of a change in pH are detailed. Electrophoresis is also shown to be a useful method to incorporate proteins. Weak alignment of the protein–micelle complex in the gel matrix is subsequently achieved using either vertical or radial compression. The magnitude of alignment can be controlled by altering the gel concentration, the acrylamide/bisacrylamide ratio, and the compression ratio. The alignment tensor can be altered relative to uncharged polyacrylamide gels by copolymerizing samples with acrylamide/acrylic acid to incorporate negative charges in the strained polyacrylamide gel to provide an alternate orientation

The paper has been published in the Journal of Magnetic Resonance.

Posted: Oct. 7, 2004  

Graduate student Mike Kaiser from Professor Len Mueller's lab at UCR, works on a triple resonance single axis goiniometer solid-state NMR probe.  


 

 

Posted: Oct. 5, 2004

Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR Spectroscopy

David S. Thiriot, Alexander A. Nevzorov, Lena Zagyanskiy, Chin H. Wu and Stanley J. Opella
 
Abstract:
The atomic resolution structure of Pf1 coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles in solution is compared to the structures previously determined by X-ray fiber and neutron diffraction, the structure of its membrane-bound form, and the structure of fd coat protein. These structural comparisons provide insights into several biological properties, differences between class I and class II filamentous bacteriophages, and the assembly process. The six N-terminal amino acid residues adopt an unusual "double hook" conformation on the outside of the bacteriophage particle. The solid-state NMR results indicate that at 30 °C, some of the coat protein subunits assume a single, fully structured conformation, and some have a few mobile residues that provide a break between two helical segments, in agreement with structural models from X-ray fiber and neutron diffraction, respectively. The atomic resolution structure determined by solid-state NMR for residues 7–14 and 18–46, which excludes the N-terminal double hook and the break between the helical segments, but encompasses more than 80% of the backbone including the distinct kink at residue 29, agrees with that determined by X-ray fiber diffraction with an RMSD value of 2.0 Å. The symmetry and distance constraints determined by X-ray fiber and neutron diffraction enable the construction of an accurate model of the bacteriophage particle from the coordinates of the coat protein monomers.

The paper has been published in the Journal of Molecular Biology 341 (3): 869-879 AUG 13 2004.
 

Posted: Oct. 5, 2004
 
Prof. Brian Sykes from the University of Alberta working hard on a 19F solid-state NMR probe
 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Posted: Oct. 4, 2004
 
Graduate student Crystal Lau and Prof. Frances Separovic from the University of Melbourne look on as Opella lab Postdoctoral researcher Anna De Angelis (right) discusses bicelles.
 


 

 

 

 

 

 

 

 

 

 

 

Posted: Sept. 28, 2004
 

Professor Gerry Boss, Professor Stanley Opella and Resource staff work with students Payton Chu, Estefanie Esteves, Steve Pao and Paul Theilmann on a blood oxygenation NMR project for their ECE 191 class.
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Posted: Feb. 05, 2004
Functional Characterization and NMR Spectroscopy on Full-Length Vpu from HIV-1 Prepared by Total Chemical Synthesis

Gerd G. Kochendoerfer, David H. Jones, Sangwon Lee, Myrta Oblatt-Montal, Stanley J. Opella, and Mauricio Montal
 
Abstract:
Vpu is an 81-residue integral membrane protein encoded in the HIV-1 genome that is of considerable interest because it plays important roles in the release of virus particles from infected cells and in the degradation of the cellular receptor. We report here the total chemical synthesis of full-length Vpu(1-81) as well as a site-specifically 15N-labeled analogue, Vpu(2-81), using native chemical ligation methodologies and also report a structural and functional comparison of these constructs with recombinant protein obtained via bacterial expression. The structures of the synthetic and expressed polypeptides were similar in lipid micelles using solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in aligned hydrated lipid bilayers indicated that their overall topologies were also very comparable. Further, the channel activity of the synthetic protein was found to be analogous to that previously characterized for the recombinant protein. We have thus demonstrated that using solid phase peptide synthesis and chemical ligation it is feasible to obtain large quantities of a purified and homogeneous membrane protein in a structurally and functionally relevant form for future structural and characterization studies.
 
Posted: Oct. 15, 2003
Three-Dimensional Structure of the Channel-forming Domain of Virus Protein "u" (Vpu) from HIV-1

Park, S.H., Mrse, A.A., Nevzorov, A.A., Mesleh, M.F., Oblatt-Montal, M., Montal, M., and Opella, S.J.

Abstract:
The three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) of HIV-1 was determined by NMR spectroscopy in micelle and bilayer samples. Vpu2–30+ is a 36-residue polypeptide that consists of residues 2–30 from the N terminus of Vpu and a six-residue "solubility tag" at its C terminus that facilitates the isolation, purification, and sample preparation of this highly hydrophobic minimal channel-forming domain. Nearly all of the resonances in the two-dimensional 1H/15N HSQC spectrum of uniformly 15N labeled Vpu2–30+ in micelles are superimposable on those from the corresponding residues in the spectrum of full-length Vpu, which indicates that the structure of the trans-membrane domain is not strongly affected by the presence of the cytoplasmic domain at its C terminus. The two-dimensional 1H/15N PISEMA spectrum of Vpu2–30+ in lipid bilayers aligned between glass plates has been fully resolved and assigned. The "wheel-like" pattern of resonances in the spectrum is characteristic of a slightly tilted membrane-spanning helix. Experiments were also performed on weakly aligned micelle samples to measure residual dipolar couplings and chemical shift anisotropies. The analysis of the PISA wheels and Dipolar Waves obtained from both weakly and completely aligned samples show that Vpu2–30+ has a trans-membrane -helix spanning residues 8–25 with an average tilt of 13°. The helix is kinked slightly at Ile17, which results in tilts of 12° for residues 8–16 and 15° for residues 17–25. A structural fit to the experimental solid-state NMR data results in a three-dimensional structure with precision equivalent to an RMSD of 0.4 Å. Vpu2–30+ exists mainly as an oligomer on PFO-PAGE and forms ion-channels, a most frequent conductance of 96(±6) pS in lipid bilayers. The structural features of the trans-membrane domain are determinants of the ion-channel activity that may be associated with the protein's role in facilitating the budding of new virus particles from infected cells.

The paper has been published in the Journal of Molecular Biology 333, 409-424 (2003).
 
Posted: Sept 5, 2003

Abstract:
A pulse sequence for high resolution separated local field spectroscopy based on "magic sandwich" elements is demonstrated on a single crystal sample. Simulations and experimental results show that this pulse sequence has a reduced frequency offset dependence compared to PISEMA (polarization inversion spin exchange at the magic angle). As a result, it has a larger effective range of homonuclear decoupling, reduced zero-frequency spectral distortions, and more reliable scale factors for individual resonances. In addition, it is easier to setup on commercial spectrometers.

The paper has been published in the Journal of Magnetic Resonance 164, 182-186 (2003).
 
Posted: Sept 5, 2003
Magnex Employee of the Month

Congratulations to the "Magnex Employee of the Month"!

 

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

Posted: March 7th, 2006

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Posted: Nov 1, 2005

Journal of Magnetic Resonance
Sang Ho Park, Anthony A. Mrse, Alexander A. Nevzorov, Anna A. De Angelis and Stanley J. Opella
 
Abstract: Rotational diffusion of membrane proteins in aligned phospholipidnext term bilayers by solid-state NMR spectroscopy

Solid-state NMR experiments on mechanically aligned bilayer and magnetically aligned bicelle samples demonstrate that membrane proteins undergo rapid rotational diffusion about the normal in phospholipid bilayers. Narrow single-line resonances are observed from 15N labeled sites in the trans-membrane helix of the channel-forming domain of the protein Vpu from HIV-1 in phospholipid bilayers with their normals at angles of 0°, 20°, 40°, and 90°, and bicelles with their normals at angles of 0° and 90° with respect to the direction of the applied magnetic field. This could only occur if the entire polypeptide undergoes rotational diffusion about the bilayer normal. Comparisons between experimental and simulated spectra are consistent with a rotational diffusion coefficient (DR) of approximately 105 s−1.

This paper was published at ScienceDirect in the Journal of Magnetic Resonance:

Posted: Aug 16, 2005

Journal of Molecular Biology
Sang Ho Park and Stanley J. Opella

Abstract: Tilt Angle of a Trans-membrane Helix is Determined by Hydrophobic Mismatch

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
 

Posted: May 12, 2005

The Journal of Peptide Research
N. Sinha, C.V. Grant, K.S. Rotondi, L. Feduik-Rotondi, L.M. Gierasch, S.J. Opella

Abstract: Peptides and the development of double- and triple-resonance solid-state NMR of aligned samples*

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

Posted: April 27, 2005

Structural basis of the temperature transition of Pf1 bacteriophage
David S. Thiriot, Alexander A. Nevzorov, and Stanley J. Opella

Abstract:

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,
 

Posted: April 6, 2005

NMR Experiments on Aligned Samples of Membrane Proteins
A. A. De Angelis, D. H. Jones, C. V. Grant, S. H. Park, M. F. Mesleh and S. J. Opella

Abstract:

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

Posted: April 5, 2005

Abstract:

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: March 28, 2005

NMR Workshop was a Success!
Thanks to everyone who attended the NMR Spectroscopy Workshop on 3/15.

Original Flyer

Posted: Feb 22, 2005

**Magnex Employee of the Month**
The Resource is supported by the National Institute for Biomedical Imaging and Bioengineering. Grant P41EB002031.
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