Biomedical subjects

Alexander A Nevzorov

Publications and source records attributed to Alexander A Nevzorov.

14 recordsLinked to original sources

PubMed2007

Three-dimensional experiment for solid-state NMR of aligned protein samples in high field magnets.

A pulse sequence that yields three-dimensional (1)H chemical shift/(1)H-(15)N heteronuclear dipolar coupling/(15)N chemical shift solid-state NMR spectra is demonstrated on a uniformly (15)N labeled membrane protein in magnetically aligned phospholipid bilayers. Based on SAMPI4, the pulse sequence yields high resolution in all three dimensions at a (1)H resonance frequency of 900 MHz with the relatively low rf field strength (33 kHz) available for a lossy aqueous sample with a commercial spectrometer and probe. The (1)H chemical shift frequency dimension is shown to select among amide resonances, which will be useful in studies of larger polytopic membrane proteins where the resonances overlap in two-dimensional spectra. Moreover, the (1)H chemical shift, which can be measured from these spectra, provides an additional orientationally dependent frequency as input for structure calculations.

Capsid Proteins↗

PubMed2006

Selective averaging for high-resolution solid-state NMR spectroscopy of aligned samples.

Solid-state NMR experiments benefit from being performed at high fields, and this is essential in order to obtain spectra with the resolution and sensitivity required for applications to protein structure determination in aligned samples. Since the amount of rf power that can be applied is limited, especially for aqueous protein samples, the most important pulse sequences suffer from bandwidth limitations resulting from the same spread in chemical shift frequencies that aids resolution. SAMPI4 is a pulse sequence that addresses these limitations. It yields separated local field spectra with narrower and more uniform linewidths over the entire spectrum than the currently used PISEMA and SAMMY experiments. In addition, it is much easier to set up on commercial spectrometers and can be incorporated as a building block into other multidimensional pulse sequences. This is illustrated with a two-dimensional HETCOR experiment, where it is crucial to transfer polarization from the amide protons to their directly bonded nitrogens over a wide range of chemical shift frequencies. A quantum-mechanical treatment of the spin Hamiltonians under high-power rf pulses is presented which gives the scaling factor for SAMPI4 as well as the durations of the rf pulses to achieve optimal decoupling.

Algorithms↗

PubMed2006

Structure determination of a membrane protein with two trans-membrane helices in aligned phospholipid bicelles by solid-state NMR spectroscopy.

The structure of the membrane protein MerFt was determined in magnetically aligned phospholipid bicelles by solid-state NMR spectroscopy. With two trans-membrane helices and a 10-residue inter-helical loop, this truncated construct of the mercury transport membrane protein MerF has sufficient structural complexity to demonstrate the feasibility of determining the structures of polytopic membrane proteins in their native phospholipid bilayer environment under physiological conditions. PISEMA, SAMMY, and other double-resonance experiments were applied to uniformly and selectively (15)N-labeled samples to resolve and assign the backbone amide resonances and to measure the associated (15)N chemical shift and (1)H-(15)N heteronuclear dipolar coupling frequencies as orientation constraints for structure calculations. (1)H/(13)C/(15)N triple-resonance experiments were applied to selectively (13)C'- and (15)N-labeled samples to complete the resonance assignments, especially for residues in the nonhelical regions of the protein. A single resonance is observed for each labeled site in one- and two-dimensional spectra. Therefore, each residue has a unique conformation, and all protein molecules in the sample have the same three-dimensional structure and are oriented identically in planar phospholipid bilayers. Combined with the absence of significant intensity near the isotropic resonance frequency, this demonstrates that the entire protein, including the loop and terminal regions, has a well-defined, stable structure in phospholipid bilayers.

Amino Acid Sequence↗

PubMed2006

Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles.

The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu(2-30+), a 36-residue polypeptide containing residues 2-30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional (1)H/(15)N PISEMA spectra of uniformly (15)N-labeled Vpu(2-30+) obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a (1)H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane alpha-helix spanning residues 8-25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent (15)N chemical shift and (1)H-(15)N dipolar coupling frequencies. Tilt angles of 30 degrees and 21 degrees are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers.

Amino Acid Sequence↗

PubMed2005

Rotational diffusion of membrane proteins in aligned phospholipid 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 degrees, 20 degrees, 40 degrees, and 90 degrees, and bicelles with their normals at angles of 0 degrees and 90 degrees 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 10(5)s-1.

Diffusion↗

PubMed2005

Structural basis of the temperature transition of Pf1 bacteriophage.

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 degrees C was compared with the structure previously determined at 30 degrees 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.

Amino Acid Sequence↗

PubMed2004

High-resolution NMR spectroscopy of membrane proteins in aligned bicelles.

High-resolution solid-state NMR spectra can be obtained from uniformly (15)N-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 orientational information necessary for protein structure determination.

Lipid Bilayers↗

PubMed2004

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

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 degrees 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 A. 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.

Bacteriophage Pf1↗

PubMed2004

Structure determination of aligned samples of membrane proteins by NMR spectroscopy.

The paper briefly reviews the process of determining the structures of membrane proteins by NMR spectroscopy of aligned samples, describes the integration of recent developments in the interpretation of spectra of aligned proteins and illustrates the application of these methods to the trans-membrane helical domain of a protein. The emerging methods of interpreting the spectral parameters from aligned samples of isotopically labeled proteins provide opportunities for simultaneously assigning the spectra and determining the structures of the proteins, and also for comparing the results from solid-state NMR experiments on completely aligned samples with those of solution NMR experiments on weakly aligned samples.

Amino Acid Sequence↗

PubMed2003

Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1.

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. Vpu(2-30+) 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 Vpu(2-30+) 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 Vpu(2-30+) 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 Vpu(2-30+) has a trans-membrane alpha-helix spanning residues 8-25 with an average tilt of 13 degrees. The helix is kinked slightly at Ile17, which results in tilts of 12 degrees for residues 8-16 and 15 degrees 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 A. Vpu(2-30+) 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.

HIV-1↗

PubMed2003

Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy.

The atomic resolution structure of fd coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles differs from that previously determined by x-ray fiber diffraction. Most notably, the 50-residue protein is not a single curved helix, but rather is a nearly ideal straight helix between residues 7 and 38, where there is a distinct kink, and then a straight helix with a different orientation between residues 39 and 49. Residues 1-5 have been shown to be mobile and unstructured, and proline 6 terminates the helix. The structure of the coat protein in virus particles, in combination with the structure of the membrane-bound form of the same protein in bilayers, also recently determined by solid-state NMR spectroscopy, provides insight into the viral assembly process. In addition to their roles in molecular biology and biotechnology, the filamentous bacteriophages continue to serve as model systems for the development of experimental methods for determining the structures of proteins in biological supramolecular assemblies. New NMR results include the complete sequential assignment of the two-dimensional polarization inversion spin-exchange at the magic angle spectrum of a uniformly 15N-labeled 50-residue protein in a 1.6 x 107 Da particle in solution, and the calculation of the three-dimensional structure of the protein from orientational restraints with an accuracy equivalent to an rms deviation of approximately 1A.

Bacteriophage M13↗

PubMed2003

Structural fitting of PISEMA spectra of aligned proteins.

An algorithm for fitting protein structures to PISEMA spectra is described, and its application to helical proteins in aligned samples is demonstrated using both simulated and experimental results. The formulation of the algorithm in terms of rotation operators yields compact recursion relations that provide a fast and effective way of obtaining peptide plane orientations from chemical and torsion angle constraints. The algorithm in combination with experimental solid-state NMR data results in a method for determining the backbone structures of proteins, since it yields the orientation of a helix as a whole, including its tilt and twist angles, and describes kinks and curves with atomic resolution. Although the algorithm can be applied in an "assignment-free" manner to spectra of uniformly labeled proteins, the precision of the structural fitting is improved by the addition of assignment information, for example the identification of resonances by residue type from spectra of selectively labeled proteins.

Algorithms↗

PubMed2003

End-to-end correlation for a C-12 hydrocarbon chain.

The 19F nuclear spin-lattice relaxation rate constants were measured as a function of magnetic field strength for 1,12-diaminododecane labeled at one end with a nitroxide radical and at the other with a trifluoromethyl group. The magnetic relaxation dispersion profile (MRD) reports the spectral density function appropriate to the end-to-end correlation function for the doubly labeled molecule. After extrapolation to zero concentration to eliminate the intermolecular relaxation contribution to relaxation, the resulting intramolecular MRD profile was compared with several model approaches. The rotational model for the spectral density functions as included in the Solomon-Bloembergen-Morgan equations does not describe the data well. The earlier model of Freed for nuclear spin relaxation induced by a freely diffusing paramagnetic co-solute is not rigorous for this case because the paramagnet is tethered to the observed nuclear spin and only a restricted space in the immediate vicinity of the nuclear spin is accessible for pseudo-translational diffusion of one end of the molecule with respect to the other. A generalization of the Torrey model for magnetic relaxation by translational diffusion developed by Nevzorov and Freed, which includes the effect of restrictions imposed by the finite length of the chain, describes the experiment within experimental errors. A simple modification of the Hwang-Freed model that does not specifically include the dynamical effects of the finite tether also provides a good approximation to the data when the tether chain is sufficiently long.

Carbon Isotopes↗

PubMed2003

A "magic sandwich" pulse sequence with reduced offset dependence for high-resolution separated local field spectroscopy.

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.

Computer Simulation↗