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S J Opella

Publications and source records attributed to S J Opella.

At least 19 recordsLinked to original sources
PubMed2005

Application of Maximum Entropy reconstruction to PISEMA spectra.

Maximum Entropy reconstruction is applied to two-dimensional PISEMA spectra of stationary samples of peptide crystals and proteins in magnetically aligned virus particles and membrane bilayers. Improvements in signal-to-noise ratios were observed with minimal distortion of the spectra when Maximum Entropy reconstruction was applied to non-linearly sampled data in the indirect dimension. Maximum Entropy reconstruction was also applied in the direct dimension by selecting sub-sets of data from the free induction decays. Because the noise is uncorrelated in the spectra obtained by Maximum Entropy reconstruction of data with different non-linear sampling schedules, it is possible to improve the signal-to-noise ratios by co-addition of multiple spectra derived from one experimental data set. The combined application of Maximum Entropy to data in the indirect and direct dimensions has the potential to lead to substantial reductions in the total amount of experimental time required for acquisition of data in multidimensional NMR experiments.

Bacteriophages↗
PubMed2005

NMR experiments on aligned samples of membrane proteins.

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.

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

Peptides and the development of double- and triple-resonance solid-state NMR of aligned samples.

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.

Magnetic Resonance Spectroscopy↗
PubMed2003

Myristoylation as a general method for immobilization and alignment of soluble proteins for solid-state NMR structural studies.

N-terminal myristoylation of the immunoglobulin-binding domain of protein G (GB1) from group G Streptococcus provides the means to bind the protein to aligned phospholipid bilayers for solid-state NMR structural studies. The myristoylated protein is immobilized by its interactions with bilayers, and the sample alignment enables orientationally dependent 15N chemical shifts and 1H-15N-dipolar couplings to be measured. Spectra calculated for the average solution NMR structure of the protein at various orientations with respect to the magnetic field direction were compared to the experimental spectrum. The best fit identified the orientation of the myristoylated protein on the lipid bilayers, and demonstrated that the protein adopts a similar structure in both its myristoylated and non-myristoylated forms, and that the structure is not grossly distorted by its interaction with the phosholipid bilayer surface or by its location in the restricted aqueous space between bilayer leaflets. The protein is oriented such that its charged sides face the phosphatidylcholine headgroups of the lipids with the single amphiphilic helix running parallel to the bilayer surface.

Bacterial Proteins↗
PubMed2002

The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor.

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane alpha-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 A at its narrowest, to 8.6 A at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.

Animals↗
PubMed2002

Structure determination of membrane proteins by NMR spectroscopy.

Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination.

Animals↗
PubMed2001

Structure and topology of a peptide segment of the 6th transmembrane domain of the Saccharomyces cerevisae alpha-factor receptor in phospholipid bilayers.

A detailed analysis of the structure of an 18-residue peptide AQSLLVPSIIFILAYSLK [M6(252-269, C252A)] in 1,2-dimyristoyl-sn-glycero-phosphocholine bilayers was carried out using solid state NMR and attenuated total reflection Fourier transform infrared spectroscopy. The peptide corresponds to a portion of the 6th transmembrane domain of the alpha-factor receptor of Saccharomyces cerevisiae. Ten homologs of M6(252-269, C252A) were synthesized in which individual residues were labeled with (15)N. One- and two-dimensional solid state NMR experiments were used to determine the chemical shifts and (1)H-(15)N dipolar coupling constants for the (15)N-labeled peptides in oriented dimyristoylphosphatidylcholine bilayers on stacked glass plates. These parameters were used to calculate the structure and orientation of M6(252-269, C252A) in the bilayers. The results indicate that the carboxyl terminal residues (9-14) are alpha-helical and oriented with an angle of about 8 degrees with respect to the bilayer normal. Independently, an attenuated total reflection Fourier transform infrared spectroscopy analysis on M6(252-269, C252A) in a 1,2-dimyristoyl-sn-glycero-phosphocholine bilayer concluded that the helix tilt angle was about 12.5 degrees. The results on the structure of M6(252-269, C252A) in bilayers are in good agreement with the structure determined in trifluoroethanol/water solutions (B. Arshava et al. Biopolymers, 1998, Vol. 46, pp. 343-357). The present study shows that solid state NMR spectroscopy can provide high resolution information on the structure of transmembrane domains of a G protein-coupled receptor.

Amino Acid Sequence↗
PubMed2001

Structural studies of the HIV-1 accessory protein Vpu in langmuir monolayers: synchrotron X-ray reflectivity.

Vpu is an 81 amino acid integral membrane protein encoded by the HIV-1 genome with a N-terminal hydrophobic domain and a C-terminal hydrophilic domain. It enhances the release of virus from the infected cell and triggers degradation of the virus receptor CD4. Langmuir monolayers of mixtures of Vpu and the phospholipid 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (DLgPC) at the water-air interface were studied by synchrotron radiation-based x-ray reflectivity over a range of mole ratios at constant surface pressure and for several surface pressures at a maximal mole ratio of Vpu/DLgPC. Analysis of the x-ray reflectivity data by both slab model-refinement and model-independent box-refinement methods firmly establish the monolayer electron density profiles. The electron density profiles as a function of increasing Vpu/DLgPC mole ratio at a constant, relatively high surface pressure indicated that the amphipathic helices of the cytoplasmic domain lie on the surface of the phospholipid headgroups and the hydrophobic transmembrane helix is oriented approximately normal to the plane of monolayer within the phospholipid hydrocarbon chain layer. At maximal Vpu/DLgPC mole ratio, the tilt of the transmembrane helix with respect to the monolayer normal decreases with increasing surface pressure and the conformation of the cytoplasmic domain varies substantially with surface pressure.

Amino Acid Sequence↗
PubMed2001

Structure of a malaria parasite antigenic determinant displayed on filamentous bacteriophage determined by NMR spectroscopy: implications for the structure of continuous peptide epitopes of proteins.

The NANP repeating sequence of the circumsporozoite protein of Plasmodium falciparum was displayed on the surface of fd filamentous bacteriophage as a 12-residue insert (NANP)(3) in the N-terminal region of the major coat protein (pVIII). The structure of the epitope determined by multidimensional solution NMR spectroscopy of the modified pVIII protein in lipid micelles was shown to be a twofold repeat of an extended and non-hydrogen-bonded loop based on the sequence NPNA, demonstrating that the repeating sequence is NPNA, not NANP. Further, high resolution solid-state NMR spectra of intact hybrid virions containing the modified pVIII proteins demonstrate that the peptides displayed on the surface of the virion adopt a single, stable conformation; this is consistent with their pronounced immunogenicity as well as their ability to mimic the antigenicity of their native parent proteins.

Amino Acid Sequence↗
PubMed2000

A solid-state NMR index of helical membrane protein structure and topology.

The secondary structure and topology of membrane proteins can be described by inspection of two-dimensional (1)H-(15)N dipolar coupling/(15)N chemical shift polarization inversion spin exchange at the magic angle spectra obtained from uniformly (15)N-labeled samples in oriented bilayers. The characteristic wheel-like patterns of resonances observed in these spectra reflect helical wheel projections of residues in both transmembrane and in-plane helices and hence provide direct indices of the secondary structure and topology of membrane proteins in phospholipid bilayers. We refer to these patterns as PISA (polarity index slant angle) wheels. The transmembrane helix of the M2 peptide corresponding to the pore-lining segment of the acetylcholine receptor and the membrane surface helix of the antibiotic peptide magainin are used as examples.

Animals↗
PubMed2000

Three-dimensional solid-state NMR spectroscopy is essential for resolution of resonances from in-plane residues in uniformly (15)N-labeled helical membrane proteins in oriented lipid bilayers.

Uniformly (15)N-labeled samples of membrane proteins with helices aligned parallel to the membrane surface give two-dimensional PISEMA spectra that are highly overlapped due to limited dispersions of (1)H-(15)N dipolar coupling and (15)N chemical shift frequencies. However, resolution is greatly improved in three-dimensional (1)H chemical shift/(1)H-(15)N dipolar coupling/(15)N chemical shift correlation spectra. The 23-residue antibiotic peptide magainin and a 54-residue polypeptide corresponding to the cytoplasmic domain of the HIV-1 accessory protein Vpu are used as examples. Both polypeptides consist almost entirely of alpha-helices, with their axes aligned parallel to the membrane surface. The measurement of three orientationally dependent frequencies for Val17 of magainin enabled the three-dimensional orientation of this helical peptide to be determined in the lipid bilayer.

Animals↗
PubMed2000

Lanthanide ions bind specifically to an added "EF-hand" and orient a membrane protein in micelles for solution NMR spectroscopy.

Twelve amino acid residues corresponding to an "EF-hand" calcium-binding site were added to the N-terminus of a protein, providing a specific lanthanide ion binding that weakly orients the protein in solution. A comparison of spectra of the protein with and without the EF-hand residues demonstrates that the structure of the native protein is not perturbed by this modification, since there are minimal chemical shift changes. With a lanthanide but not calcium bound to the EF-hand, the protein is weakly oriented by the magnetic field, since residual dipolar couplings can be measured. Since the signs and magnitudes of the couplings varied with the type of lanthanide, this demonstrated the ability to obtain multiple orientations of the protein in solution. The sample is a membrane protein in lipid micelles that disrupted the commonly employed bicelle and filamentous phage solutions; therefore, the addition of a specific metal binding site in the form of an EF-hand may provide a general approach to orienting proteins where the addition of external agents is problematic. An additional benefit is that the lanthanide ions perturb the protein resonances in ways that provide unique orientational and distance constraints.

Amino Acid Sequence↗
PubMed1999

Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1.

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic alpha-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly (15)N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane alpha-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu(2-51) (residues 2-51), which contains the transmembrane alpha-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu(28-81) (residues 28-81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2-81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 +/- 3 pS and 12 +/- 3 pS in 0.5 M KCl and 29 +/- 3 pS and 12 +/- 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu(2-51), which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu(28-81), which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.

Amino Acid Sequence↗
PubMed1999

Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structures of eel calcitonin (CT) and two glycosylated CT derivatives, [Asn(GlcNAc)3]-CT (CT-GlcNAc) and [Asn(Man6-GlcNAc2)3]-CT (CT-M6), in micelles were determined by solution NMR spectroscopy. The topologies of these peptides associated with oriented lipid bilayers were determined with solid-state NMR. All of the peptides were found to have an identical conformation in micelles characterized by an amphipathic alpha-helix consisting of residues Ser5 through Leu19 followed by an unstructured region at the C-terminus. The overall conformation of the peptide moiety was not affected by the glycosylation. Nevertheless, comparison of the relative exchange rates of the Leu12 amide proton might suggest the possibility that fluctuations of the alpha-helix are reduced by glycosylation. The presence of NOEs between the carbohydrate and the peptide moieties of CT-GlcNAc and CT-M6 and the amide proton chemical shift data suggested that the carbohydrate interacted with the peptide, and this might account for the conformational stabilization of the alpha-helix. Both the unmodified CT and the glycosylated CT were found to have orientations with their helix axes parallel to the plane of the lipid bilayers by solid-state NMR spectroscopy.

Acetylglucosamine↗
PubMed1999

Effects of temperature and Y21M mutation on conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd.

Solid-state NMR spectroscopy was used to analyze the conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd. Both one and two-dimensional solid-state NMR spectra of magnetically aligned samples of fd bacteriophage reveal that an increase in temperature and a single site substitution (Tyr21 to Met, Y21M) reduce the conformational heterogeneity observed throughout wild-type pVIII. The NMR results are consistent with previous studies indicating that conformational flexibility in the hinge-bend segment that links the amphipathic and hydrophobic helices in the membrane-bound form of the protein plays an essential role during phage assembly, which involves a major change in the tertiary, but not secondary, structure of the coat protein.

Capsid↗
PubMed1999

Three-dimensional 13C shift/1H-15N coupling/15N shift solid-state NMR correlation spectroscopy.

Triple-resonance experiments capable of correlating directly bonded and proximate carbon and nitrogen backbone sites of uniformly 13C- and 15N-labeled peptides in stationary oriented samples are described. The pulse sequences integrate cross-polarization from 1H to 13C and from 13C to 15N with flip-flop (phase and frequency switched) Lee-Goldburg irradiation for both 13C homonuclear decoupling and 1H-15N spin exchange at the magic angle. Because heteronuclear decoupling is applied throughout, the three-dimensional pulse sequence yields 13C shift/1H-15N coupling/15N shift correlation spectra with single-line resonances in all three frequency dimensions. Not only do the three-dimensional spectra correlate 13C and 15N resonances, they are well resolved due to the three independent frequency dimensions, and they can provide up to four orientationally dependent frequencies as input for structure determination. These experiments have the potential to make sequential backbone resonance assignments in uniformly 13C- and 15N-labeled proteins.

Carbon Isotopes↗