Biomedical subjects

T A Cross

Publications and source records attributed to T A Cross.

At least 73 records · Page 4Linked to original sources

PubMed1993

A method for studying the structure of uniaxially aligned biopolymers using solid state 15N-nmr: application to Bombyx mori silk fibroin fibers.

Recent advances in the application of solid state nmr spectroscopy to uniformly aligned biopolymers have opened a window through which to view the detailed structure of biological macromolecules that are unable to be seen with standard techniques for structure determination such as x-ray diffraction. Atomic resolution structural details are obtained from solid state nmr data in the form of bond orientations, which yield the relative positions of specific atoms within the molecule. For static aligned systems such as fibers, in which rapid reorientation about the axis of alignment does not occur, it has generally been necessary to perform trial and error line-shape simulations to extract structural details from nmr spectra arising from a single type of nuclear spin interaction. In the present work, a new method is developed in which solid state 15N-nmr spectra obtained from uniaxially aligned molecules placed with the axis of alignment both parallel and perpendicular to the magnetic field are analyzed to yield the orientations of specific molecular bonds. Analytical expressions are derived that utilize spectral features read from 15N chemical shift anisotropy line shapes to calculate a discrete number of possible orientations for a specific site. The 15N-1H dipolar interaction is employed to further narrow the number of unique orientations possible for a given site. With this method, a neighborhood of possible orientations is quickly determined, and full line-shape simulations within this region of allowed space can be performed to refine the limits of orientation. This technique demonstrates the use of a single type of isotopic label to determine the orientation of a specific molecular group such as a peptide plane within a protein. Results from the application of this method to the Bombyx mori silk fibroin protein provide structural detail that is consistent with currently accepted structural models based on fiber diffraction studies.

Animals↗

PubMed1993

Orientational constraints as three-dimensional structural constraints from chemical shift anisotropy: the polypeptide backbone of gramicidin A in a lipid bilayer.

Chemical shifts observed from samples that are uniformly aligned with respect to the magnetic field can be used as very high-resolution structural constraints. This constraint takes the form of an orientational constraint rather than the more familiar distance constraint. The accuracy of these constraints is dependent upon the quality of the tensor characterization. Both tensor element magnitudes and tensor orientations with respect to the molecular frame need to be considered. Here these constraints have been used to evaluate models for the channel conformation of gramicidin A. Of the three models used, the one experimentally derived model of gramicidin in sodium dodecyl sulfate micelles fits the data least well.

Amino Acid Sequence↗

PubMed1993

High-resolution structure and dynamic implications for a double-helical gramicidin A conformer.

The high-resolution structure of a dimeric conformer of gramicidin A, a 15-residue polypeptide, has been determined in the mixed-solvent system of benzene and ethanol by 2D NMR techniques. NOEs, coupling constants and hydrogen-bond information were used to generate 744 experimental constraints for the dimer. Stereoassignment of most beta-methylene groups was achieved by analysis of 3J alpha beta, d alpha beta(i,i), dN beta(i,i) and dN beta(i + 1,i) distances, and consideration of the initial backbone structure determinations. Stereoassignment of several leucine methyl groups was accomplished via a distance geometry/simulated annealing routine, used for structure determination and refinement. The relatively static backbone structure was determined first and held rigid while side-chain conformations were calculated. This procedure is evaluated versus standard NMR structure determination protocols. The backbone is an antiparallel intertwined double helix, with 5.6-5.7 residues per turn, a total dimer length of 36-37 A, and a pore width of 2.5-3.0 A (van der Waals to van der Waals). The structure and dynamics of the side chains are discussed in depth, with careful attention for both the convergence of structures and the residual constraint violations per residue. Side-chain positions impart substantial amphipathic character to the helix, which could influence the conformational change that takes place upon membrane insertion of this channel-forming polypeptide.

Amino Acid Sequence↗

PubMed1993

2H NMR determination of the global correlation time of the gramicidin channel in a lipid bilayer.

A detailed experimental description of molecular dynamics requires an accurate description of the global correlation time. For the gramicidin cation-selective channel the local dynamics of the polypeptide backbone are thought to play a very significant role in the functional process of this channel which occurs on the 10-100-ns time scale. By solid-state NMR spectroscopy an experimental description of the local dynamics including a description of the axis about which the motions occur, the amplitude of the motions, whether they are diffusional or discontinuous and the frequency of the motions is possible. Initial interpretations of the NMR data for this polypeptide backbone in fully hydrated lipid bilayers suggests that the time scale for the local motions is the same as the kinetic time scale (North and Cross, 1993). Here the global correlation time is found from d4-Ala3 and d4-Ala5-gramicidin A powder pattern spectra to be 36 microseconds at 309 K. This surprisingly long correlation time may reflect the high viscosity of the peptide environment or possibly the higher molecular weight of a peptide-lipid aggregate. Furthermore, this reassessment of the global correlation time supports the initial interpretation of relaxation data for the local motions.

Biophysical Phenomena↗

PubMed1992

A conformational rearrangement in gramicidin A: from a double-stranded left-handed to a single-stranded right-handed helix.

A conformational transition is described for the polypeptide, gramicidin A, in which a dimer that forms a left-handed intertwined antiparallel helix is converted to a single-stranded amino terminus to amino terminus right-handed helix. The starting structure is determined here by solution NMR methods while reference is made to the well-established folding motif of gramicidin in a lipid bilayer for the ultimate conformation of this transition. Furthermore, an organic solvent system of benzene and ethanol in which gramicidin has a unique conformation is identified. This conformation is shown to be very similar to that derived from X-ray diffraction of crystals prepared from a similar solvent system.

Amino Acid Sequence↗

PubMed1992

Structure of an isolated gramicidin A double helical species by high-resolution nuclear magnetic resonance.

A conformational species of gramicidin A has been isolated in dioxane by high pressure liquid chromatography and characterized by circular dichroism and two-dimensional proton nuclear magnetic resonance. Double-quantum filtered two-dimensional correlation spectroscopy, two-dimensional homonuclear Hartman Hahn spectroscopy and two-dimensional nuclear Overhauser effect spectra at 500 MHz were used to obtain virtually complete proton assignments and produce 192 distance constraints. Protocols to determine the state of aggregation, monomer-specific assignment of nuclear Overhauser enhancement values, hydrogen bonding pattern and helix handedness are described. A distance geometry/simulated annealing routine was used to generate well-defined backbone and side-chain structures. The species isolated is a right-handed intertwined double helix, with approximately 5.7 residues per turn. Unique values for helical dimensions are also specified.

Amino Acid Sequence↗

PubMed1991

Experimental determination of torsion angles in the polypeptide backbone of the gramicidin A channel by solid state nuclear magnetic resonance.

An analytical method for the determination of torsion angles from solid state 15N nuclear magnetic resonance (n.m.r.) spectroscopic data is demonstrated. Advantage is taken of the 15N-1H and 15N-13C dipolar interactions as well as the 15N chemical shift interaction in oriented samples. The membrane-bound channel conformation of gramicidin A has eluded an atomic resolution structure determination by more traditional approaches. Here, the torsion angles for the Ala3 site are determined by obtaining the n.m.r. data for both the Gly2-Ala3 and Ala3-Leu4 peptide linkages. Complete utilization of the orientational constraints derived from these orientation-dependent nuclear spin interactions in restricting the conformational space is most effectively achieved by utilizing spherical trigonometry. Two possible sets of torsion angles for the Ala3 site are obtained (phi, psi = -129 degrees, 153 degrees and -129 degrees, 122 degrees), both of which are consistent with a right-handed beta-helix. Other functional and computational evidence strongly supports the set for which the carbonyl oxygen atom of the Ala3-Leu4 linkage is rotated into the channel lumen.

Gramicidin↗

PubMed1991

Solid-state nuclear magnetic resonance derived model for dynamics in the polypeptide backbone of the gramicidin A channel.

The dynamics of the backbone of the gramicidin A transmembrane cation channel in dimyristoylphosphatidylcholine bilayers have been investigated using solid state 15N nuclear magnetic resonance (n.m.r.) spectroscopy. With the temperature-dependent fluidity of the bilayer, the rates of motions in the helical gramicidin channel can be modulated. It is shown that in the gel phase, all substantial motions of the channel are slow on the timescale of the n.m.r. experiment (3.5 kHz). The use of oriented samples in which the axis of global channel rotation is aligned parallel to the magnetic field enables separation of global and local dynamics. Spectra obtained from oriented bilayer samples containing single-site 15N-labeled gramicidin at 8 degrees C are analyzed to yield a spatial model for local backbone motion. This model includes the axis of motion, the mean orientation, and the maximum amplitude of displacement for individual peptide planes. Specific sites in the first turn of the amino terminus were investigated, with emphasis on the Ala3 and Leu4 linkages, for which the orientation of the 15N chemical shift tensor with respect to the molecular frame has been determined. The effect of two well-characterized bilayer defect structures, parabolic focal conics and oily streaks, is included in the spectral simulations. It is found that only relatively small amplitude motions are possible at the two sites, with amplitudes of not more than +/- 8 degrees and +/- 15 degrees for the Ala3 and Leu4 sites, respectively. Detailed characterization of the bilayer surface geometry in the oriented samples is presently the major limiting factor in the use of this technique for probing the spatial extent of local motions in integral membrane proteins.

Amino Acid Sequence↗

PubMed1991

Molecular dynamics computations and solid state nuclear magnetic resonance of the gramicidin cation channel.

This paper reports on a coupled approach to determining the structure of the gramicidin A ion channel, utilizing solid state nuclear magnetic resonance (NMR) of isotopically labeled gramicidin channels aligned parallel to the magnetic field direction, and molecular dynamics (MD). MD computations using an idealized right-handed beta-helix as a starting point produce a refined molecular structure that is in excellent agreement with atomic resolution solid state NMR data. The data provided by NMR and MD are complementary to each other. When applied in a coordinated manner they provide a powerful approach to structure determination in molecular systems not readily amenable to x-ray diffraction.

Amino Acid Sequence↗

PubMed1990

Optimizing and characterizing alignment of oriented lipid bilayers containing gramicidin D.

31P NMR spectroscopy and optical microscopy have been used to characterize samples of gramicidin D in oriented lipid bilayers. Correlations have been made between the defect structures observed under crossed polarizers by optical microscopy and characteristic features of 31P NMR spectra. The sample preparation protocol has been improved using these techniques to achieve minimal dispersion of the bilayer normal and minimal amounts of unoriented sample. The molar ratio of gramicidin to dimyristoyl-phosphatidylcholine, the extent of hydration, and the cosolubilizing solvent system were used as the protocol variables. While hydration level and solvent system had profound effects on the sample orientation the molar ratio did not. However, the 31P chemical shift anisotropy is very sensitive to the molar ratio and can be used as an in situ method for determining the molar ratio.

Dimyristoylphosphatidylcholine↗

PubMed1989

Gramicidin cation channel: an experimental determination of the right-handed helix sense and verification of beta-type hydrogen bonding.

Due to the difficulty of obtaining protein/lipid cocrystals for diffraction studies, structural research on intrinsic membrane proteins and polypeptides has been largely restricted to indirect experimental techniques. Hence, many fundamental questions associated with peptide/lipid systems remain unanswered. In particular, the handedness of the gramicidin A transmembrane ion channel incorporated into lipid bilayers has been an open question for nearly two decades. In this study, solid-state 15N NMR spectroscopy is employed to probe directly the secondary structure of the polypeptide backbone. Recent determinations of the 15N chemical shift anisotropy tensor with respect to the molecular frame enable the quantitative evaluation of the 15N chemical shift resonances obtained from oriented dimyristoylphosphatidylcholine (DMPC) bilayer samples containing specific site 15N labeled gramicidin. This direct structural approach verifies the beta-sheet hydrogen-bonding pattern proposed by Urry [Urry, D. W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676] and determines that in our DMPC bilayer preparations the gramicidin channel is right-handed. Additional structural information is provided by the 15N chemical shift data in the form of orientational constraints on the C alpha-C alpha axis orientation of individual peptides relative to the helix axis. The significance of these solid-state NMR results lies in the direct determination of the helix sense and the verification of the beta-type hydrogen bonding, in the development of the solid-state NMR methods for obtaining such information, and in emphasizing the importance of having direct structural data at atomic resolution.

Chemical Phenomena↗

PubMed1989

Solid phase peptide synthesis of 15N-gramicidins A, B, and C and high performance liquid chromatographic purification.

Four single-site 15N-labeled molecules of gramicidin have been synthesized using the 9-fluorenylmethoxycarbonyl method of solid phase peptide synthesis. Formylvaline was coupled as the N-terminal amino acid, and the peptide was cleaved from the resin with ethanolamine. Each synthesized gramicidin was purified in one step by semipreparative reverse phase high performance liquid chromatography and obtained in overall yields as high as 86%. The peptide was characterized by comparison with natural gramicidin using amino acid analysis, u.v. spectroscopy, and analytical high performance liquid chromatography.

Chromatography, High Pressure Liquid↗

PubMed1988

Solid-state 15N-NMR evidence that gramicidin A can adopt two different backbone conformations in dimyristoylphosphatidylcholine model membrane preparations.

Using [15N-Val7]gramicidin A it is shown by solid state 15N-NMR that in dimyristoylphosphatidylcholine model membrane preparations evidence is obtained for two different backbone conformations of gramicidin. One of these conformations is the familiar channel state while a second conformation possesses very different dynamic and structural characteristics. The relative amounts of the conformations depend upon the solvent used to initially codissolve peptide and lipid. Furthermore, by incubation of the samples at modestly elevated temperatures a conversion can be induced from the non-channel to the channel state in a lipid environment.

Circular Dichroism↗

PubMed1988

Solvent history dependence of gramicidin A conformations in hydrated lipid bilayers.

Reconstituted lipid bilayers of dimyristoylphosphatidylcholine (DMPC) and gramicidin A' have been prepared by cosolubilizing gramicidin and DMPC in one of three organic solvent systems followed by vacuum drying and hydration. The conformational state of gramicidin as characterized by 23Na NMR, circular dichroism, and solid state 15N NMR is dependent upon the cosolubilizing solvent system. In particular, two conformational states are described; a state in which Na+ has minimal interactions with the polypeptide, referred to as a nonchannel state, and a state in which Na+ interacts very strongly with the polypeptide, referred to as the channel state. Both of these conformations are intimately associated with the hydrophobic core of the lipid bilayer. Furthermore, both of these states are stable in the bilayer at neutral pH and at a temperature above the bilayer phase transition temperature. These results with gramicidin suggest that the conformation of membrane proteins may be dictated by the conformation before membrane insertion and may be dependent upon the mechanism by which the insertion is accomplished.

Circular Dichroism↗

PubMed1988

Solid-phase peptide synthesis and solid-state NMR spectroscopy of [Ala3-15N][Val1]gramicidin A.

[Ala3-15N][Val1]Gramicidin A has been prepared by solid-phase peptide synthesis and studied by solid-state 15N nuclear magnetic resonance spectroscopy. The synthesis of desformyl[Ala3-15N][Val1]gramicidin A employed N-hydroxysuccinimide esters of 9-fluorenylmethoxycarbonyl-N alpha-amino acids and completely avoided the use of acid. Since deblocking was done with piperidine and the peptide was removed from the resin by treatment with ethanolamine, this synthetic protocol prevented oxidation of the indole rings of this tryptophan-rich peptide and reduced truncations produced by acid hydrolysis. After formylation and purification by anion-exchange and high-pressure liquid chromatography, the peptide was obtained in an overall yield of 30%. Solid-state 15N nuclear magnetic resonance spectra of this peptide and uniformly labeled [15N]gramicidin A' oriented in hydrated lipid bilayers have been obtained, allowing unambiguous assignment of the [15N]Ala3 resonance in the latter. The solid-state 15N nuclear magnetic resonance experiments provide evidence that [Val1]gramicidin A is rotating about an axis that is perpendicular to the plane of the lipid bilayer and that the N--H axis is nearly parallel with the rotational axis. This study demonstrates that site-specifically labeled [15N]gramicidin A analogs prepared by solid-phase peptide synthesis are valuable tools in the study of the solid-state nuclear magnetic resonance spectra of samples in oriented lipid bilayers.

Amino Acid Sequence↗

PubMed1987

Solid-state 15N NMR of oriented lipid bilayer bound gramicidin A'.

Highly oriented samples of lipid and gramicidin A' (8:1 molar ratio) have been prepared with the samples extensively hydrated (approximately 70% water v/w). These preparations have been shown to be completely in a bilayer phase with a transition temperature of 28 degrees C, and evidence is presented indicating that the gramicidin is in the channel conformation. An estimate of the disorder in the alignment of the bilayers parallel with the glass plates used to align the bilayers can be made from the asymmetry of the nuclear magnetic resonances (NMR). Such an analysis indicates a maximal range of disorder of +/- 3 degrees. Uniformly 15N-labeled gramicidin has been biosynthesized by Bacillus brevis grown in a media containing 15N-labeled Escherichia coli cells as the only nitrogen source. When prepared with labeled gramicidin, the oriented samples result in high-resolution 15N NMR spectra showing 12 resonances for the 20 nitrogen sites of the polypeptide. The frequency of the three major multiple resonance peaks has been interpreted to yield the approximate orientation of the N-H bonds in the peptide linkages with respect to the magnetic field. These bond orientations are only partially consistent with the extant structural models of gramicidin.

Calorimetry, Differential Scanning↗

PubMed1986

Cerebral metabolic studies in situ by 31P-nuclear magnetic resonance after hypothermic circulatory arrest.

Cerebral high energy phosphates were studied in the intact rabbit brain using nuclear magnetic resonance spectroscopy. The effect of hypothermia on degradation kinetics in total ischemia due to circulatory arrest was examined, measuring phosphocreatine, adenosine triphosphate, and inorganic phosphate as a function of time at three different temperatures (35, 24, 21 degrees C). Phosphocreatine- and ATP-decays followed single exponential functions at all three temperatures. The half-life times increased by approximately a factor of three upon lowering the temperature from 35 to 21 degrees C with activation energies of 15-20 kcal/mol, which corresponds to values of Q10 between 2.4 and 3.2. In the temperature range studied, no critical temperature was found below which metabolism would stop completely. We conclude that nuclear magnetic resonance spectroscopy allows, in the intact animal, quantitative assessment of the influence of hypothermia on energy metabolism in the brain. This influence is a major concern in the field of cardiac surgery in infants and children who are often operated in total circulatory arrest under deep hypothermia.

Adenosine Triphosphate↗

PubMed1985

Protein structure by solid state nuclear magnetic resonance. Residues 40 to 45 of bacteriophage fd coat protein.

The three-dimensional structure of part of the coat protein in the filamentous bacteriophage fd is described by nuclear magnetic resonance (n.m.r.). Residues 40 to 45 are in a somewhat distorted alpha-helix. This n.m.r. approach for determining protein structure relies on the spectral manifestations of chemical shift and heteronuclear dipolar couplings in a symmetrical assembly of protein subunits oriented parallel to the applied magnetic field. The angles between individual peptide linkages and the filament axis of the virion constitute the basic source of structural information. These angles are directly related to x, y, z co-ordinates for describing the protein structure.

Amino Acid Sequence↗