Biomedical subjects

T A Cross

Publications and source records attributed to T A Cross.

At least 19 recordsLinked to original sources

PubMed2001

Comparison of gramicidin A and gramicidin M channel conductance dispersities.

To explore the possible role of Trp side chains in gramicidin channel conductance dispersity, we studied the dispersity of gramicidin M (gM), a gramicidin variant in which all four tryptophan residues are replaced with phenylalanine residues, and its enantiomer, gramicidin M(-) (gM(-)), and compared them to that of gramicidin A (gA). The conductances of highly purified gM and gM(-) were studied in alkali metal solutions at a variety of concentrations and voltages, in seven different types of lipid, and in the presence of detergent. Like gA channels, the most common gM channel conductance forms a narrow band. However, unlike gA channels, where the remaining 5-30% of channel conductances are broadly distributed below (and slightly above) the main band, in gM there is a narrow secondary band with <50% of the main peak conductance. This secondary peak was prominent in NaCl and KCl, but significantly diminished in CsCl and RbCl. Under some conditions, minor components can be observed with conductances yet lower than the secondary peak. Interconversions between the primary conductance state and these yet lower conductance states were observed. The current-voltage relations for both primary and secondary gM channel types have about the same curvature. The mean lifetime of the secondary channel type is below one third that of the primary type. The variants represent state deviations in the peptide or adjacent lipid structure.

Electric Conductivity↗

PubMed2001

Complete cross-validation and R-factor calculation of a solid-state NMR derived structure.

Cross-validation of a solid-state NMR-derived membrane polypeptide structure is demonstrated. An initial structure has been achieved directly from solid-state NMR derived orientational restraints based on a variety of anisotropic nuclear spin interactions. Refining the molecular structure involves setting up a penalty function that incorporates all available solid-state NMR experimental data and an energy function. A validation method is required to choose the optimal weighting factor for the total penalty function to balance the contribution from the experimental restraints and the energy function. Complete cross-validation has been used to avoid over-fitting the orientational restraints. Such cross-validation involves partitioning of the experimental data into a test set and a working set followed by checking the free R-value during the refinement process. This approach is similar to the method used in crystallography and solution NMR. Optimizing the weighting factor on the penalty function by cross-validation will increase the quality of the refined structure from solid-state NMR data. The complete cross-validation and R-factor calculation is demonstrated using experimental solid-state NMR data from gramicidin A, a monovalent cation channel in lipid bilayers.

Journal Article↗

PubMed2001

PISEMA powder patterns and PISA wheels.

Resonance patterns observed in 2D PISEMA (polarization inversion spin exchange at magic angle) spectra from a transmembrane alpha-helix have been demonstrated to yield structural details of the protein. This paper presents a mathematical discussion of the PISEMA powder spectrum as the image in the frequency plane of a quadratic function from the sphere of unit vectors. The simplicity of this function allows easy calculation of the powder spectrum. Based on this analysis of powder patterns, four degeneracies are discussed which arise in determining possible orientations associated with PISA spectra. This paper also gives parametric equations for PISA wheels, which are specific patterns observed in PISEMA spectra of oriented peptides. These wheels are useful both in assigning the resonances and in determining the orientation of the helix with respect to the magnetic field. The union of these PISA wheels gives the entire powder spectrum.

Influenza A virus↗

PubMed2001

Noncontact dipole effects on channel permeation. IV. Kinetic model of 5F-Trp(13) gramicidin A currents.

Nonlinear least squares fitting was used to assign rate constants for the three-barrier, two-site, double-occupancy, single-filing kinetic model for previously reported current-voltage relations of (5F-Indole)Trp(13) gramicidin A and gramicidin A channels (, 75:2830-2844). By judicious coupling of parameters, it was possible to reduce the parameter space from 64 parameters to 24, and a reasonable fit consistent with other experimental data was obtained. The main features of the fit were that fluorination increased the rate constant for translocation by a factor of 2.33, consistent with a free energy change in the translocation barrier of -0.50 kcal/mol, and increased first-ion binding affinity by a factor of 1.13, primarily by decreasing the first-ion exit rate constant. The translocation rate constant was 5.62 times slower in diphytanoyl phosphatidylcholine (DPhPC) bilayers than in monoolein (GMO) bilayers (coupled for the four combinations of peptide and salt), suggesting a 44.2-mV difference in the projection of the interfacial dipole into the channel. Thus fluorination caused increased currents in DPhPC bilayers, where a high interfacial dipole potential makes translocation more rate limiting because the translocation barrier was reduced, and decreased currents in GMO bilayers, where ion exit or entry is rate limiting because these barriers were increased.

Algorithms↗

PubMed2001

Noncontact dipole effects on channel permeation. V. Computed potentials for fluorinated gramicidin.

Experimental and theoretical calculations indicate that the dipole moment of the four Trp side chains in gramicidin A (gA) channels modify channel conductance through long-range electrostatic interactions. Electrostatic ion/side-chain interaction energies along the channel were computed with CHARMM using ab initio atom charges for native and 4-, 5-, or 6-fluorinated Trp side chains. The bulk water reaction to the polar side chains was included using the method of images as implemented by, and channel waters in idealized structures were included. Ion/Trp interaction energies were approximately -0.6 kcal/mol throughout the channel for all four of the native Trp pairs. Channel waters produced a modest reduction in the magnitude of interactions, essentially offsetting images representing the bulk water outside the channel. The effects of side-chain fluorination depended on ring position and, to a lesser extent, residue number. Compared with native Trp, 5-fluorination reduces the translocation barrier with minor effects on the exit barrier. In contrast, 6-fluorination primarily reduces exit barrier. 4-Fluorination produces a more complex double-well energy profile. Effects of measured side-chain movements resulting from fluorination or change in lipid bilayer were negligible whereas thermal side chain librations cause large effects, especially in the region of the ion-binding sites.

Binding Sites↗

PubMed2001

Structure of the transmembrane region of the M2 protein H(+) channel.

The transmembrane domain of the M2 protein from influenza A virus forms a nearly uniform and ideal helix in a liquid crystalline bilayer environment. The exposure of the hydrophilic backbone structure is minimized through uniform hydrogen bond geometry imposed by the low dielectric lipid environment. A high-resolution structure of the monomer backbone and a detailed description of its orientation with respect to the bilayer were achieved using orientational restraints from solid-state NMR. With this unique information, the tetrameric structure of this H(+) channel is constrained substantially. Features of numerous published models are discussed in light of the experimental structure of the monomer and derived features of the tetrameric bundle.

Influenza A virus↗

PubMed2000

Influence of transmembrane peptides on bilayers of phosphatidylcholines with different acyl chain lengths studied by solid-state NMR.

The molecular orientation in a lipid membrane of the peptide fragment VEYAGIALFFVAAVLTLWSMLQYLSAAR (phosphatidylglycerophosphate synthase (Pgs) peptide E) of an integral membrane protein, Pgs, in Escherichia coli has been investigated by solid-state 15N nuclear magnetic resonance (NMR) on macroscopically aligned lipid bilayers. The secondary structure of the peptide in lipid vesicles was determined by circular dichroism spectroscopy. Furthermore, the phase behaviour of the Pgs peptide E/dierucoylphosphatidylcholine (DEruPC)/water system was determined by (2)H, (31)P and 15N solid-state NMR spectroscopy. The phase behaviour obtained was then compared to that of the Pgs peptide E solubilised in dioleoylphosphatidylcholine and water that was previously studied by Morein et al. [Biophys. J. 73 (1997) 3078-3088]. This was aimed to answer the question whether a difference in the length of the hydrophobic part of this peptide and the hydrophobic thickness of the lipid bilayer (hydrophobic mismatch) will affect the phase behaviour. The peptide mostly has a transmembrane orientation and is in an alpha-helical conformation. An isotropic phase is formed in DEruPC with high peptide content (peptide/lipid molar ratio (p/l) > or =1:15) and high water content (> or =50%, w/w) at 35 degrees C. At 55 and 65 degrees C an isotropic phase is induced at high water content (> or =50%, w/w) at all peptide contents studied (no isotropic phase forms in the lipid/water system under the conditions in this study). At high peptide contents (p/l> or =1:15) an isotropic phase forms at 20 and 40% (w/w) of water at 55 and 65 degrees C. A comparison of the phase behaviour of the two homologous lipid systems reveals striking similarities, although the thicknesses of the two lipid bilayers differ by 7 A. This suggests that the rationalisation of the phase behaviour in terms of the hydrophobic mismatch is not applicable to these systems. The C-terminus of Pgs peptide E is amphiphilic and a considerable part of the peptide is situated outside the hydrophobic part of the bilayer, a property of the peptide that to a large extent will affect the lipid/peptide phase behaviour.

Amino Acid Sequence↗

PubMed2000

Helix tilt of the M2 transmembrane peptide from influenza A virus: an intrinsic property.

Solid-state NMR has been used to study the influence of lipid bilayer hydrophobic thickness on the tilt of a peptide (M2-TMP) representing the transmembrane portion of the M2 protein from influenza A. Using anisotropic (15)N chemical shifts as orientational constraints, single-site isotopically labeled M2-TMPs were studied in hydrated dioleoylphosphatidylcholine (DOPC) and dimyristoylphosphatidylcholine (DMPC) lipid bilayers oriented between thin glass plates. These chemical shifts provide orientational information for the molecular frame with respect to the magnetic field in the laboratory frame. When modeled as a uniform ideal alpha-helix, M2-TMP has a tilt of 37(+/-3) degrees in DMPC and 33(+/-3) degrees in DOPC with respect to the bilayer normal in these lipid environments. The difference in helix tilt between the two environments appears to be small. This lack of a substantial change in tilt further suggests that significant interactions occur between the helices, as in an oligomeric state, to prevent a change in tilt in thicker lipid bilayers.

Anisotropy↗

PubMed2000

Imaging membrane protein helical wheels.

Resonance patterns have been observed in 2D solid-state NMR spectra of the transmembrane segment of M2 protein from Influenza A virus in oriented samples reflecting the helical wheel of this alpha-helix. The center of this pattern uniquely defines the helical tilt with respect to the bilayer normal without a need for resonance assignments. The distribution of resonances from amino acid specific labels around the "PISA wheel" defines the rotational orientation of the helix and yields preliminary site-specific assignments. With assignments high-resolution structural detail, such as differences in tilt and rotational orientation along the helical axis leading to an assessment of helical coiling, can be obtained.

Influenza A virus↗

PubMed2000

Atomic refinement using orientational restraints from solid-state NMR.

We describe a procedure for using orientational restraints from solid-state NMR in the atomic refinement of molecular structures. Minimization of an energy function can be performed through either (or both) least-squares minimization or molecular dynamics employing simulated annealing. The energy, or penalty, function consists of terms penalizing deviation from "ideal" parameters such as covalent bond lengths and terms penalizing deviation from orientational data. Thus, the refinement strives to produce a good fit to orientational data while maintaining good stereochemistry. The software is in the form of a module for the popular refinement package CNS and is several orders of magnitude faster than previous software for refinement with orientational data. The short computer time required for refinement removes one of the difficulties in protein structure determination with solid-state NMR.

Gramicidin↗

PubMed2000

Transmembrane domain of M2 protein from influenza A virus studied by solid-state (15)N polarization inversion spin exchange at magic angle NMR.

The M2 protein from the influenza A virus forms a proton channel in the virion that is essential for infection. This tetrameric protein appears to form a four-helix bundle spanning the viral membrane. Here the solid-state NMR method, 2D polarization inversion spin exchange at magic angle (PISEMA), has been used to obtain multiple constraints from specifically amino acid-labeled samples. The improvement of spectral resolution from 2D PISEMA over 1D methods and 2D separated local field methods is substantial. The reliability of the method is validated by comparison of anisotropic chemical shift and heteronuclear dipolar interactions from single site labeled samples. The quantitative interpretation of the high-resolution constraints confirms the helix tilt to be within the range of previous experimental determinations (32 degrees -38 degrees ). The binding of the channel inhibitor, amantadine, results in no change in the backbone structure at position Val(27,28), which is thought to be a potential binding site for the inhibitor.

Amino Acid Sequence↗

PubMed2000

Inter- and intramolecular distance measurements by solid-state MAS NMR: determination of gramicidin A channel dimer structure in hydrated phospholipid bilayers.

Distance constraints are an important complement to orientational constraints. While a high-resolution monomer structure of the ion channel forming polypeptide, gramicidin A, has been solved with 120 orientational constraints, the precise geometry of the dimer interface has not been characterized. Here, using both 13C and 15N labeled gramicidin A samples in hydrated phospholipid bilayers, both inter- and intramolecular distances have been measured with a recently developed simultaneous frequency and amplitude modulation (SFAM) solid-state NMR scheme. Using this approach 15N-13C1 residual dipolar couplings across a hydrogen bond as small as 20+/-2 Hz have been characterized. While such distances are on the order of 4.2+/-0.2 A, the spectroscopy is complicated by rapid global motion of the molecular structure about the bilayer normal and channel axis. Consequently, the nominal 40 Hz dipolar coupling is averaged depending on the orientation of the internuclear vector with respect to the motional axis. The intermolecular distance confirmed the previously described monomeric structure, while the intramolecular distance across the monomer-monomer interface defined this junction and confirmed the previous model of this interface.

Bacillus↗

PubMed1999

Water: foldase activity in catalyzing polypeptide conformational rearrangements.

Polypeptide conformer interconversion in a low dielectric environment is shown to be highly dependent on water concentration. Water increases this rate by 10(3), apparently by catalyzing hydrogen bond exchange, and thereby presenting functional properties analogous to that of a foldase. This catalytic effect is demonstrated on the interconversion of a parallel gramicidin dimer into an antiparallel dimer. A Hill coefficient of 6.5 is observed, illustrating the highly cooperative nature of the process. Protein folding in nonpolar environs, such as the hydrophobic core of a protein or the hydrophobic domain of a lipid bilayer, may be contingent on and rate-limited by the scarcity of water.

Dioxanes↗

PubMed1999

Modulating dipoles for structure-function correlations in the gramicidin A channel.

Dipoles of the tryptophan indole side chains have a direct impact on ion conductance in the gramicidin channel. Here, fluorination of the indoles (both 5- and 6-fluoro) is used to manipulate both the orientations and the magnitudes of the dipoles. The orientations and positions with respect to the channel axis were determined using (2)H solid state NMR of uniformly aligned lipid bilayer preparations. By exchange of the remaining four protons in the indole ring for deuterium, comparison could be made to d(5)-indole spectra that have previously been recorded for each of the four indoles of gramicidin A. After making the assignments which were aided by the observation of (19)F-(2)H dipolar interactions, we found that fluorination caused only minor changes in side chain conformation. With the high-resolution structural characterization of the fluorinated indoles in position 11, 13, and 15, the electrostatic interactions with a cation at the channel and bilayer center can be predicted and the influence of the modified dipoles on ion conductance estimated. The importance of the long-range electrostatic interaction was recently documented with the observation of alpha-helical dipoles oriented toward the bilayer center on the ion conductance pathway for the Streptomyces K(+) channel. We present direct measurements of the orientation of gramicidin channel F-Trp positions for use in analysis of dipole effects on channel permeation.

Bacillus↗

PubMed1999

Validation of the single-stranded channel conformation of gramicidin A by solid-state NMR.

The monovalent cation selective channel formed by a dimer of the polypeptide gramicidin A has a single-stranded, right-handed helical motif with 6.5 residues per turn forming a 4-A diameter pore. The structure has been refined to high resolution against 120 orientational constraints obtained from samples in a liquid-crystalline phase lipid bilayer. These structural constraints from solid-state NMR reflect the orientation of spin interaction tensors with respect to a unique molecular axis. Because these tensors are fixed in the molecular frame and because the samples are uniformly aligned with respect to the magnetic field of the NMR spectrometer, each constraint restricts the orientation of internuclear vectors with respect to the laboratory frame of reference. The structural motif of this channel has been validated, and the high-resolution structure has led to precise models for cation binding, cation selectivity, and cation conductance efficiency. The structure is consistent with the electrophysiological data and numerous biophysical studies. Contrary to a recent claim [Burkhart, B. M., Li, N., Langs, D. A., Pangborn, W. A. & Duax, W. L. (1998) Proc. Natl. Acad. Sci. USA 95, 12950-12955], the solid-state NMR constraints for gramicidin A in a lipid bilayer are not consistent with an x-ray crystallographic structure for gramicidin having a double-stranded, right-handed helix with 7.2 residues per turn.

Gramicidin↗

PubMed1999

Cation transport: an example of structural based selectivity.

Through the high-resolution structure of the gramicidin A channel in lamellar phase lipids and the characterization of specific ion peptide interactions, fundamental principles for ion channel selectivity and conductance efficiency are illustrated with atomic resolution detail. Delocalized cation binding in the first turn of the helix reduces the unfavorable entropy contribution upon binding. Stepwise dehydration minimizes the energy barrier for cation entry and provides valence selectivity in this channel. Three or more water molecules in the monovalent cation binding site result in flexibility in the cation solvation environment causing weak cation size selectivity. Lack of cation induced structural modification avoids the formation of a significant energy barrier, thus permitting efficient cation transport.

Amino Acid Sequence↗

PubMed1999

The influenza virus M2 ion channel protein: probing the structure of the transmembrane domain in intact cells by using engineered disulfide cross-linking.

The influenza A virus M2 integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes, and it also modulates the pH of the trans-Golgi network in virus-infected cells. M2 protein is a homo-oligomer of 97 residues with a single transmembrane (TM) domain whose residues encompass the pore region of the channel and the biologically active form of the channel is a homotetramer. To understand the structural arrangement of the TM domains, each residue of the TM domain was changed in turn to cysteine, and oxidative disulfide cross-linking used to identify residues in close proximity. Oxidative treatment of M2 protein in membranes using iodine resulted in maximum cross-linking at TM domain residues 27, 34, and 41. Oxidation of M2 protein in membranes using the catalyst Cu(II)(1,10-phenanthroline)3 resulted in cross-linking of many TM domain residues when the reaction was allowed to proceed at 37 degreesC, suggesting that rotational movements of the TM domains in the membrane can occur. However, analysis of the kinetics of disulfide-linked dimer formation showed that TM domain residues 27, 30, 34, 37, and 41 formed most rapidly. Furthermore, when oxidation was performed at 4 degreesC, maximum cross-linking occurred at TM domain residues 27, 30, 34, 37, and 41. These positions correspond to the a and d positions of a heptad repeat. Thus these biochemical data are consistent with the TM domain region of the M2 tetramer forming a four-helix bundle. Analysis of the disulfide bonds that formed when oxidation of M2 protein in membranes was performed at pH 5.2 showed greatly reduced cross-linking at TM domain residues 40, 42, and 43 than that found at pH 7.4. This pH-dependent change in cross-linking of residues toward the cytoplasmic side of the TM domain parallels with the activation of the M2 ion channel at low pH.

Amino Acid Sequence↗

PubMed1999

13C selective polarization and spin diffusion in a lipid bilayer-bound polypeptide by solid-state NMR.

While (15)N solid-state NMR has proven to be very advantageous for the development of structural biological methods, (13)C spectroscopy has increased sensitivity and spectral dispersion. However, large natural abundance signals and homonuclear dipolar interactions pose significant problems. Here we have used a pair of (13)C-labeled sites in a lipid-solubilized polypeptide to show the selective polarization can be used in combination with spin diffusion to achieve simplified spectra. Both unoriented and oriented samples have been used, with the latter providing a well-resolved homonuclear dipolar splitting.

Carbon Isotopes↗