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T A Cross

Publications and source records attributed to T A Cross.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1999

Solid-state NMR and hydrogen-deuterium exchange in a bilayer-solubilized peptide: structural and mechanistic implications.

Hydrogen-deuterium exchange has been monitored by solid-state NMR to investigate the structure of gramicidin M in a lipid bilayer and to investigate the mechanisms for polypeptide insertion into a lipid bilayer. Through exchange it is possible to observe 15N-2H dipolar interactions in oriented samples that yield precise structural constraints. In separate experiments the pulse sequence SFAM was used to measure dipolar distances in this structure, showing that the dimer is antiparallel. The combined use of orientational and distance constraints is shown to be a powerful structural approach. By monitoring the hydrogen-deuterium exchange at different stages in the insertion of peptides into a bilayer environment it is shown that dimeric gramicidin is inserted into the bilayer intact, i.e., without separating into monomer units. The exchange mechanism is investigated for various sites and support for a relayed imidic acid mechanism is presented. Both acid and base catalyzed mechanisms may be operable. The nonexchangeable sites clearly define a central core to which water is inaccessible or hydroxide or hydronium ion is not even momentarily stable. This provides strong evidence that this is a nonconducting state.

Binding Sites↗
PubMed1999

Solid-state nuclear magnetic resonance investigation of protein and polypeptide structure.

Solid-state nuclear magnetic resonance (NMR) is rapidly emerging as a successful and important technique for protein and peptide structural elucidation from samples in anisotropic environments. Because of the diversity of nuclei and nuclear spin interactions that can be observed, and because of the broad range of sample conditions that can be studied by solid-state NMR, the potential for gaining structural constraints is great. Structural constraints in the form of orientational, distance, and torsional constraints can be obtained on proteins in crystalline, liquid-crystalline, or amorphous preparations. Great progress in the past few years has been made in developing techniques for obtaining these constraints, and now it has also been clearly demonstrated that these constraints can be assembled into uniquely defined three-dimensional structures at high resolution. Although much progress toward the development of solid-state NMR as a routine structural tool has been documented, the future is even brighter with the continued development of the experiments, of NMR hardware, and of the molecular biological methods for the preparation of labeled samples.

Anisotropy↗
PubMed1999

The influence of plasma on basal and ACTH-stimulated in vitro adrenocortical steroidogenesis.

Early descriptions of in vitro ACTH bioassays all emphasised the need to use extracted plasma samples due to interference by an unidentified component. The aim of these studies was to elucidate the effects of whole plasma on ACTH steroidogenic activity in vitro and to identify the responsible factor. A sensitive in vitro dispersed bovine adrenocortical cell bioassay was established. The addition of 10% ACTH-depleted human pooled plasma to the incubation media resulted in basal steroidogenesis equivalent to that achieved with 10(-9) M ACTH1-24 and potentiated the steroidogenic activity of 10(-9) M ACTH1-24 by 7.8-fold. This potentiation was dependent on the concentration of both ACTH and plasma in the media, but did not result from the mitogenic effect of plasma. A pituitary source was excluded and the potentiating activity was not extractable by Vycor glass. Column chromatography demonstrated two peaks of activity corresponding to molecular weights of 650 and 220x10(3) Da. These peaks did not correspond to the plasma binding of 125I-ACTH which resulted from non-specific binding to albumin. Lipoprotein-deficient serum had no effect on either basal or ACTH-stimulated steroidogenesis, but both were restored by the addition of purified lipoproteins. However, novel findings demonstrated a differential effect of low (LDL) and high (HDL) density lipoproteins on basal and ACTH-stimulated steroid production; thus, LDL exerted a greater effect on the former, whilst HDL potentiated the steroidogenic activity of added ACTH more than LDL. The addition of the lipoproteins to lipoprotein-deficient serum restored its basal and ACTH potentiating effects, the cholesterol concentrations of the chromatographic fractions exactly paralleling their ACTH potentiating effect. These findings suggest that not only are lipoproteins the plasma factor(s) which potentiates ACTH steroidogenic activity in in vitro bioassays, but also that they exert differential effects on basal and ACTH-stimulated steroid production.

Adrenocorticotropic Hormone↗
PubMed1999

Correlations of structure, dynamics and function in the gramicidin channel by solid-state NMR spectroscopy.

The high resolution structure of the gramicidin A channel has been determined in a lamellar phase environment using solid-state NMR spectroscopy. While the fold is similar to previous characterizations, channel function is exquisitely dependent on structural detail. There is essentially no structural change upon cation binding and no significant change in dynamics. The cations appear to be adequately solvated in their binding site by no more than two carbonyls and no fewer than three water molecules at any one time. The relatively large number of water molecules allows for geometric flexibility and little selectivity among monovalent cations. However, the dehydration energies of cations clearly explain the selectivity for monovalent versus divalent cations. Moreover, the binding site is shown to be delocalized, resulting in a shallow potential energy well so that efficient cation conductance can be realized. The potential energy barrier at the bilayer centre has been shown to be rate limiting under certain circumstances through a correlation between conductance and the electrostatic interactions between cations at the gramicidin monomer-monomer junction and the indole dipole moments at the lipid-water interface. The dynamics are functionally important. The time-scale for carbonyl fluctuations about the C alpha-C alpha axis and kinetic rates for cation movement in the channel are the same, suggesting a correlation between molecular dynamics and kinetics. These functional correlations will be described in light of the recent K+ channel structure and the biological challenge to achieve both selectivity and efficiency.

Amino Acid Sequence↗
PubMed1998

Cation binding induced changes in 15N CSA in a membrane-bound polypeptide.

Cation binding to the monovalent cation selective channel, gramicidin A, is shown to induce changes in the dipolar and chemical shift observables from uniformly aligned samples. While these changes could be the result of structural or dynamic changes, they are shown to be primarily induced by through-bond polarizability effects when cations are solvated by the carbonyl oxygens of the peptide backbone. Upon cation binding partial charges are changed throughout the peptide plane, inducing large changes in the 13C1 chemical shifts, smaller changes in the 15N chemical shifts, and even smaller effects for the 15N-13C1 and 15N-2H dipolar interactions. These conclusions are substantiated by characterizing the 15N chemical shift tensors in the presence and absence of cations in fast-frozen lipid bilayer preparations of gramicidin A.

Anti-Bacterial Agents↗
PubMed1998

Noncontact dipole effects on channel permeation. I. Experiments with (5F-indole)Trp13 gramicidin A channels.

Gramicidin A (gA), with four Trp residues per monomer, has an increased conductance compared to its Phe replacement analogs. When the dipole moment of the Trp13 side chain is increased by fluorination at indole position 5 (FgA), the conductance is expected to increase further. gA and FgA conductances to Na+, K+, and H+ were measured in planar diphytanoylphosphatidylcholine (DPhPC) or glycerylmonoolein (GMO) bilayers. In DPhPC bilayers, Na+ and K+ conductances increased upon fluorination, whereas in GMO they decreased. The low ratio in the monoglyceride bilayer was not reversed in GMO-ether bilayers, solvent-inflated or -deflated bilayers, or variable fatty acid chain monoglyceride bilayers. In both GMO and DPhPC bilayers, fluorination decreased conductance to H+ but increased conductance in the mixed solution, 1 M KCl at pH 2.0, where K+ dominates conduction. Eadie-Hofstee plot slopes suggest similar destabilization of K+ binding in both lipids. Channel lifetimes were not affected by fluorination in either lipid. These observations indicate that fluorination does not change the rotameric conformation of the side chain. The expected difference in the rate-limiting step for transport through channels in the two bilayers qualitatively explains all of the above trends.

Biophysical Phenomena↗
PubMed1997

Protein stability and conformational rearrangements in lipid bilayers: linear gramicidin, a model system.

The replacement of four tryptophans in gramicidin A by four phenylalanines (gramicidin M) causes no change in the molecular fold of this dimeric peptide in a low dielectric isotropic organic solvent, but the molecular folds are dramatically different in a lipid bilayer environment. The indoles of gramicidin A interact with the anisotropic bilayer environment to induce a change in the molecular fold. The double-helical fold of gramicidin M, as opposed to the single-stranded structure of gramicidin A, is not compatible with ion conductance. Gramicidin A/gramicidin M hybrid structures have also been prepared, and like gramicidin M homodimers, these dimeric hybrids appear to have a double-helical fold, suggesting that a couple of indoles are being buried in the bilayer interstices. To achieve this equilibrium structure (i.e., minimum energy conformation), incubation at 68 degrees C for 2 days is required. Kinetically trapped metastable structures may be more common in lipid bilayers than in an aqueous isotropic environment. Structural characterizations in the bilayers were achieved with solid-state NMR-derived orientational constraints from uniformly aligned lipid bilayer samples, and characterizations in organic solvents were accomplished by solution NMR.

Dimerization↗
PubMed1997

Transmembrane four-helix bundle of influenza A M2 protein channel: structural implications from helix tilt and orientation.

The transmembrane portion of the M2 protein from the Influenza A virus has been studied in hydrated dimyristroylphosphotidylcholine lipid bilayers with solid-state NMR. Orientational constraints were obtained from isotopically labeled peptide samples mechanically aligned between thin glass plates. 15N chemical shifts from single site labeled samples constrain the molecular frame with respect to the magnetic field. When these constraints are applied to the peptide, modeled as a uniform alpha-helix, the tilt of the helix with respect to the bilayer normal was determined to be 33 degrees +/- 3 degrees. Furthermore, the orientation about the helix axis was also determined within an error of +/- 30 degrees. These results imply that the packing of this tetrameric protein is in a left-handed four-helix bundle. Only with such a large tilt angle are the hydrophilic residues aligned to the channel axis.

Amino Acid Sequence↗
PubMed1997

Protein structural analysis from solid-state NMR-derived orientational constraints.

High-resolution orientational constraints from solid-state NMR spectroscopy of uniformly aligned biological macromolecules provide a great structural analysis problem. Several approaches to this problem have been made in the past. Here a vector algebra method is developed that provides analytical solutions for the torsion angles and a concise and simple view of the structural possibilities. Numerical instabilities in this approach are easily predicted. Insight into how the structural ambiguities arise in the first place and how they can be reduced in number is demonstrated with this new approach.

Gramicidin↗