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T A Cross

Publications and source records attributed to T A Cross.

At least 55 records · Page 3Linked to original sources
PubMed1997

Solid-state nuclear magnetic resonance characterization of gramicidin channel structure.

The method of using orientational constraints derived from solid-state NMR for structural characterization of polypeptides in heterogeneous environments has now been demonstrated. A very high resolution structure has been achieved that has led to greater functional understanding of this channel. Much can be done to improve this structural technique to make it more efficient and more generally applicable. Others as well as ourselves are applying this approach to membrane proteins. Although solid-phase synthesis and specific site isotopic labeling has been essential for the development described here, one of the primary challenges is to be able to use amino acid-specific and uniform labeling of peptides and proteins by biosynthetic means for isotopic incorporation. This will allow for the study of many more proteins and significantly large proteins. Unlike solution NMR structural methods, there are no intrinsic molecular weight limitations. In fact, as the molecular weight increases the molecular motion will become less and the spectroscopic properties will improve. The major limitation will be sensitivity: as the molecular weight increases the number of moles will decrease in the samples, causing sensitivity to decrease. Advances in field strength and NMR technology help to address this problem. With larger molecules and more isotopically labeled sites resolution could also be a problem; however, the two- and three-dimensional methods demonstrated by Opella and co-workers clearly show the potential for enormous resolving power. In the 15N dimension alone it is shown that the resolution is greater than in solution NMR. Although challenges such as spectral assignments have yet to be completely solved, several approaches have been described, and the prospects are excellent for solving this and other problems facing the development of this novel approach for structural elucidation. Although there is an attempt to get away from solid-phase synthesis to solve larger molecular weight structures, peptide synthesis will continue to be important for generating single- and double-site labeled model compounds for characterizations of spin interaction tensors. Such characterizations will continue to be a very important aspect of this structural approach.

Gramicidin↗
PubMed1997

High resolution and high fields in biological solid state NMR.

2H solid state NMR spectra of a polypeptide in an oriented membrane environment is demonstrated to have an orientational resolution of 0.3 degree. Such data results in high resolution structural constraints. Similar spectra are demonstrated at 23.2 T using a resistive magnet at the National High Magnetic Field Laboratory.

Anti-Bacterial Agents↗
PubMed1996

Monovalent cation transport: lack of structural deformation upon cation binding.

Cations often deform the structure of regulatory proteins to affect a functional response, but for other protein functions a more passive effect is desired. For instance, it is shown here that in the conductance of Na+ by the gramicidin channel there appears to be no significant structural deformation of either the side chains or backbone upon Na+ binding in the channel. This is based on 15N and 13C chemical shifts, 2H quadrupolar interactions, and 15N-2H dipolar interactions obtained by solid-state NMR spectroscopy of uniformly aligned lipid bilayer preparations of the gramicidin channel in the presence and absence of Na+. This conclusion is despite some significant changes in the 15N alpha and 13C1 chemical shift values which are argued here to be the result of indirect polarization effects upon cation binding rather than reflections of structural and dynamic changes. The lack of structural deformation implies that Na+ moves to the carbonyl oxygens lining the pore of this channel for solvation rather than the carbonyl groups moving in toward the channel axis. This forces the cations onto a helical path following the positions of the carbonyl oxygens around the channel pore. Furthermore, an ideal binding site geometry for Na+ in the channel is avoided. Instead, adequate binding energy is provided by the channel to compensate for the loss of hydration energy when the cations enter the channel. The avoidance of strong binding ensures that efficient transport of the cations through the channel can be realized.

Amino Acid Sequence↗
PubMed1996

Conformational trapping in a membrane environment: a regulatory mechanism for protein activity?

Functional regulation of proteins is central to living organisms. Here it is shown that a nonfunctional conformational state of a polypeptide can be kinetically trapped in a lipid bilayer environment. This state is a metastable structure that is stable for weeks just above the phase transition temperature of the lipid. When the samples are incubated for several days at 68 degrees C, 50% of the trapped conformation converts to the minimum-energy functional state. This result suggests the possibility that another mechanism for functional regulation of protein activity may be available for membrane proteins: that cells may insert proteins into membranes in inactive states pending the biological demand for protein function.

Magnetic Resonance Spectroscopy↗
PubMed1996

Macromolecular structural elucidation with solid-state NMR-derived orientational constraints.

The complete structure determination of a polypeptide in a lipid bilayer environment is demonstrated built solely upon orientational constraints derived from solid-state NMR observations. Such constraints are obtained from isotopically labeled samples uniformly aligned with respect to the B(0) field. Each observation constrains the molecular frame with respect to B(0) and the bilayer normal, which are arranged to be parallel. These constraints are not only very precise ( a few tenths of a degree), but also very accurate. This is clearly demonstrated as the backbone structure is assembled sequentially and the i to i + 6 hydrogen bonds in this structure of the gramicidin channel are shown on average to be within 0.5 A of ideal geometry. Similarly, the side chains are assembled independently and in a radial direction from the backbone. The lack of considerable atomic overlap between side chains also demonstrates the accuracy of the constraints. Through this complete structure, solid-state NMR is demonstrated as an approach for determining three-dimensional macromolecular structure.

Animals↗
PubMed1996

Hydrogen exchange in the lipid bilayer-bound gramicidin channel.

Hydrogen exchange experiments for a membrane-bound polypeptide could lead to interesting functional and structural insights. Here, hydrogen/deuterium exchange, saturation transfer and differential relaxation experiments have been performed on oriented lipid bilayer-bound polypeptide samples to measure the exchange lifetimes. The polypeptide, gramicidin A, forms a monovalent cation selective channel across membranes. The pH dependent results suggest that the indole N epsilon 1-H groups show base catalyzed hydrogen exchange, but that the backbone amide sites are not base catalyzed, consistent with the exclusion of anions from this channel. Furthermore, the recently described [1] orientational distribution of the individual peptide carbonyls (i.e. carbonyls either tipped slightly in toward or away from the channel axis) is consistent with the observed difference in odd- and even-numbered amide residue exchange lifetimes.

Deuterium↗
PubMed1995

Tryptophan dynamics and structural refinement in a lipid bilayer environment: solid state NMR of the gramicidin channel.

Tryptophans in the gramicidin channel are important for defining the conformation and the orientation with respect to the bilayer normal and for facilitating cation conductance. Here, high-resolution structure and dynamics of these rings are characterized by solid state NMR. Both oriented and unoriented lipid bilayer preparations are used. Fast frozen lipid bilayer preparations of unoriented samples have been used to obtain static characterizations of nuclear spin interaction tensors. The temperature dependence of these unoriented samples and the spectral features of fast frozen oriented samples were used to experimentally define the local motions of the individual indole rings. Local motions were shown to have amplitudes as high as +/- 29 degrees, and the motions were dominated by libration about the chi 2 axis. The high-resolution structure has been achieved by interpreting seven precise (+/- 0.3 degree) orientational constraints from 2H and 15N NMR for each indole ring in light of the motionally averaged interaction tensors. Each of the four indoles is restricted to a unique orientation of the ring with respect to the bilayer normal and one of two possible rotameric states. The side chain torsion angles for each residue are very similar, generating similar electric dipole moment orientations with respect to the channel.

Amino Acid Sequence↗
PubMed1995

Tryptophan hydrogen bonding and electric dipole moments: functional roles in the gramicidin channel and implications for membrane proteins.

The known high-resolution structure and dynamics characterization of the lipid bilayer-bound polypeptide gramicidin A provides a unique opportunity to study structure-function and dynamics-function correlations in a model membrane protein. In particular, the indoles have a variety of very important functional roles in this cation channel that will undoubtedly be recognized in membrane proteins. That indoles and phenols are oriented at the hydrophobic-hydrophilic interface of lipid bilayers is already well-recognized in membrane proteins. The most buried indole of the gramicidin channel, Trp9, is shown by 15N solid state NMR to be exposed to the hydrophilic surface through hydrogen exchange. Here the importance of the indole dipole moments is described for cation conductance. Preparation of samples with high concentrations of Na+ is shown by high-resolution orientational constraints derived from 2H NMR to have no structural effect on the indole side chain conformations. These dipoles stabilize cations in the binding sites near the channel entrance and substantially reduce the potential energy barrier at the bilayer center. This latter finding conclusively documents that the rate-limiting step in cation conductance by this channel involves the barrier at the bilayer center. Furthermore, dynamics of the indole rings cause significant fluctuations in the energy of stabilization at the binding site that may result in a rapid mechanism for gating the channel.

Electrochemistry↗
PubMed1995

Correlations between function and dynamics: time scale coincidence for ion translocation and molecular dynamics in the gramicidin channel backbone.

The polypeptide backbone of gramicidin lines the channel lumen, which maintains a single file string of water molecules and the occasional cation. Whether or not local motions of the backbone are important for facilitating cation transport has been the subject of debate. Here it is demonstrated that motions of the backbone occur on the same time scale as cation translation through this transmembrane channel. To characterize the frequency of the local motions, field-dependent 15N T1 relaxation times were interpreted in light of an independently determined motional model that defines the motions as occurring about the C alpha-C alpha axis. To overdamp these local motions, several models that involve extensive correlations of the molecular motions were considered. One of these schemes for correlated motions suggests a unidirectional reaction path that would minimize the time necessary for the cation to pass through the low-dielectric center of the bilayer.

Amino Acid Sequence↗
PubMed1995

Lipid-peptide interface: valine conformation and dynamics in the gramicidin channel.

High-resolution dynamic and structural characterizations have been achieved for each of the valine side chains of the gramicidin channel while solubilized in hydrated lipid bilayers. The characterizations have been achieved by 2H NMR spectra of both oriented and unoriented samples obtained at 36 and 5 degrees C, respectively. Powder patterns displaying intermediate time frame averaging provide dynamic information, and quadrupole splittings from aligned samples provide orientational constraints for the side chain structure. Librational amplitudes for each site throughout the side chain have also been characterized. Val6 and Val8 are shown to be fixed in rotameric states, potentially constraining two of the indole rings and the functionally important indole dipole moment orientations. Val1 and Val7 undergo three-state jump motions. The jump frequencies increase from the microsecond to nanosecond time frame upon increasing the temperature through the lipid phase transition. For the same temperature range, there is no evidence for changes in conformational state populations. Despite small differences in the substate populations for the two residues, the motions may be loosely coupled as indicated by the high-resolution structure.

Gramicidin↗
PubMed1995

Low-temperature solid-state 15N NMR characterization of polypeptide backbone librations.

The local molecular dynamics of gramicidin A in dimyristoylphosphatidylcholine bilayers were probed by a combination of low-temperature solid-state 15N NMR and rapid freezing of samples using liquid propane. It has previously been shown that this approach leads to sharp discontinuities in powder-pattern spectra of the gramicidin channel in lipid bilayers, and hence, an accurate determination of tensor element magnitudes is achieved. The static-tensor-element magnitudes determined here using hydrated bilayer samples at low temperatures are different from the values obtained from dry-powder samples at 308 K. However, for the one site studied, the orientation of the 15N chemical-shift tensor in the molecular frame is the same for both types of samples. Averaging of the chemical-shift tensor between 200 and 263 K is shown to be anisotropic and consistent with a motional axis parallel with the C alpha-C alpha axis for adjacent residues. The librational amplitudes determined here range from +/- 14 degrees to +/- 22 degrees at 263 K and are significantly larger than the nanosecond librations characterized by relaxation studies. The nanosecond motions are thought to represent correlated motions, while the librations described here are the sum of correlated (nanosecond) and uncorrelated (picosecond) motions. The picosecond librational amplitudes become considerably larger and more isotropic above 0 degree for the residues near the bilayer surface.

Amino Acid Sequence↗
PubMed1994

Polypeptide conformational space. Dynamics by solution NMR disorder by X-ray crystallography.

A symmetric dimer of a polypeptide in an organic solvent is shown to be a useful molecular system for observing the effects of crystal packing and solution dynamics on structure determination by X-ray crystallography and solution NMR methods. Organic solvents are not ideal models of a lipid environment, but they represent a better model environment than does water, in fact, for modeling some properties of the lipid environment, organic solvents work very well. In particular, it is a good model for assessing the influence of lipids on local polypeptide dynamics and conformational rearrangements. The solution NMR structure of gramicidin in the mixed solvent of benzene and ethanol is compared to the crystal structure formed from a benzene/ethanol azeotrope. High resolution structural characterization leads to unique correlations of disorder in the crystal to dynamics in solution. Furthermore, crystal packing effects lead to significant asymmetries in both the polypeptide backbone and in the side-chains.

Amino Acid Sequence↗
PubMed1994

Structural analysis of highly oriented poly(p-phenylene-terephthalamide) by 15N solid-state nuclear magnetic resonance.

The structure of an aromatic polyamide, poly(p-phenylene-terephthalamide) (PPTA), was studied in the solid state using 15N nuclear magnetic resonance (NMR) spectroscopy. Spectra of uniaxially aligned molecules placed with the axis of alignment both parallel with and perpendicular to the applied magnetic field were analyzed to yield the orientations of specific molecular bonds with respect to the fiber axis. The 15N chemical shift tensor was characterized by simulating powder pattern spectra of both PPTA and a model compound, benzanilide. Chemical shift and dipolar coupled chemical shift line shapes were calculated through Euler angle transformations from the principal axis system (PAS) reference frame to the fiber axis system (FAS) frame. The orientations of NH and NC' bonds in PPTA are determined as well as the orientational distribution of the PPTA fiber axis. The structural parameters determined for PPTA are compared with those obtained by X-ray diffraction.

Magnetic Resonance Spectroscopy↗
PubMed1994

Side-chain structure and dynamics at the lipid-protein interface: Val1 of the gramicidin A channel.

High resolution dynamics and structural information has been resolved from 2H solid-state NMR spectra of the Val-1 side-chain of the gramicidin channel in a lipid bilayer. Both powder pattern lineshapes and spectra from uniformly aligned samples of gramicidin in lipid bilayers have been analyzed to achieve a fully consistant interpretation of the data. Torsional motions about the C alpha C beta axis (chi 1) are shown to be three-state jumps in which the occupancy of the states is given by the ratio, 75:15:10 for the chi 1 angles of 184 degrees:304 degrees:64 degrees. The dominant conformer is also the most common conformation observed for valines in well defined protein structures. The distribution of conformational substates that represents the chi 1 dynamics appears to be largely independent of the lipid phase transition and the hydration of the sample. However, there is evidence that the residence time between jumps is dependent on the lipid phase transition. Although this time is shown to be approximately 1 microseconds below the phase transition temperature, it is in the fast exchange limit above the transition temperature.

Amino Acid Sequence↗
PubMed1993

Rapidly-frozen polypeptide samples for characterization of high definition dynamics by solid-state NMR spectroscopy.

A method is described for defining anisotropic local dynamics in polypeptides by solid-state NMR. To avoid conformational heterogeneity introduced by large hexagonal ice crystals in low temperature hydrated samples, a fast-freezing technique is used for sample preparation. For a demonstration of this approach, the backbone librational motions of the gramicidin A channel conformation are studied in hydrated DMPC bilayers. The static 15N chemical shift tensor is characterized at 123 K for the Ala3 site. The temperature dependence of this tensor yields a determination of the librational amplitude and anisotropy of the motionally sampled space. This amplitude represents the sum of nanosecond and picosecond time-frame motions, both of which have a significant amplitude.

Alanine↗
PubMed1993

High-resolution conformation of gramicidin A in a lipid bilayer by solid-state NMR.

Solid-state nuclear magnetic resonance spectroscopy of uniformly aligned preparations of gramicidin A in lipid bilayers has been used to elucidate a high-resolution dimeric structure of the cation channel conformation solely on the basis of the amino acid sequence and 144 orientational constraints. This initial structure defines the helical pitch as single-stranded, fixes the number of residues per turn at six to seven, specifies the helix sense as right-handed, and identifies the hydrogen bonds. Refinement of this initial structure yields reasonable hydrogen-bonding distances with only minimal changes in the torsion angles.

Amino Acid Sequence↗
PubMed1993

Tryptophans in membrane proteins: indole ring orientations and functional implications in the gramicidin channel.

Orientational constraints generated from solid-state NMR of uniformly aligned gramicidin A in hydrated lipid bilayers have been used to determine the indole ring orientations for the four tryptophans of the gramicidin A monomer with respect to the bilayer normal and the channel axis. 15N epsilon 1 labeled tryptophan has been incorporated into gramicidin at positions 9, 11, 13, and 15. The chemical shift tensor orientation has been oriented with respect to the N-H bond via doubly labeled sample in which the 15N epsilon 1-1H has been exchanged for 2H. By observation of the dipolar coupled 15N chemical shift powder pattern of the amino acid, sigma cc has been shown to be perpendicular to the plane of the ring and that sigma aa makes an angle of 25 degrees with respect to the N-H bond. The indole ring orientations were obtained from a consideration of both the chemical shift and the 15N-1H dipolar interaction. These four rings have very similar orientations with respect to the bilayer normal as given by the range of angles between the bilayer and ring normals (64-67 degrees). Furthermore, the N-H bond orientations with respect to the bilayer normal varies by only 10 degrees among the four sites. This orientational analysis has been based on an assumption that large amplitude librational motions in the hydrated bilayer samples are not averaging the nuclear spin interactions. This assumption was verified by analyzing the 2H quadrupole spectra of d5-Trp11-labeled gramicidin A in oriented preparations. The orientations predicted for the five C-H bonds in the indole ring from the 15N data agreed (root-mean-square deviation of 3.7 degrees) with the observed orientations from quadrupole splittings of the C-D bonds in the ring. From the orientation of the indole rings with respect to the bilayer normal and a polypeptide backbone conformation, the four typtophans of the gramicidin monomer are oriented with respect to the backbone of the channel conformation. The similarity among the indole orientations with respect to the bilayer normal is therefore consistent with the electrophysiological results that the individual replacement of the indole rings with phenyl rings results in a incremental decrease in the conductance of the channels formed. The indole orientations with respect to the backbone as defined by the side-chain torsion angles is not uniquely determined but yields a discrete set of possible values.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗