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Constraints on the proximity of the carboxyl carbons of the Asp-85 and Asp-212 side chains to the 14-carbon of the retinal chromophore have been established for the bR(555), bR(568), and M(412) states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via (13)C-(13)C magnetization exchange, which was observed in two-dimensional RF-driven recoupling (RFDR) and spin diffusion experiments. A comparison of relative RFDR cross-peak intensities with simulations of the NMR experiments yields distance measurements of 4.4 +/- 0.6 and 4.8 +/- 1.0 A for the [4-(13)C]Asp-212 to [14-(13)C]retinal distances in bR(568) and M(412), respectively. The spin diffusion data are consistent with these results and indicate that the Asp-212 to 14-C-retinal distance increases by 16 +/- 10% upon conversion to the M-state. The absence of cross-peaks from [14-(13)C]retinal to [4-(13)C]Asp-85 in all states and between any [4-(13)C]Asp residue and [14-(13)C]retinal in bR(555) indicates that these distances exceed 6.0 A. For bR(568), the NMR distance constraints are in agreement with the results from recent diffraction studies on intact membranes, while for the M state the NMR results agree with theoretical simulations employing two bound waters in the region of the Asp-85 and Asp-212 residues. The structural information provided by NMR should prove useful for refining the current understanding of the role of aspartic acid residues in the proton-pumping mechanism of bR.

15N solid-state NMR (SSNMR) spectra of guanidyl-15N-labeled bacteriorhodopsin (bR) show perturbation of an arginine residue upon deprotonation of the retinal Schiff base during the photocycle. At the epsilon position, an upfield shift of 4 ppm is observed while the eta nitrogens develop a pair of 'wing' peaks separated by 24 ppm. Proton-driven spin diffusion between the two 'wing' peaks indicates that they arise from a single Arg residue. An unusually asymmetric environment for this residue is indicated by comparison with guanidyl-15N chemical shifts in a series of arginine model compounds. The 'wing' peaks are tentatively assigned to Arg-82 on the basis of the SSNMR investigations of the alkaline and neutral dark-adapted forms of the D85N bacteriorhodopsin mutant. Another, less asymmetric pair of eta signals, that is not affected by Schiff base deprotonation or D85 mutation, is tentatively assigned to Arg-134. The results are discussed in relation to existing models of bR structure and function.

To enforce vectorial proton transport in bacteriorhodopsin (bR), it is necessary that there be a change in molecular structure between deprotonation and reprotonation of the chromophore-i.e., there must be at least two different M intermediates in the functional photocycle. We present here the first detection of multiple M intermediates in native wild-type bacteriorhodopsin by solid-state NMR. Illumination of light-adapted [zeta-15N-Lys]-bR at low temperatures shifts the 15N signal of the retinal Schiff base (SB) downfield by about 150 ppm, indicating a deprotonated chromophore. In 0.3 M Gdn-HCl at pH 10.0, two different M states are obtained, depending on the temperature during illumination. The M state routinely prepared at the lower temperature, Mo, decays to the newly observed M state, Mn, and the N intermediate, as the temperature is increased. Both relax to bR568 at 0 degreesC. A unique reaction sequence is derived: bR568-->Mo-->(Mn+N)-->bR568. Mo and Mn have similar chemical shifts at [12-13C]ret, [14-13C]ret, and [epsilon-13C]Lys216, indicating that Mn, like Mo, has a 13-cis and C=N anti chromophore. However, a small splitting in the [14-13C]ret signal of Mo reveals that it has at least two substates. The 7 ppm greater shielding of the SB nitrogen in Mn compared to Mo suggests an increase in basicity and/or hydrogen bonding. Probing the peptide backbone of the protein, via [1-13C]Val labeling, reveals a substantial structural change between Mo and Mn including the relaxation of perturbations at some sites and the development of new perturbations at other sites. The combination of the change in the protein structure and the increase in the pKa of the SB suggests that the demonstrated Mo-->Mn transition may function as the "reprotonation switch" required for vectorial proton transport.

The L550 intermediate in the bacteriorhodopsin (bR) photocycle has drawn much attention with respect to the mechanism of light-driven proton transport because it selectively releases the Schiff base (SB) proton to the extracellular proton channel in the L-->M transition. Here we extend our solid-state NMR studies of bR photocycle intermediates to the L state. Under conditions that stabilize L550, a new SB signal is detected in the 15N NMR spectrum which disappears upon thermal relaxation. This signal is in the range for a protonated SB, but downfield from the SB signals of bR568 and N520. Since steric interactions would have the opposite effect on shielding, the data argue against a 13,14-dicis chromophore in L550. Comparison with the 15N chemical shifts of halide salts of protonated Schiff bases (pSB's) of retinal suggests that the interaction of the SB with its counterion is significantly stronger in L550 than in N520 (which in turn is stronger than in bR568). This is consistent with models of the early photocycle in which the electrostatic interaction between the SB and its counterion constitutes an important constraint. Although the L counterion interaction is comparable to that of a 6-s-trans,13-cis chloride salt, the visible spectrum is strongly red-shifted from the lambdamax = 491 nm of the chloride. This suggests some double bond strain in L550, particularly about the C=N bond. This strain is apparently gone in the N intermediate, which has a normal relationship between the 15N chemical shift and lambdamax. Such a relaxed chromophore is consistent with orientation of the SB proton toward the cytoplasmic surface in the N intermediate.

Electron paramagnetic resonance spectroscopy at 139.5 GHz has been used to study p21 ras complexed with Mn(II) and guanosine 5'-(beta, gamma-imidotriphosphate), an analog of GTP. The p21 sample studied was selectively labeled with [17O gamma]threonine to a final enrichment of 30%. A Mn(II)-17O hyperfine interaction was observed, but the value of the coupling constant, 0.11 +/- 0.04 mT, is the smallest such value yet reported. Ab initio calculations indicate that this value is consistent with direct coordination of the threonine hydroxyl group and provide an estimate for the Mn(II)-17O bond length of 2.7 A. The measured hyperfine coupling constant and associated bond length starkly contrast with typical values for Mn(II)-17O coordination complexes, namely, approximately 0.25 mT and approximately 2.2 A, respectively. This contrast underscores the peculiar weakness of this Mn(II)-O interaction in p21 and persuasively argues that the nucleotide-induced conformational change, which is known to encompass the region of p21 involving Thr35, is not driven by Mn(II) coordination of the Thr35 hydroxyl group.

As a molecular switch, the ras protein p21 undergoes structural changes that couple recognition sites on the protein surface to the guanine nucleotide-divalent metal ion binding site. X-ray crystallographic studies of p21 suggest that coordination between threonine-35 and the divalent metal ion plays an important role in these conformational changes. Recent ESEEM studies of p21 in solution, however, place threonine-35 more distant from the metal and were interpreted as weak or indirect coordination of this residue. We report high frequency (139.5 GHz) EPR spectroscopy of p21.Mn(II) complexes of two guanine nucleotides that probes the link between threonine-35 and the divalent metal ion. By analysis of high-frequency EPR spectra, we determine the number of water molecules in the first coordination sphere of the manganous ion to be four in p21.Mn(II).GDP, consistent with prior low-frequency EPR and X-ray crystallographic studies. In the complex of p21 with a GTP analog, p21.Mn(II).GMPPNP, we determine the hydration number to be 2, also consistent with crystal structures. This result rules out indirect coordination of threonine-35 in the solution structure of p21.Mn(II).GMPPNP, and implicates direct, weak coordination of this residue as suggested by Halkides et al. [(1994) Biochemistry 33,4019]. The 17O hyperfine coupling constant of H2(17)O is determined as 0.25 mT in the GDP from and 0.28 mT in the GTP form. These values are similar to reported values for 17O-enriched aquo ligands and some phosphato ligands in Mn(II) complexes. The high magnetic field strength (4.9 T) employed in these 139.5 GHz EPR measurements leads to a narrowing of the Mn(II) EPR lines that facilitates the determination of 17O hyperfine interactions.

Previous solid state 13C NMR studies of bacteriorhodopsin (bR) have inferred the C = N configuration and the protonation state of the retinal-lysine Schiff base (SB) linkage from the [13-13C]-retinal, [14-13C]retinal, and [epsilon-13C]lysine-216 chemical shifts in the bR555, bR568, and M412 states. Here we determine the C = N configuration and the protonation state of the N photointermediate that is cryotrapped along with the M photointermediate at high salt concentrations (0.1 M NaCl) and high pH (10.0). We obtained 13C and 15N SSN MR spectra of [epsilon-15N]lysine bR and [12-13C]- and [13-13C]retinal bR for samples illuminated under the above conditions. Two species are observed, both of which decay to bR568 upon warming. One species has chemical shifts identical to those obtained previously for M thermally trapped in guanidine.HCl at high pH (Smith et al., 1989a; Farrar et al., 1993). In the other species, the [epsilon-15N]lysine and 13-13C chemical shifts indicate that the SB is protonated, the 12-13C shift indicates a 13 = 14 cis configuration, and the previously published [14-13C]- and [epsilon-13C]lysine shifts indicate a C = N anti configuration. These results are consistent with other studies of the N photointermediate.

Solid-state deuterium NMR spectroscopy is used to examine the dynamic behavior of 18-CD3 methyl groups in microcrystalline 6-s-cis-retinoic acid (triclinic) and 6-s-trans-retinoic acid (monoclinic) model compounds, as well as in the membrane protein bacteriorhodopsin (bR), regenerated with CD3-labeled retinal. Temperature dependent quadrupolar echo line shapes and T1 anisotropy measurements were used to characterize activation energies for 3-fold hopping motion of the methyl groups. These data provide supporting evidence that the conformation of the retinal chromophore in bR is 6-s-trans. The 6-s-cis conformer is characterized by strong eclipsing interactions between the 8-C proton and the 18-C methyl group protons; the 18-CD3 group shows an activation energy barrier for methyl 3-fold hopping of 14.5 +/- 1 kJ/mol. In contrast, the 18-CD3 group in the 6-s-trans isomer shows a considerably lower activation energy barrier of 5 +/- 1 kJ/mol. In bR, it is possible to obtain an approximate activation energy of 9 kJ/mol. This data is inconsistent with a 6-s-cis conformer but is consistent with the existence of a 6-s-trans-retinal Schiff base in bR with some interaction with the protein matrix. These results suggest that methyl rotor motions can be used to probe the van der Waals contact between a ligand and a protein binding pocket. The 6-s-trans conformer of the [16,17-(CD3)2]retinal in frozen hexane exhibits a major kinetic component with an activation energy barrier of of 14 -/+ 2 kJ/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

A detailed study on the structure, dynamics, and thermodynamic behavior of phosphatidylcholine/cholesterol (PC/CHOL) mixtures was undertaken using differential scanning calorimetry (DSC) and solid-state nuclear magnetic resonance (NMR) spectroscopy. DSC thermograms of mixtures of cholesterol (CHOL) with 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-phosphatidylcholine (DSPC), and 1,2-diarachidoyl-sn-phosphatidylcholine (DAPC) showed a broadening of the first-order gel-->liquid crystalline transition and a decrease in the transition enthalpy, indicating a gradual loss of cooperativity for high CHOL concentrations. DPPC and DSPC were labeled with 13C at the carbonyl group of the sn-2 chain and 2H was introduced into the middle of the sn-2 chain at the 6- and 12-position for DPPC and DSPC, respectively. The 13C and 2H NMR spectra of each labeled lipid were studied as a function of temperature and CHOL concentration. The residual quadrupole splitting in the 2H NMR spectra, delta nu Q perpendicular, was analyzed as a function of temperature and composition. For CHOL concentrations less than 30 mol %, a precipitous change in delta nu Q perpendicular occurs near the chain melting temperature of the phospholipid. Further increases in CHOL concentration broaden the transition and shift the midpoint to higher temperature, indicating the presence of a new phase at higher CHOL contents. Moreover, at a given temperature, delta nu Q perpendicular increases with increasing cholesterol content, which indicates a more ordered structure. The 13C NMR spectra in the gel state consisted of a superposition of two components which can be attributed to both gel-like and fluid phospholipid domains in the bilayer. This two-component spectrum can be simulated quantitatively with a two-parameter chemical exchange model, which permits the fraction of each form and the exchange rate to be determined as a function of temperature and composition. At high CHOL contents the line width of the fluid component broadens, suggesting an increase in the exchange rate between the domains. These results were interpreted in terms of a temperature composition diagram with one region L beta', two regions LGI and LGII, and one liquid crystalline region L alpha, with LG denoting "liquid-gel" type phases. Liquid-gel phases correspond to phases with increased order in the hydrocarbon chains (in comparison to that of the pure PC bilayer in the L alpha phase) combined with fast limit axial diffusion that averages the 13C NMR spectrum to a "fluidlike" line.(ABSTRACT TRUNCATED AT 400 WORDS)

We have used solid-state 13C NMR to study the structure of the adduct resulting from the inactivation of the enzyme transglutaminase by 3-halo-4,5-dihydroisoxazoles. These inhibitors were conceived on the assumption that they would inhibit transglutaminase by attack of an enzyme active site cysteine thiol on the imine carbon of the dihydroisoxazole ring. The tetrahedral intermediate formed could then break down with the loss of the halide group and the subsequent formation of a stable imino thioether adduct. We have compared the 13C CPMAS spectra of the chloro-, bromo-, and (ethylthio)dihydroisozazole inhibitors, and the results indicate that the chemical shift of the C-3 carbon is sensitive to the nature of the heteroatom. Subtraction of the natural-abundance 13C solid-state NMR spectrum of the enzyme from that of the enzyme inactivated by C-3-labeled chlorodihydroisoxazole reveals a broad peak at 156 ppm. The chemical shift of this peak is very close to that observed for a model 3-ethylthio compound and suggests the formation of a stable imino thioether enzyme adduct. Similar results were obtained for lyophilized enzyme adducts and for frozen solutions of the enzyme adduct in the absence and presence of Ca2+. We have also compared these results with those obtained by solution NMR on an aqueous solution of the enzyme-inhibitor complex. The 13C-labeled C-3 resonance was not observed in this case.

Rotational resonance, a new solid-state NMR technique for determining internuclear distances, is used to measure a distance in the active site of bacteriorhodopsin (bR) that changes in different states of the protein. The experiments are targeted to the active site of bR through 13C labeling of both the retinal chromophore and the Lys side chains of the protein. The time course of the rotor-driven magnetization exchange between a pair of 13C nuclei is then observed to determine the dipolar coupling and therefore the internuclear distance. Using this approach, we have measured the distance from [14-13C]retinal to [epsilon-13C]Lys216 in dark-adapted bR in order to examine the structure of the retinal-protein linkage and its role in coupling the isomerizations of retinal to unidirectional proton transfer. This distance depends on the configuration of the intervening C=N bond. The 3.0 +/- 0.2 A distance observed in bR555 demonstrates that the C=N bond is syn, and the 4.1 +/- 0.3 A distance observed in bR568 demonstrates that the C=N bond is anti. These direct distance determinations independently confirm the configurations previously deduced from solid-state NMR chemical shift and resonance Raman vibrational spectra. The spectral selectivity of rotational resonance allows these two distances to be measured independently in a sample containing both bR555 and bR568; the presence of both states and of 25% lipid in the sample demonstrates the use of rotational resonance to measure an active site distance in a membrane protein with an effective molecular mass of about 85 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

The formation of insoluble proteinaceous deposits is characteristic of many diseases which are collectively known as amyloidosis. There is very little molecular-level structural information available regarding the amyloid deposits due to the fact that the constituent proteins are insoluble and noncrystalline. Therefore, traditional protein structure determination methods such as solution NMR and X-ray crystallography are not applicable. We report herein the application of the solid-state NMR technique rotational resonance (R2) to the accurate measurement of carbon-to-carbon distances in the amyloid formed from a synthetic fragment (H2N-LeuMetValGlyGlyValValIleAla-CO2H) of the amyloid-forming protein of Alzheimer's disease (AD). This sequence has been implicated in the initiation of amyloid formation. Two distances measured by R2 indicate that an unusual structure, probably involving a cis amide bond, is present in the aggregated peptide amyloid. This structure is incompatible with the accepted models of fibril structure. A relationship between this structure and the stability of the amyloid is proposed.

31P and 15N chemical shifts and 31P-15N bond lengths have been measured with solid-state NMR techniques in two inhibitors of thermolysin, carbobenzoxy-Glyp-L-Leu-L-Ala (ZGpLA) and carbobenzoxy-L-Phep-L-Leu-L-Ala (ZFpLA), both as free lithium salts and when bound to the enzyme. Binding of both inhibitors to thermolysin results in large changes in the 31P chemical shifts. These changes are more dramatic for the tighter binding inhibitor ZFpLA, where a approximately 20 ppm downfield movement of the 31P isotropic chemical shift (sigma iso) is observed. This shift is due to changes in the shift tensor elements sigma 11 and sigma 22, while sigma 33 remains essentially constant. We observed a similar pattern for ZGpLA, but only a approximately 5 ppm change occurs in sigma iso. The changes in the 15N chemical shifts for both inhibitors are small upon binding, amounting to downfield shifts of 2 and 4 ppm for ZGpLA and ZFpLA, respectively. This indicates that there are no changes in the protonation state of the 15N in either the ZFpLA- or the ZGpLA-thermolysin complex. NMR distance measurements yield a P-N bond length rP-N = 1.68 +/- 0.03 A for the tight binding inhibitor ZFpLA both in its free lithium salt form and in its thermolysin-ZFpLA complex, a distance that is much shorter than the 1.90-A distance reported by X-ray crystallography studies [Holden et al. (1987) Biochemistry 26, 8542-8553].(ABSTRACT TRUNCATED AT 250 WORDS)

Solid-state 13C NMR spectra have been obtained of bovine rhodopsin and isorhodopsin regenerated with retinal selectively 13C labeled along the polyene chain. In rhodopsin, the chemical shifts for 13C-5, 13C-6, 13C-7, 13C-14, and 13C-15 correspond closely to the chemical shifts observed in the 11-cis protonated Schiff base (PSB) model compound. Differences in chemical shift relative to the 11-cis PSB chloride salt are observed for positions 8 through 13, with the largest deshielding (6.2 ppm) localized at position 13. The localized deshielding at C-13 supports previous models of the opsin shift in rhodopsin that place a protein perturbation in the vicinity of position 13. Spectra obtained of isorhodopsin regenerated with 13C-labeled 9-cis-retinals reveal large perturbations at 13C-7 and 13C-13. The similar deshielding of the 13C-13 resonance in both pigments supports the presence of a protein perturbation near position 13. However, the chemical shifts at positions 7 and 12 in isorhodopsin are not analogous to those observed in rhodopsin and suggest that the binding site interactions near these positions are different for the two pigments. The implications of these results for the mechanism of the opsin shift in these proteins are discussed.

The visible absorption of bacteriorhodopsin (bR) is highly sensitive to pH, the maximum shifting from 568 nm (pH 7) to approximately 600 nm (pH 2) and back to 565 nm (pH 0) as the pH is decreased further with HCl. Blue membrane (lambda max greater than 600 nm) is also formed by deionization of neutral purple membrane suspensions. Low-temperature, magic angle spinning 13C and 15N NMR was used to investigate the transitions to the blue and acid purple states. The 15N NMR studies involved [epsilon-15N]lysine bR, allowing a detailed investigation of effects at the Schiff base nitrogen. The 15N resonance shifts approximately 16 ppm upfield in the neutral purple to blue transition and returns to its original value in the blue to acid purple transition. Thus, the 15N shift correlates directly with the color changes, suggesting an important contribution of the Schiff base counterion to the "opsin shift". The results indicate weaker hydrogen bonding in the blue form than in the two purple forms and permit a determination of the contribution of the weak hydrogen bonding to the opsin shift at a neutral pH of approximately 2000 cm-1. An explanation of the mechanism of the purple to blue to purple transition is given in terms of the complex counterion model. The 13C NMR experiments were performed on samples specifically 13C labeled at the C-5, C-12, C-13, C-14, or C-15 positions in the retinylidene chromophore. The effects of the purple to blue to purple transitions on the isotropic chemical shifts for the various 13C resonances are relatively small. It appears that bR600 consists of at least four different species. The data confirm the presence of 13-cis- and all-trans-retinal in the blue form, as in neutral purple dark-adapted bR. All spectra of the blue and acid purple bR show substantial inhomogeneous broadening which indicates additional irregular distortions of the protein lattice. The amount of distortion correlates with the variation of the pH, and not with the color change.

Calorimetric, X-ray diffraction, and 31P nuclear magnetic resonance (NMR) studies of aqueous dispersions of 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) gel phases at low temperatures (-60 to 22 degrees C) show thermal, structural, and dynamic differences when compared to aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) gel phases at corresponding temperatures. Differential scanning calorimetry of DHPC dispersions demonstrates a reversible, low-enthalpy "subtransition" at 4 degrees C in contrast to the conditionally reversible, high-enthalpy subtransition observed at 17 degrees C for annealed DPPC bilayers. X-ray diffraction studies indicate that DHPC dispersions form a lamellar gel phase with dav congruent to 46 A both above and below the "subtransition". It is suggested that the reduced dav observed for DHPC (46 A as compared to 64 A in DPPC) is due to an interdigitated lamellar gel phase which exists at all temperatures below the pretransition at 35 degrees C. 31P NMR spectra of DHPC gel-phase bilayers show an axially symmetric chemical shift anisotropy powder pattern which remains sharp down to -20 degrees C, suggesting the presence of fast axial diffusion. In contrast, 31P spectra of DPPC bilayers indicate this type of motion is frozen out at approximately 0 degrees C.

Magic angle sample spinning (MASS) 13C NMR spectra have been obtained of bovine rhodopsin regenerated with retinal prosthetic groups isotopically enriched with 13C at C-5 and C-14. In order to observe the 13C retinal chromophore resonances, it was necessary to employ low temperatures (-15-----35 degrees C) to restrict rotational diffusion of the protein. The isotropic chemical shift and principal values of the chemical shift tensor of the 13C-5 label indicate that the retinal chromophore is in the twisted 6-s-cis conformation in rhodopsin, in contrast to the planar 6-s-trans conformation found in bacteriorhodopsin. The 13C-14 isotropic shift and shift tensor principal values show that the Schiff base C = N bond is anti. Furthermore, the 13C-14 chemical shift (121.2 ppm) is within the range of values (120-123 ppm) exhibited by protonated (C = N anti) Schiff base model compounds, indicating that the C = N linkage is protonated. Our results are discussed with regard to the mechanism of wavelength regulation in rhodopsin.

In reconstituted vesicles above the lipid phase transition temperature, bacteriorhodopsin (BR) undergoes rotational diffusion about an axis perpendicular to the plane of the bilayer [Cherry, R. J., Muller, U., & Schneider, G. (1977) FEBS Lett. 80, 465]. This diffusion narrows the 13C NMR powder line shape of the BR peptide carbonyls. In contrast, BR in native purple membrane is relatively immobile and exhibits a rigid-lattice powder line shape. By use of the principal values of the rigid-lattice chemical shift tensor and the motionally narrowed line shape from the reconstituted system, the range of Euler angles of the leucine peptide groups relative to the diffusion axis has been calculated. The experimentally observed line shape is inconsistent with those expected for structures which consist entirely of either alpha helix or beta sheet perpendicular to the membrane or beta sheet tilted at angles up to about 60 degrees from the membrane normal. However, for two more complex structural models, the predicted line shapes agree well with the experimental one. These are, first, a structure consisting entirely of alpha1 helices tilted at 20 degrees from the membrane normal and, second, a combination of 60% alpha II helix perpendicular to the membrane plane and 40% antiparallel beta sheet tilted at 10-20 degrees from the membrane normal. The results also indicate that the peptide backbone of bacteriorhodopsin in native purple membrane is extremely rigid even at 40 degrees. The experiments presented here demonstrate a new approach, using solid-state nuclear magnetic resonance (NMR) methods, for structural studies of transmembrane proteins in fluid membrane environments, either natural or reconstituted.(ABSTRACT TRUNCATED AT 250 WORDS)

Solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray powder diffraction were used to investigate the mechanism of trehalose (TRE) stabilization of lipid bilayers. Calorimetric investigation of dry TRE-stabilized bilayers reveals a first-order phase transition (L kappa----L lambda) at temperatures similar to the L beta'----(P beta')----L alpha transition of hydrated lipid bilayers. X-ray diffraction studies show that dry mixtures of TRE and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) have a lamellar structure with excess crystalline TRE being present. The L kappa phase shows typical gel-phase X-ray diffraction patterns. In contrast, the L lambda-phase diffraction patterns indicate disordered hydrocarbon chains. 2H NMR of specifically 2H chain-labeled DPPC confirmed that the acyl chains are disordered in the L lambda phase over their entire lengths. 2H spectra of the choline headgroup show hindered molecular motions as compared to dry DPPC alone, and 13C spectra of the sn-2-carbonyl show rigid lattice powder patterns indicating very little motion at the headgroup and interfacial regions. Thus, the sugar interacts extensively with the hydrophilic regions of the lipid, from the choline and the phosphate moieties in the headgroup to the glycerol and carbonyls in the interfacial region. We postulate that the sugar and the lipid form an extensive hydrogen-bonded network with the sugar acting as a spacer to expand the distance between lipids in the bilayer. The fluidity of the hydrophobic region in the L lambda phase together with the bilayer stabilization at the headgroup contributes to membrane viability in anhydrobiotic organisms.

Our previous solid-state 13C NMR studies on bR have been directed at characterizing the structure and protein environment of the retinal chromophore in bR568 and bR548, the two components of the dark-adapted protein. In this paper, we extend these studies by presenting solid-state NMR spectra of light-adapted bR (bR568) and examining in more detail the chemical shift anisotropy of the retinal resonances near the ionone ring and Schiff base. Magic angle spinning (MAS) 13C NMR spectra were obtained of bR568, regenerated with retinal specifically 13C labeled at positions 12-15, which allowed assignment of the resonances observed in the dark-adapted bR spectrum. Of particular interest are the assignments of the 13C-13 and 13C-15 resonances. The 13C-15 chemical resonance for bR568 (160.0 ppm) is upfield of the 13C-15 resonance for bR548 (163.3 ppm). This difference is attributed to a weaker interaction between the Schiff base and its associated counterion in bR568. The 13C-13 chemical shift for bR568 (164.8 ppm) is close to that of the all-trans-retinal protonated Schiff base (PSB) model compound (approximately 162 ppm), while the 13C-13 resonance for bR548 (168.7 ppm) is approximately 7 ppm downfield of that of the 13-cis PSB model compound. The difference in the 13C-13 chemical shift between bR568 and bR548 is opposite that expected from the corresponding 15N chemical shifts of the Schiff base nitrogen and may be due to conformational distortion of the chromophore in the C13 = C14-C15 bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

31P and 2H solid-state NMR studies of dry trehalose (TRE) and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) mixtures are reported. 31P spectra are consistent with a rigid head group above and below the calorimetric phase transition for both dry DPPC and a dry 2:1 TRE/DPPC mixture. In addition, 2H spectra of DPPC labeled at the 7-position of the sn-2 chain (2[7,7-2H2]DPPC) show exchange-narrowed line shapes with a width of 120 kHz over the temperature range 25-75 degrees C. These line shapes can be simulated with a model involving two-site jumps of the deuteron. In contrast, the 2H NMR spectrum of a dry 2:1 TRE/2[7,7-2H2]DPPC mixture above the phase transition (Tc = 46 degrees C) is narrowed by a factor of approximately 4 to a width of 29 kHz. Simulation of this spectrum requires a model involving four-site jumps of the deuteron and is indicative of highly disordered lipid acyl chains similar to those found in the L alpha-phases of hydrated lipids. Thus, TRE/DPPC mixtures above their transition temperatures exist in a new type of liquid crystalline like phase, which we term a lambda-phase. The observation of the dynamic properties of this new phase indicates the mechanism by which anhydrobiotic organisms maintain the integrity of their membranes upon dehydration.

Inhibition of alanine racemase from the Gram-positive bacterium Bacillus stearothermophilus by (1-aminoethyl) phosphonic acid (Ala-P) proceeds via a two-step reaction pathway in which reactivation occurs very slowly. In order to determine the mechanism of inhibition, we have recorded low-temperature, solid-state 15N NMR spectra from microcrystals of the [15N]Ala-P-enzyme complex, together with spectra of a series of model compounds that provide the requisite database for the interpretation of the 15N chemical shifts. Proton-decoupled spectra of the microcrystals exhibit a line at approximately 150 ppm, which conclusively demonstrates the presence of a protonated Ala-P-PLP aldimine and thus clarifies the structure of the enzyme-inhibitor complex. We also report the pH dependence of Ala-P binding to alanine racemase.

Solid-state 13C NMR spectra of the M photocycle intermediate of bacteriorhodopsin (bR) have been obtained from purple membrane regenerated with retinal specifically 13C labeled at positions 5, 12, 13, 14, and 15. The M intermediate was trapped at -40 degrees C and pH = 9.5-10.0 in either 100 mM NaCl [M (NaCl)] or 500 mM guanidine hydrochloride [M (Gdn-HCl)]. The 13C-12 chemical shift at 125.8 ppm in M (NaCl) and 128.1 ppm in M (Gdn-HCl) indicates that the C13 = C14 double bond has a cis configuration, while the 13C-13 chemical shift at 146.7 ppm in M (NaCl) and 145.7 ppm in M (Gdn-HCl) demonstrates that the Schiff base is unprotonated. The principal values of the chemical shift tensor of the 13C-5 resonance in both M (NaCl) and M (Gdn-HCl) are consistent with a 6-s-trans structure and a negative protein charge localized near C-5 as was observed in dark-adapted bR. The approximately 5 ppm upfield shift of the 13C-5 M resonance (approximately 140 ppm) relative to 13C-5 bR568 and bR548 (approximately 145 ppm) is attributed to an unprotonated Schiff base in the M chromophore. Of particular interest in this study were the results obtained from 13C-14 M. In M (NaCl), a dramatic upfield shift was observed for the 13C-14 resonance (115.2 ppm) relative to unprotonated Schiff base model compounds (approximately 128 ppm). In contrast, in M (Gdn-HCl) the 13C-14 resonance was observed at 125.7 ppm. The different 13C-14 chemical shifts in these two M preparations may be explained by different C = N configurations of the retinal-lysine Schiff base linkage, namely, syn in NaCl and anti in guanidine hydrochloride.

High-resolution, solid-state 15N NMR has been used to study the chemical shift anisotropies of the Schiff bases in bacteriorhodopsin (bR) and in an extensive series of model compounds. Using slow-spinning techniques, we are able to obtain sufficient rotational sideband intensity to determine the full 15N chemical shift anisotropy for the Schiff base nitrogen in bR548 and bR568. Comparisons are made between all-trans-bR568 and N-all-trans-retinylidene butylimine salts with halide, phenolate, and carboxylate counterions. It is argued that for the model compounds the variation in 15N chemical shift reflects the variation in (hydrogen) bond strength with the various counterions. The results suggest that carboxylates and tyrosinates may form hydrogen bonds of comparable strength in a hydrophobic environment. Thus, the hydrogen bonding strength of a counterion depends on factors that are not completely reflected in the solution pKa of its conjugate acid. For the model compounds, the two most downfield principal values of the 15N chemical shift tensor, sigma 22 and sigma 33, vary dramatically with different counterions, whereas sigma 11 remains essentially unaffected. In addition, there exists a linear correlation between sigma 22 and sigma 33, which suggests that a single mechanism is responsible for the variation in chemical shifts present in all three classes of model compounds. The data for bR568 follow this trend, but the isotropic shift is 11 ppm further upfield than any of the model compounds. This extreme value suggests an unusually weak hydrogen bond in the protein.

Solid-state 13C magic-angle sample spinning (MASS) NMR has been used to study lyophilized dark-adapted purple membrane containing 13C-labeled retinals. C-10-, C-11-, and C-12-labeled derivatives each showed two lines, assigned to the coexisting 13-cis and all-trans isomers. The isotropic chemical shifts, particularly of C-11, indicate that the Schiff base is protonated. Shift anisotropies are also similar to those of model compounds, indicating that this part of the chromophore is rigid and immobile and possesses the same degree of in-plane bending as crystalline retinal derivatives. Purple membrane samples labeled on the C-19- and C-20-methyl groups both give single lines from the retinal, upfield shifted by 2.1 and 1.0 ppm, respectively, from model compounds. In all cases, high-quality spectra were obtained from approximately 50-mg samples in modest signal-averaging times. These results suggest that it is now practical to exploit the enormous potential of MASS NMR for structural studies of 13C-labeled membrane proteins.

A series of 2H NMR inversion recovery experiments in the L alpha phase of the cerebroside N-palmitoylgalactosylsphingosine (NPGS) have been performed. In these liquid crystalline lipid bilayers we have observed substantial anisotropy in the spin-lattice relaxation of the CD2 groups in the acyl chains. The form and magnitude of the anisotropy varies with position in the chain, being positive in the upper region, decreasing to zero at the 4-position, and reversing sign at the lower chain positions. It is also shown that addition of cholesterol to the bilayer results in profound changes in the anisotropy. These observations are accounted for by a simple motional model of discrete hops among nine sites, which result from the coupling of two modes of motion--long-axis rotational diffusion and gauche-trans isomerization. This model is employed in quantitative simulations of the spectral line shapes and permits determination of site populations and motional rates. These results, plus preliminary results in sphingomyelin and lecithin bilayers, illustrate the utility of T1 anisotropy measurements as a probe of dynamics in L alpha-phase bilayers.

Angular-dependent 31P NMR spectra of oriented biaxial monodomain DPPC.H2O multilayers are employed to study head-group conformation in this phospholipid. The results indicate that the O-P-O plane of the phosphate, where the O's are the nonesterified oxygens of the phosphodiester, is tilted at 47 +/- 5 degrees with respect to the bilayer normal. This PO4 orientation could result in the choline moiety being extended parallel to the bilayer plane, and it will explain the breadth of the axially symmetric 31P powder spectrum observed for DPPC in excess water. This work is the first direct observation of this conformation for lecithins and it illustrates the utility of high-resolution solid-state NMR in structural studies of disordered systems.

Phosphorus-31 magic angle sample spinning NMR spectra have been employed to investigate the structure and composition of the mineral deposits in chicken bone. Three different pulse sequences, Bloch decay, cross-polarization, and dipolar suppression, were employed to obtain spectra from bone specimens of varying age. These were compared to the spectra obtained from a variety of crystalline and noncrystalline synthetic calcium phosphate solids used as reference standards. The results suggest that the most suitable model for the major solid calcium phosphate mineral phase in bone is a hydroxyapatite containing approximately 5-10% CO32- and approximately 5-10% HPO42- groups, the latter in a brushite-like configuration. From the NMR line shapes it was deduced that the fraction of HPO42- groups was highest in the youngest bone and decreased progressively with increasing age of the specimen.

Histidine enriched in 15N in the imidazole nitrogens was incorporated into the catalytic triad of alpha-lytic protease, and high-resolution solid-state 15N NMR spectra of lyophilized enzyme powders were recorded. The lyophilized powders were prepared from aqueous solutions with pH values ranging from 4.9 to 9.3. The behavior of the 15N resonances as a function of "pH" in these solid samples closely parallels that observed previously in the corresponding solution-state study, with the exception that in the powders proton exchange at His-57 is slow on the NMR time scale whereas in solutions it is fast. Thus, the 15N isotropic shifts demonstrate that the N pi-H tautomer of His-57 predominates in powders prepared at high pH and that N pi(H) participates in a strong hydrogen bond, as the hydrogen-bond donor, in powders prepared at both high pH and low pH. The simplest interpretation of these results is that the active site catalytic triad structure of Asp-His-Ser is maintained in these lyophilized powders. Because Asp-102 and His-57 are sequentially separated, their interaction in these lyophilized powders suggests that the tertiary structures of alpha-lytic protease in the powder and in solution are very similar. The 15N isotropic shifts further indicate that His-57 located within the intact triad in lyophilized enzyme powders has what can be taken as a normal "pKa" for a histidyl residue, undergoing a transition from the protonated to the neutral state with a midpoint between pH 6.0 and 7.0.

Solid-state 13C magic angle sample spinning NMR spectroscopy has been used to study the ionone ring portion of the chromophore of bacteriorhodopsin. Spectra were obtained from fully hydrated samples regenerated with retinals 13C labeled at positions C-5, C-6, C-7, C-8, and C-18 and from lyophilized samples regenerated with retinals labeled at C-9 and C-13. C-15-labeled samples were studied in both lyophilized and hydrated forms. Three independent NMR parameters (the downfield element of the C-5 chemical shift tensor, the C-8 isotropic chemical shift, and the C-18 longitudinal relaxation time) indicate that the chromophore has a 6-s-trans conformation in the protein, in contrast to the 6-s-cis conformation that is energetically favored for retinoids in solution. We also observe an additional 27 ppm downfield shift in the middle element of the C-5 shift tensor, which provides support for the existence of a negatively charged protein residue near C-5. Evidence for a positive charge near C-7, possibly the counterion for the negative charge, is also discussed. On the basis of these results, we present a new model for the retinal binding site, which has important implications for the mechanism of the "opsin shift" observed in bacteriorhodopsin.