Phospholipids chiral at phosphorus. Absolute configuration of chiral thiophospholipids and stereospecificity of phospholipase D

Separate diastereomers of 1,2-dipalmitoyl-sn-glycero-3- thiophosphoethanolamine ( DPPsE ) were prepared in 97% diastereomeric purity and characterized by 31P, 13C, and 1H nuclear magnetic resonance (NMR). The isomers hydrolyzed by phospholipases A2 and C specifically were designated as isomer B (31P NMR delta 59.13 in CDCl3 + Et3N ) and isomer A (59.29 ppm), respectively, analogous to the isomers B and A of 1,2-dipalmitoyl-sn-glycero-3- thiophosphocholine ( DPPsC ) [ Bruzik , K., Jiang , R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486]. Phospholipase D from cabbage was shown to be specific to isomer A of DPPsC in transphosphatidylation . The product DPPsE was shown to be isomer A. The absolute configuration of chiral DPPsE at phosphorus was elucidated by bromine-mediated desulfurization in H2 18O to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphoethanolamine ( [18O]DPPE) followed by 31 P NMR analysis [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The absolute configuration of chiral DPPsC was elucidated by desulfurization in H2 18O mediated by bromine or cyanogen bromide to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphocholine ( [18O]DPPC), which was then converted to [18O]DPPE by phospholipase D with retention of configuration [ Bruzik , K., & Tsai, M.-D. (1984) Biochemistry (preceding paper in this issue)]. The results indicate that isomer A of both DPPsE and DPPsC is SP whereas isomer B is RP.     

31P and 2H NMR studies of structure and motion in bilayers of phosphatidylcholine and phosphatidylethanolamine

The structural and motional properties of mixed bilayers of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) have been examined by using wide-line 31P, 14N, and 2H NMR. 2H and 14N NMR data showed that in mixed bilayers containing both PC and PE the conformations of the head-group moieties are essentially identical with those observed for bilayers containing a single phospholipid species. Equimolar amounts of cholesterol induce also only a small change in head-group conformation. 31P T1 relaxation measurements (at 300 MHz) at various temperatures of bilayers containing phospholipids with a mixture of phosphocholine and phosphoethanolamine head-groups and unsaturated fatty acid residues revealed in all cases a clearly defined minimum corresponding to the condition omega O tau C-1 approximately 1. For all phospholipid mixtures studied, the 31P T1 relaxation was homogeneous over the whole powder spectrum and could be fitted to a single-exponential decay. The 31P vs temperature profiles were analyzed by a simple correlation model following the analysis of Seelig et al. (1981) [Seelig, J., Tamm, L., Hymel, L., & Fleischer, S. (1981) Biochemistry 20, 3922-3932]. Rotational diffusion of the phosphate moiety in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was slower than that of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and the activation energy was increased by a factor of 1.7 to 31.4 kJ mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of cholesterol on the polar region of phosphatidylcholine and phosphatidylethanolamine bilayers

The structural changes in the polar head group region of unsonicated bilayer membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine produced by addition of cholesterol have been determined using deuterium and phosphorus-31 NMR. Incorportion of up to 50 mol percent cholesterol produces little change in the phosphorus-31 chemical shielding anisotropies, compared with the values in pure bilayers above the phase transition temperatures, while some of the deuterium quadrupole splittings are reduced by almost a factor of two. Adjustment of the head group torsion angles by only a few degrees accounts for the observed spectral changes. Addition of cholesterol therefore has opposite effects on the hydrocarbon and polar regions of membranes: although cholesterol makes the hydrocarbon region gel-like, with an increased probability of trans conformations, the conformation of the polar head groups is very similar to that found in the liquid crystalline phase of pure phospholipid bilayers.     

Carbon-13 nuclear magnetic resonance spectroscopy of lipids: differential line broadening due to cross-correlation effects as a probe of membrane structure

We have obtained proton-coupled carbon-13 nuclear magnetic resonance (NMR) spectra of a variety of lipid-water and lipid-drug-water systems, at 11.7 T, as a function of temperature, using the "magic-angle" sample-spinning (MAS) NMR technique. The resulting spectra show a wide range of line shapes, due to interferences between dipole-dipole and dipole-chemical shielding anisotropy interactions. The differential line-broadening effects observed are particularly large for aromatic and olefinic (sp2) carbon atom sites. Coupled spectra of the tricyclic antidepressants desipramine and imipramine, in 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophases, show well-resolved doublets having different line shapes for each of the four aromatic methine groups, due to selective averaging of the four C-H dipolar interactions due to rapid motion about the director (or drug C2) axis. 2H NMR spectra of [2,4,6,8-2H4]desipramine (and imipramine) in the same 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophase exhibit quadrupole splittings of approximately 0-2 and approximately 20 kHz, indicating an approximate magic-angle orientation of the C2-2H(1H) and C8-2H(1H) vectors with respect to an axis of motional averaging, in accord with the 13C NMR results. Selective deuteration of imipramine confirms these ideas. Spectra of digalactosyl diglyceride [primarily 1,2-di[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl ]-3- (alpha-D-galactopyranosyl-1-6-beta-D-galactopyranosyl)-sn-glycerol]-H2O (in the L alpha phase) show a large differential line broadening for C9 but a reduced effect for C10, consistent with the results of 2H NMR of specifically 2H-labeled phospholipids [Seelig, J., & Waespe-Saracevic, N. (1978) Biochemistry 17, 3310-3315].(ABSTRACT TRUNCATED AT 250 WORDS)     

14N NMR of lipid bilayers: effects of ions and anesthetics

The interaction of divalent and trivalent metal cations, ferricyanide, a lipophilic ion (tetraphenylborate), and a local anesthetic (tetracaine) with the phosphocholine head group of egg lecithin was investigated by using wide-line 14N and 31P NMR. Measurements of the 14N quadrupolar splittings in the presence of a variety of perturbing agents demonstrated that the 14N NMR technique can be used to directly monitor ion or anesthetic binding. The 14N quadrupolar splitting (delta nu Q) is a measure of the order parameter of the C beta-N bond segment, and changes in delta nu Q as large as 3.5 kHz were observed. Moreover, a comparison of the changes in the quadrupolar splittings induced by the binding of ions or anesthetics provided a sensitive method of discriminating between these perturbing agents in their ability to alter the orientational order of the C beta-N bond segment of the phosphocholine moiety. Without exception, addition of metal ions or anesthetics always resulted in a decrease of the 14N delta nu Q. This reduction reflects a change in the average orientation or degree of motional averaging at the C beta-N bond segment position. In the case of metal ion binding, the strength of the interaction increased with the charge of the metal ion in the order Ca2+ less than Ln3+, in agreement with a previous 2H NMR study [Akutsu, H., & Seelig, J. (1981) Biochemistry 20, 7366-7373].(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipids chiral at phosphorus. Synthesis of chiral phosphatidylcholine and stereochemistry of phospholipase D

Chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphocholines (DPPC) with known configuration were synthesized by N-methylation of chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). Transphosphatidylation of (RP)- and (SP)-[18O]DPPC catalyzed by phospholipase D from cabbage gave (RP)- and (SP)-[18O]DPPE, respectively, as indicated by 31P nuclear magnetic resonance (NMR) analysis of [18O]DPPE. Therefore, phospholipase D catalyzes transphosphatidylation with overall retention of configuration at phosphorus. The steric course of hydrolysis of DPPC catalyzed by the same enzyme was elucidated by the following procedures. Hydrolysis of (RP)-[17O, 18O]DPPC by phospholipase D gave 1,2-dipalmitoyl-sn-glycero-3-[ 16O , 17O, 18O]phosphate ( [ 16O , 17O, 18O] DPPA ) with unknown configuration. The latter compound was then converted to 1-[ 16O , 17O, 18O]phospho-(R)-propane-1,2-diol by a procedure involving no P-O bond cleavage [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The configuration of the phosphopropane -1,2-diol was determined as RP by 31P NMR analysis following ring closure and methylation [ Buchwald , S. L., & Knowles, J. R. (1980) J. Am. Chem. Soc. 102, 6601-6603]. The results indicated that hydrolysis of DPPC catalyzed by phospholipase D also proceeds with retention of configuration at phosphorus. Our results therefore support a two-step mechanism involving a phosphatidyl-enzyme intermediate in the reactions catalyzed by phospholipase D from cabbage.     

Structural studies of alpha-bungarotoxin. 2. 1H NMR assignments via an improved relayed coherence transfer nuclear overhauser enhancement experiment

Complete sequence-specific assignments of the 1H NMR spectrum of bungarotoxin were reported in the previous paper [Basus, V.J., Billeter, M., Love, R.A., Stroud, R.M., & Kuntz, I.D. (1988) Biochemistry (first paper of three in this issue)]. The assignment was significantly aided by the use of the homonuclear Hartman-Hahn relayed coherence transfer nuclear Overhauser enhancement spectroscopy experiment (HRNOESY) which we present here, as a modification of relayed coherence transfer nuclear Overhauser enhancement spectroscopy (relayed NOESY) [Wagner, G. (1984) J. Magn. Reson. 57, 497]. As shown here, HRNOESY resolves problems of proton resonance overlap especially in extended chain conformations as found in beta-sheets.     

Solution studies of staphylococcal nuclease H124L. 2. 1H, 13C, and 15N chemical shift assignments for the unligated enzyme and analysis of chemical shift changes that accompany formation of the nuclease-thymidine 3',5'-bisphosphate-calcium ternary complex.

Accurate 1H, 15N, and 13C chemical shift assignments were determined for staphylococcal nuclease H124L (in the absence of inhibitor or activator ion). Backbone 1H and 15N assignments, obtained by analysis of three-dimensional 1H-15N HMQC-NOESY data [Wang, J., Mooberry, E.S., Walkenhorst, W.F., & Markley, J. L. (1992) Biochemistry (preceding paper in this issue)], were refined and extended by a combination of homo- and heteronuclear two-dimensional NMR experiments. Staphylococcal nuclease H124L samples used in the homonuclear 1H NMR studies were at natural isotopic abundance or labeled randomly with 2H (to an isotope level of 50%); nuclease H124L samples used for heteronuclear NMR experiments were labeled uniformly with 15N (to an isotope level greater than 95%) or uniformly with 13C (to an isotope level of 26%). Additional nuclease H124L samples were labeled selectively by incorporating single 15N- or 13C-labeled amino acids. The chemical shifts of uncomplexed enzyme were then compared with those determined previously for the nuclease H124L.pdTp.Ca2+ ternary complex [Wang, J., LeMaster, D. M., & Markley, J.L. (1990) Biochemistry 29, 88-101; Wang, J., Hinck, A.P., Loh, S. N., & Markley, J.L. (1990) Biochemistry 29, 102-113; Wang, J., Hinck, A.P., Loh, S.N., & Markley, J.L. (1990) Biochemistry 29, 4242-4253]. The results reveal that the binding of pdTp and Ca2+ induces large shifts in the resonances of several amino acid segments. These chemical shift changes are interpreted in terms of changes in backbone torsion angles that accompany the binding of pdTp and Ca2+; changes at the binding site appear to be transmitted to other regions of the molecule through networks of hydrogen bonds.     

Use of transferred nuclear Overhauser effect measurements to compare binding of coenzyme analogues to dihydrofolate reductase

Transferred nuclear Overhauser effect measurements have been made on complexes of NADP+ and thioNADP+ with Lactobacillus casei dihydrofolate reductase to provide information about the glycosidic bond conformations in these complexes. Both NADP+ and thioNADP+ are shown to have very similar anti conformations about their adenine glycosidic bonds when bound to the enzyme. However, their nicotinamide glycosidic bond conformations are very different: while NADP+ binds in an exclusively anti conformation, thioNADP+ binds with a distribution of syn/anti conformations very similar to that observed in nicotinamide mononucleotides in free solution (approximately 50:50). Thus for thioNADP+, binding to the enzyme does not significantly perturb the potential function for rotation about the nicotinamide glycosidic bond. Earlier NMR studies [Hyde, E. I., Birdsall, B., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1980) Biochemistry 19, 3738] had indicated that large downfield 1H shifts of the nicotinamide ring protons (0.61-1.36 ppm) are detected on binding NADP+ while only very small shifts (less than 0.1 ppm) are observed in complexes with thioNADP+. The chemical shift and conformational findings are best explained if the thionicotinamide ring extends into solution making essentially no contacts with the enzyme.     

Conformational analysis of cyclic partially modified retro-inverso enkephalin analogues by proton NMR

The conformations of five cyclic retro-inverso enkephalin analogues have been probed by proton NMR. After assignment of peaks, intramolecularly hydrogen-bonded amide protons were detected by temperature perturbation. Carbonyl hydrogen-bond acceptors were surmised from the computer simulations of minimum energy conformations of Hassan and Goodman [Hassan, M., & Goodman, M. (1986) Biochemistry (preceding paper in this issue)]. Hydrogen bonds were identified in dimethyl-d6 sulfoxide solutions and monitored as H2O was added. One hydrogen bond was observed in each of the retro-inverso-modified enkephalin analogues although in the parent analogue H-Tyr-c-(D-A2bu-Gly-Phe-Leu) two were detected. The change in solvent altered the conformations of two of the analogues.     

Identification of residues involved in a conformational change accompanying substitutions for glutamate-43 in staphylococcal nuclease

A recent paper from our laboratories [Hibler, D. W., Stolowich, N. J., Reynolds, M. A., Gerlt, J. A. Wilde, J. A., & Bolton, P. H. (1987) Biochemistry 26, 6278] described the generation of site-directed substitutions for the putative general base Glu-43 in the active site of Staphylococcal nuclease (SNase) and the use of 1H NMR spectroscopy to characterize the effect of the substitutions on the conformations of the mutant proteins. The replacements for Glu-43 (Asp, Gln, Asn, Ser, and Ala) both decreased the catalytic efficiency and changed the one- and two-dimensional NMR spectral properties of the mutant enzymes. We have prepared and studied the NMR spectral properties of several samples of deuteriated wild-type SNase that allow sequence-specific resonance assignments for several aromatic and aliphatic amino acid side chains that experience changes both in normal one-dimensional spectra and in two-dimensional NOESY spectra. Due to severe spectral congestion of resonances in the one- and two-dimensional spectra of protiated SNase, the assignments would have been difficult, if not impossible, to obtain without deuteriation of selected amino acids. The spectra we have obtained demonstrate that changes in NOE intensities involve a valine residue that is spatially adjacent to two phenylalanine residues; given the X-ray structure for SNase [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551], these residues must be Val-74, Phe-34, and Phe-76. In addition, a leucine residue experiencing changes in NOE intensities spatially adjacent to Val-74 and Phe-34 can be assigned to Leu-25.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipids chiral at phosphorus. Characterization of the sub-gel phase of thiophosphatidylcholines by use of X-ray diffraction, phosphorus-31 nuclear magnetic resonance, and Fourier transform infrared spectroscopy

A recent study using differential scanning calorimetry (DSC) showed that the thermotropic phase behavior of 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC) is sensitive to the configuration at phosphorus and that the Rp isomer displayed only a broad transition at 45.6 degrees C [Wisner, D. A., Rosario-Jansen, T., & Tsai, M.-D. (1986) J. Am. Chem. Soc. 108, 8064-8068]. We have employed X-ray diffraction, 31P NMR, and Fourier transform infrared (FT-IR) spectroscopy to characterize various phases of the isomers of DPPsC, to compare the structural differences between 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and isomers of DPPsC, and to identify structural factors responsible for the unique behavior of the RP isomer. The results from all three techniques support the previous proposal based on DSC studies that (SP)- and (RP + SP)-DPPsC undergo a subtransition, a pretransition, and a main transition analogous to those of DPPC, while (RP)-DPPsC is quite stable at the subgel phase and undergoes a direct subgel----liquid-crystalline transition at 46 degrees C. Quantitative differences between DPPC and DPPsC (i.e., the effect of sulfur substitution rather than the configurational effect) in the subgel phase have also been observed in the chain spacing, the motional averaging, and the factor group splitting (revealed by X-ray diffraction, 31P NMR, and FT-IR, respectively). In particular, DPPsC isomers are motionally rigid and show enhanced factor group splitting in the subgel phase. These results suggest that DPPsC is packed in different subcells relative to DPPC in the subgel phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipids chiral at phosphorus. Use of chiral thiophosphatidylcholine to study the metal-binding properties of bee venom phospholipase A2

It has been shown recently by 31P nuclear magnetic resonance (NMR) that phospholipase A2 (PL A2) from bee venom shows a high degree of stereoselectivity toward the "isomer B" of 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC) [Bruzik, K., Jiang, R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486]. We now report a quantitative kinetic study of PL A2 using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and (RP)-, (SP)-, and (RP + SP)-DPPsC by a spectrophotometric assay. The substrates were mixed with Triton X-100 to form mixed micelles, and steady-state kinetic theories were applied. The enzyme was activated by Ca2+, which induced a conformational change of the enzyme, as shown by UV difference spectra. The apparent dissociation constant of Ca2+/PL A2 is 2.5 mM. In the presence of Ca2+, large substrate specificity and stereospecificity in Vmax (in mumol min-1 mg-1) were observed: DPPC, 1850; (RP)-DPPsC, 7.6; (RP + SP)-DPPsC, 64; (SP)-DPPsC, 0.044. On the other hand, relatively small variation in Km was observed, which suggests that the interfacial interaction is relatively nonspecific among the substrates studied. (SP)-DPPsC and Cd2+ were shown as competitive inhibitors for the hydrolysis of DPPC by Ca2+/PL A2. Binding of Cd2+ with apo-PL A2 was also demonstrated by UV difference spectra, with a dissociation constant of 0.59 mM. Activation of apo-PL A2 by Cd2+ was unequivocally demonstrated for (SP)-DPPsC by use of 31P NMR. The Vmax values of Cd2+/PL A2 were DPPC/(RP)-DPPsC/(SP)-DPPsC = 17.6/0.069/0.0044 mumol min-1 mg-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Fourier transform infrared spectroscopic study of the molecular interaction of cholesterol with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

The temperature dependencies of the infrared spectra of pure and cholesterol-containing multibilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine were studied using Fourier transform infrared techniques. A comparison of the spectroscopic data showed the retention of a melting phenomenon at 60 mol% cholesterol content, and the retention of some all-trans conformations in the liquid-crystalline phase. It is also demonstrated that at temperatures less than 30 degrees C, the cholesterol-containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine multibilayers still contain a small amount of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, packed in an orthorhombic subcell lattice. Spectral changes were found in the absorptions characteristic of the phospholipid head groups. The addition of cholesterol results in changes in the ester bands, and demonstrates the induction by cholesterol of non-equivalent ester conformations.     

NMR studies of ion binding to Escherichia coli tRNAPhe

The effects of magnesium, spermine, and temperature on the conformation of Escherichia coli tRNAPhe have been examined by proton and phosphorus nuclear magnetic resonance spectroscopy. In the low-field proton NMR spectra we have characterized two slowly interconverting conformations of this tRNA at low magnesium ion concentrations. The relative proportion of the conformers is ion dependent but not ion specific. Magnesium affects protons in all the stems of tRNA while spermine effects are localized near the s4U-8-A-14 and G-15-C-48 tertiary bonds. The effects seen in the proton NMR spectra are compared and correlated with those observed in the phosphorus spectra to give assignments of some of the resolved signals from the phosphate groups. The phosphorus spectra are compared with those of yeast tRNAPhe [Gorenstein, D. G., Goldfield, E. M., Chen, R., Kovar, K., & Luxon, B. A. (1981) Biochemistry 20, 2141; Salemink, P. J. M., Reijerse, E. J., Mollevanger, L., & Hilbers, C. W. (1981) Eur. J. Biochem. 115, 635], and the ion effects are discussed with reference to the magnesium and spermine sites found in the crystal structures of yeast tRNAPhe [Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, S.-H. (1977) Nucleic Acids Res. 4, 2811; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64; Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315].     

Design of a helix-bundle cross-link: NMR and UV-visible spectroscopic analyses and molecular modeling of ring-oxidized retinals

In order to generate potential chemical cross-links for studying the chromophore binding site of bacteriorhodopsin and related helix-bundle proteins, MnO2 was used to oxidize all-trans-retinal's ring moiety. The structures and solution conformations of three ring-oxidized retinal analogues have been determined by using UV-visible absorption and 1H and 13C NMR spectroscopies, primarily with regard to (i) the introduction of a functional group at the ring end of the chromophore, (ii) the retention of the all-trans geometry of the polyenal side chain, and (iii) the torsional angle of the ring-polyenal bond. Analyses of their UV-visible absorption spectral parameters (lambda max, epsilon max, and vibrational fine structure) and NMR spectral parameters (1H-1H coupling constants, 1H and 13C NMR chemical shifts, and 1H homonuclear Overhauser effects) indicated the 4-oxo and the 2,3-dehydro-4-oxo derivatives both possess the twisted 6-s-cis conformation adopted by most six-membered ring analogues of retinal in solution or crystal. However, the alpha-dioxocyclopentenyl analogue exists in solution predominantly (70-80%) as the planar 6-s-trans conformer, similar to violerythrine chromophore analogues. In order to identify the minor solution forms, molecular modeling and geometry optimizations using the semiempirical molecular orbital method AM1 defined two additional symmetry-related minima at +/- 30-40 degrees in its C6-C7 torsional energy profile. Because the chromophores of bacterio- and halorhodopsins and sensory rhodopsins are bound as the 6-s-trans conformer [Harbison, G.S., Smith, S.O., Pardoen, J.A., Courtin, J.M.L., Lugtenburg, J., Herzfeld, J., Mathies, R.A., & Griffin, R.G. (1985) Biochemistry 24, 6955-6962; Baselt, D.R., Fodor, S.P.A., van der Steen, R., Lugtenburg, J., Bogomolni, R.A., & Mathies, R.A. (1989) Biophys. J. 55, 193-196], we suggest that the cyclopentenyl analogue's alpha-diketo function may be favorably positioned within the binding pocket and sufficiently reactive toward nucleophilic attack to cross-link an arginine located in or near the ring end of the chromophore cavity: Arg134 according to the current model of bacteriorhodopsin's tertiary structure [Henderson, R., Baldwin, J.M., Ceska, T.A., Zemlin, F., Beckmann, E., & Downing, K.H. (1990) J. Mol. Biol. 213, 899-929] or Arg82 as postulated from an alternate model constructed primarily to accommodate the external point charge contribution to bacteriorhodopsin's opsin shift.     

Correlation of tryptophan fluorescence intensity decay parameters with 1H NMR-determined rotamer conformations: [tryptophan2]oxytocin.

While the fluorescence decay kinetics of tyrosine model compounds [Laws, W. R., Ross, J. B. A., Wyssbrod, H. R., Beechem, J. M., Brand, L., & Sutherland, J. C. (1986) Biochemistry 25, 599-607] and the tyrosine residue in oxytocin [Ross, J. B. A., Laws, W. R., Buku, A., Sutherland, J. C., & Wyssbrod, H. R. (1986) Biochemistry 25, 607-612] can be explained in terms of heterogeneity derived from the three ground-state chi 1 rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from chi 2 rotations. In this paper, the time-resolved and steady-state fluorescence properties of [tryptophan2]oxytocin at pH 3 are presented and compared with 1H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength and a global analysis of these decay curves for common emission wavelength-independent decay constants, only three exponential terms are required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the 1H NMR-determined chi 1 rotamer populations of the indole side chain. 15N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the chi 1 rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two chi 2 populations. Depending upon the degree of correlation between chi 2 and chi 1, there may be from three to six side-chain conformations for the tryptophan residue. The combined fluorescence and NMR results are consistent with a rotamer model in which either (i) the chi 2 rotations are fast compared to the fluorescence intensity decay of the tryptophan residue, (ii) environmental factors affecting fluorescence intensity decay properties are dominated by chi 1 interactions, or (iii) the chi 2 and chi 1 rotations are highly correlated.     

The influence of cholesterol on head group mobility in phospholipid membranes.

The dielectric dispersion in the MHz range of the zwitterionic dipolar phosphocholine head groups has been measured from 0--70 degrees C for various mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. The abrupt change in the derived relaxation frequency f2 observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42 degrees C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42 degrees C no sudden changes in f2 are found at 20 or 33 mol% cholesterol, where phase boundaries have been suggested from other methods. Above 42 degrees C, however, a decrease in f2 at cholesterol contents up to 20--30 mol% is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules.     

31P NMR and X-ray diffraction study of the effect of photopolymerization on lipid polymorphism.

It was recently shown that oligolamellar vesicles of 3:1 mixtures of dioleoylphosphatidylethanolamine (DOPE) and the photopolymerizable lipid 1,2-bis[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocho line (SorbPC) are destabilized by polymerization of the SorbPC [Lamparski, H., Liman, U., Frankel, D.A., Barry, J.A., Ramaswami, V., Brown, M.F., & O'Brien, D.F. (1992) Biochemistry 31, 685-694]. The current work describes the polymorphic phase behavior of these mixtures in extended bilayers, as studied by 31P NMR spectroscopy and X-ray diffraction. In the NMR experiments, samples with varying degrees of polymerization were slowly raised in temperature, with spectra acquired every 2.5-10 degrees C. In the unpolymerized mixiture, and in those photopolymerized samples where the monomeric SorbPC was decreased by 33% and 51%, an isotropic signal grew progressively until no signal from the lamellar liquid-crystalline (L alpha) phase remained. In the highly polymerized sample with a 90% loss of monomeric SorbPC, less than 20% of the lipids underwent this transition. In none of the samples was an inverted hexagonal phase (HII) observed, under conditions of slow heating to almost 100 degrees C. The X-ray diffraction studies indicated that samples which exhibit the isotropic NMR signal corresponded to a structure exhibiting no well-defined crystalline order, which upon thermal cycling became an inverted cubic phase belonging to either the Pn3m or Pn3 space groups. The temperature of the transition to the cubic precursor decreased as the extent of polymerization increased, demonstrating that photopolymerization of these lipid bilayers can significantly alter the composition and thermotropic phase behavior of the mixture.     

Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Staphylococcal nuclease H124L is a recombinant protein produced in Escherichia coli whose sequence is identical with that of the nuclease produced by the V8 variant of Staphylococcus aureus. The enzyme-metal ion activator-nucleotide inhibitor ternary complex, nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+, was investigated by two-dimensional (2D) NMR techniques. Efficient overproduction of the enzyme facilitated the production of random fractionally deuterated protein, which proved essential for detailed NMR analysis. 1H NMR spin systems were analyzed by conventional 2D 1H[1H] methods: COSY, relayed COSY, HOHAHA, and NOESY. Assignments obtained by 1H NMR experiments were confirmed and extended by 1H-13C and 1H-15N heteronuclear NMR experiments [Wang, J., Hinck, A. P., Loh, S. N., & Markley, J. L. (1990) Biochemistry (following paper in this issue)]. Spectra of the ternary complexes prepared with protein at natural abundance and at 50% random fractional deuteration provided the information needed for sequence-specific assignments of 121 of the 149 amino acid residues. Short- and intermediate-range NOE connectivities allowed the determination of secondary structural features of the ternary complex: three alpha-helical domains and three antiparallel beta-pleated sheets with several reverse turns. A number of nonsequential long-range HN-HN and H alpha-HN connectivities revealed additional information about the spatial arrangement of these secondary structural elements. The solution structure of this ternary complex shows a close correspondence to the crystal structure of the nuclease wt-thymidine 3',5'-bisphosphate-Ca2+ ternary complex [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555].