A nuclear magnetic resonance study of the ribotrinucleoside diphophate UpUpC

500 MHz 1H, 67.89 MHz 13C and 80.97 MHz 31P-NMR studies are reported on the ribotrinucleoside diphosphate UpUpC, the triplet codon corresponding to the amino acid phenylalanine. Complete spectral assignments are given and conformational parameters for the backbone and the furanose rings are determined. All three nucleotide units show a near-balance for the N/S equilibrium with a slight preference for the N-type ribose (approximately 60%). The backbone conformation around the C3'-03' bonds show a preference for the trans domain, while the orientation around the C5'-05' bonds is predominantly trans.     

Conjugation-length dependence of the T1 lifetimes of carotenoids free in solution and incorporated into the LH2, LH1, RC, and RC-LH1 complexes: possible mechanisms of triplet-energy dissipation

In addition to the roles of antioxidant and spacer, carotenoids (Cars) in purple photosynthetic bacteria pursue two physiological functions, i.e., light harvesting and photoprotection. To reveal the mechanisms of the photoprotective function, i.e., quenching triplet bacteriochlorophyll to prevent the sensitized generation of singlet oxygen, the triplet absorption spectra were recorded for Cars, where the number of conjugated double bonds (n) is in the region of 9-13, to determine the dependence on n of the triplet lifetime. The Cars examined include those in (a) solution; (b) the reconstituted LH1 complexes; (c) the native LH2 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, Rsp. molischianum, and Rps. acidophila 10050; (d) the RCs from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, and Rsp. rubrum S1; and (e) the RC-LH1 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, Rsp. molischianum, Rps. acidophila 10050, and Rsp. rubrum S1. The results lead us to propose the following mechanisms: (i) A substantial shift of the linear dependence to shorter lifetimes on going from solution to the LH2 complex was ascribed to the twisting of the Car conjugated chain. (ii) A substantial decrease in the slope of the linear dependence on going from the reconstituted LH1 to the LH1 component of the RC-LH1 complex was ascribed to the minor-component Car forming a leak channel of triplet energy. (iii) The loss of conjugation-length dependence on going from the isolated RC to the RC component of the RC-LH1 complex was ascribed to the presence of a triplet-energy reservoir consisting of bacteriochlorophylls in the RC component.     

Cis proline mutants of ribonuclease A. I. Thermal stability.

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.     

Conformational properties of adenylyl-3' leads to 5'-adenosine in aqueous solution.

A detailed 220-MHz NMR study has been made of the conformational properties for the homodinucleotide adenylyl-3' leads to 5'-adenosine, ApA, in D2O. Unambiguous signal assignments of all proton signals were made with the aid of selectively deuterated nucleotidyl units, ApA, ApA, and D-8ApA, and complete, accurate sets of NMR parameters were derived by simulation-iteration methods. Sets of limiting chemical shifts and coupling values were also obtained for ApA and constituent monomers 3'-AMP and 5'-AMP at infinite dilution and at identical ionization states for assessment of dimerization effects. Conformational properties were evaluated quantitatively for most of the conformational bonds of ApA and these are consistent with two compact folded dynamically averaged structures, a base-stacked right helical structure, I, characterized as anti, C3'-endo, g-, w,w' (320,330 degrees), g'g', gg, C3'-endo, anti, and a more loosely base-stacked loop structure, II, with anti, C3'-endo, g-, w,w' (80 degrees, 50 degrees), g'g', gg, C3'-endo, anti orientations. Dimerization produces a number of nucleotidyl conformational changes including a shift in ribose equilibrium C2'-endo (S) in equilibrium C3'-endo (N) in favor of C3'-endo in both Ap- and -pA (60:40 vs. 35:65 in monomers), a change in glycosidic torsion angle chiCN toward 0 degrees, and a greater locking-in of rotamers along bonds involved in the phosphodiester backbone. Moreover, there is clear evidence that the transitions from S leads to N forms and chiCN leads to 0 degrees are directly related to base stacking in ApA. Finally, ApA exists in solution as an equilibrium between I, II and an unstacked form(s) with as yet undetermined conformational features. Since C4'-C5', C5'-O5', and C3'-O3' bonds possess exceptional conformational stabilities, it is proposed that destacking occurs primarily by rotation about P-O5' and/or O3'-P. Predominant factors influencing the overall ApA conformation are thus base-base interaction and flexibility about P-O5' and O3'-P, with change of ribose conformation occurring in consequence of an alteration of chiCN, the latter in turn being governed by the need for maximum eta overlap of stacked adenine rings.     

Conformation, orientation and dynamics of dodecylphosphocholine in micellar aggregate: a 3.2 ns molecular dynamics simulation study

A dodecylphosphocholine micelle of 86 monomers with 5776 water molecules has been simulated under NPT conditions for 3.2 ns using GROMACS2.0. The micelle was found to be very dynamic. Some of the C-C bonds, independent of their position in the DPC monomer, adopt gauche conformation and the trans <--> gauche transitions are quite frequent. An average of about 11% of the C-C bonds in the micelle are observed to be in the gauche conformation (i.e., |dihedral angle|< 120 degrees). The terminal methyl groups are randomly distributed all over the micelle whereas the nitrogen atom of phosphocholine headgroup atoms is restricted to the interface region. Some of the monomers were found to lie on the surface. The shape of micelle, influenced by the packing considerations, shows deviations from spherical shape. The phosphocholine headgroup is well solvated and there is no water penetration into the micelle core. The overall features of the micelle of 86 DPC monomers conforms to the lattice model of micelle proposed by Dill and Flory [Dill K A, Flory P J (1981) Proc Natl Acad Sci USA 78, 676-680] and is similar to DPC micelles of smaller aggregate sizes except for the positional preference of the C-C bonds for the gauche conformation and the trans<-->gauche transition times [Tieleman D P, van der Spoel D, Berendsen H J C (2000) J Phys Chem B 104, 6380-6388; Wymore T, Gao X F, Wong T C (1999) J Mol Struct (Theochem) 485-486, 195-210]. It appears that packing considerations play a predominant role in determining the shape and dynamics of the micelle.     

EPR characterisation of the triplet state in photosystem II reaction centers with singly reduced primary acceptor QA

The triplet states of photosystem II core particles from spinach were studied using time-resolved cw EPR technique at different reduction states of the iron-quinone complex of the reaction center primary electron acceptor. With doubly reduced primary acceptor, the well-known photosystem II triplet state characterised by zero-field splitting parameters |D| = 0.0286 cm⁻¹, |E| = 0.0044 cm⁻¹ was detected. When the primary acceptor was singly reduced either chemically or photochemically, a triplet state of a different spectral shape was observed, bearing the same D and E values and characteristic spin polarization pattern arising from RC radical pair recombination. The latter triplet state was strongly temperature dependent disappearing at T = 100 K, and had a much faster decay than the former one. Based on its properties, this triplet state was also ascribed to the photosystem II reaction center. A sequence of electron-transfer events in the reaction centers is proposed that explains the dependence of the triplet state properties on the reduction state of the iron-quinone primary acceptor complex.     

Angular disorder in actin: is it consistent with general principles of protein structure?

Harold Erickson has recently provided a useful analysis of helical structures having one class versus two classes of intersubunit bonds. His analysis is based upon an assumption that the subunits themselves are essentially unchanged upon bond formation (polymerization). He shows that such a structure having two classes of bonds (i.e. one in which each subunit interacts with four of its neighbors rather than two) can explain some of the features of actin. While he acknowledges that for actin there could be a conformational change and that, in principle, it could explain such features, he argues that the allowed magnitude of such a conformational change is inadequate. Since kinetics and thermodynamics cannot distinguish between the energy derived from the formation of a bond from that due to a conformational change, the question of whether the features of F-actin are derived from a conformational change or a system of two classes of bonds or both must be answered with high-resolution structural information. Recent studies by K. C. Holmes and others suggest that the second possibility might be closest to the truth. The heart of our disagreement is not whether Erickson's thermodynamic analysis is correct, given rigid subunits, but whether all protein polymers are characterized by rigid subunits with rigid intersubunit contacts. Erickson maintains that the observation of an angular disorder of 12 degrees per subunit within the actin filament conflicts with his formalism of rigid subunit interfaces and must therefore result from the erroneous interpretation of measurements. He presents an alternative model to explain the observations. His model, however, does not account for the observations and we will argue that, ultimately, like the resolution of the matter of the number of classes of bonds and the extent of their contact, the amount of angular disorder will require higher-resolution structural studies.     

Influence of metal ions and a local anesthetic on the conformation of the choline group of phosphatidylcholine bilayers studied by Raman spectroscopy

A Raman band assigned to the 'totally' symmetric stretching vibration of the choline C-N bonds is relatively strong and sensitive to the conformation of the choline backbone (Akutsu, H. (1981) Biochemistry 20, 7359-7366). By monitoring this Raman band, the influence of Eu3+, La3+, Ca2+ and a local anesthetic, dibucaine, on the conformation of the choline group was examined for the bilayers of dipalmitoylphosphatidylcholine and those of deuterated one at the choline methyl group (-N(C2H3)3). NMR lanthanide-shift studies proposed that the interaction with metal ions induces a conformational change from the gauche to the trans form in the O-C-C-N+ backbone of the choline group. However, present Raman work clearly showed that neither metal ions nor anesthetics induce such a conformational change. Therefore, a structural change in the polar group detected by 2H-NMR on addition of metal ions should not include a significant conformational change in the choline group as well. Deuterated phosphatidylcholine used here was proved to be more suitable for the direct detection of the amount of the trans conformation by Raman spectroscopy than the nondeuterated one. The spectra of the deuterated compound in the gel and liquid-crystalline states confirmed that the trans conformation of the choline group does not appear at all in both states.     

Heat induced protein denaturation in the particulate fraction of HeLa S3 cells: effect of thermotolerance.

In this study we investigated the effect of heat on the proteins of the particulate fraction (PF) of HeLa S3 cells using electron spin resonance (ESR) and thermal gel analysis (TGA). ESR detects overall conformational changes in proteins, while TGA detects denaturation (aggregation due to formation of disulfide bonds) in specific proteins. For ESR measurements the -SH groups of the proteins were labelled with a maleimido bound spin label (4-maleimido-tempo). The sample was heated inside the ESR spectrometer at a rate of 1 degree C/min. ESR spectra were made every 2-3 degrees C between 20 degrees C and 70 degrees C. In the PF of untreated cells conformational changes in proteins were observed in three temperature stretches: between 38 and 44 degrees C (transition A, TA); between 47 and 53 degrees C (transition B, TB); and above 58 degrees C (transition C, TC). With TGA, using the same heating rate, we identified three proteins (55, 70, and 90 kD) which denatured during TB. No protein denaturation was observed during TA, while during TC denaturation of all remaining proteins in the PF occurred. When the ESR and TGA measurements were done with the PF of (heat-induced) thermotolerant cells, TA was unchanged while TB and TC started at higher temperatures. The temperature shift for the onset of these transitions correlated with the degree of thermotolerance that was induced in the cells. These results suggest that protection against heat-induced denaturation of proteins in the PF is involved in heat induced thermotolerance.     

Top-down control analysis of the effect of temperature on ectotherm oxidative phosphorylation

Top-down control and elasticity analysis was conducted on mitochondria isolated from the midgut of the tobacco hornworm (Manduca sexta) to assess how temperature affects oxidative phosphorylation in a eurythermic ectotherm. Oxygen consumption and protonmotive force (measured as membrane potential in the presence of nigericin) were monitored at 15, 25, and 35 degrees C. State 4 respiration displayed a Q(10) of 2.4-2.7 when measured over two temperature ranges (15-25 degrees C and 25-35 degrees C). In state 3, the Q(10)s for respiration were 2.0 and 1.7 for the lower and higher temperature ranges, respectively. The kinetic responses (oxygen consumption) of the substrate oxidation system, proton leak, and phosphorylation system increased as temperature rose, although the proton leak and substrate oxidation system showed the greatest thermal sensitivity. Whereas there were temperature-induced changes in the activities of the oxidative phosphorylation subsystems, there was no change in the state 4 membrane potential and little change in the state 3 membrane potential. Top-down control analysis revealed that control over respiration did not change with temperature. In state 4, control of respiration was shared nearly equally by the proton leak and the substrate oxidation system, whereas in state 3 the substrate oxidation system exerted over 90% of the control over respiration. The proton leak and phosphorylation system account for <10% of the temperature-induced change in the state 3 respiration rate. Therefore, when the temperature is changed, the state 3 respiration rate is altered primarily because of temperature's effect on the substrate oxidation system.     

Conformational response of tartaric acid to derivatization: role of 1,3-dipole-dipole interactions

The four-carbon chain in (R,R)-tartaric acid derivatives is predominantly antiperiplanar (trans) in the acid, its salts, esters, and NH-amides, while (-)-synclinal (gauche) conformer is the most abundant in N,N'-tetraalkyltartramides. Trialkylsilylation or tert-butylation of the hydroxy groups at C2 and C3 does not appear to affect the conformational preference of NH-tartramides, but it does change the conformational equilibrium in the case of tartrates (toward (-)-gauche) and N,N'-tetraalkyltartramides (toward trans), as judged from the NMR data. X-ray diffraction data point to the stabilizing role of antiparallel dipole-dipole interactions due to the 1,3-CO/CH bonds. These interactions can be found in the trans and (-)-gauche conformers but are not possible for the (+)-gauche conformers of (R,R)-tartaric acid derivatives. This rationalizes small proportion of (+)-gauche conformers in tartaric acid derivatives and points to a significance of 1,3-dipole-dipole interactions. The conformation around the C1-C2 (and C3-C4) bond is different in tartrates (O-C-C=O, syn) and tartramides (O-C-C=O, anti); the CD data (n-pi* band) show that O-silylation or O-tert-butylation brings about conformational changes around the C1-C2 bond in the case of N,N'-tetraalkyldiamides only.     

Conformational changes in yeast tRNATyr revealed by EPR spectra of spin-labelled N6-(delta2-isopentenyl)-adenosine residue.

Temperature-induced conformational changes in the anticodon region of yeast tRNATyr were studied by EPR spectroscopy. The spin label 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl was attached to the N6-(delta2-isopentenyl)-adenosine residue in tRNATyr, previously made reactive by iodination. The labelled tRNATyr gave an asymmetrical triplet spectrum typical of rapidly tumbling nitroxide, with a rotational correlation time (tauc) of 0.65 ns. Spin-labelled tRNATyr was exposed to heating and cooling in three different buffers each with or without MgCl2. In each case the Arrhenius plot of --log tauc vs. inverse absolute temperature gave two straight lines, intersecting at a critical temperature (tcr). Above tcr, the anisotropy of the spectrum was not reduced and the activation energy of motion increased, indicating that the transition is associated with a conformational change of the macromolecule. Transitions in 0.05 M potassium phosphate (pH 8.0) and 0.02 M Tris - HC1 (pH 7.0) were observed at potassium phosphate (pH 8.0) and 0.02 M Tris - Hc1 (pH 7.0) were observed at approx. 37 degrees C. When 0.01 M mgCl2 was present in these buffers, transitions were shifted to 46 degrees and 53 degrees C, respectively. Transitions in 0.01 M sodium cacodylate were observed at temperatures which are significantly lower. Since all these transitions occur at temperatures considerably below those required to melt the helical regions of tRNA, and at least approximately 10 degrees C below those reported to break tertiary interactions, it is supposed that they reflect some reorientation of the anticodon region, e.g. a change in tilt of the bases.     

A tribute to Ulrich Heber (1930–2016) for his contribution to photosynthesis research: understanding the interplay between photosynthetic primary reactions,metabolism and the environment

Upon high light excitation in photosynthetic bacteria, various triplet states of pigments can accumulate leading to harmful effects. Here, the generation and lifetime of flash-induced carotenoid triplets (³Car) have been studied by observation of the quenching of bacteriochlorophyll (BChl) fluorescence in different strains of photosynthetic bacteria including Rvx. gelatinosus (anaerobic and semianaerobic), Rsp. rubrum, Thio. roseopersicina, Rba. sphaeroides 2.4.1 and carotenoid- and cytochrome-deficient mutants Rba. sphaeroides Ga, R-26, and cycA, respectively. The following results were obtained: (1) ³Car quenching is observed during and not exclusively after the photochemical rise of the fluorescence yield of BChl indicating that the charge separation in the reaction center (RC) and the carotenoid triplet formation are not consecutive but parallel processes. (2) The photoprotective function of ³Car is not limited to the RC only and can be described by a model in which the carotenoids are distributed in the lake of the BChl pigments. (3) The observed lifetime of ³Car in intact cells is the weighted average of the lifetimes of the carotenoids with various numbers of conjugated double bonds in the bacterial strain. (4) The lifetime of ³Car measured in the light is significantly shorter (1–2 μs) than that measured in the dark (2–10 μs). The difference reveals the importance of the dynamics of ³Car before relaxation. The results will be discussed not only in terms of energy levels of the ³Car but also in terms of the kinetics of transitions among different sublevels in the excited triplet state of the carotenoid.     

Absolute configuration of Rp-uridine 3'',5''-cyclic phosphorothioate.

Triethylammonium uridine-3',5'-cyclic phosphorothioate crystallizes in space group P2(1)2(1)2(1), a = 7.177(1), b = 13.155(6), c = 21.114(7) A, C15H26N3O7PS, MW 423.4, Z = 4, dx = 1.41g/cm3. The crystal structure was solved by direct methods on the basis of 1493 counter X-ray diffraction data (CuK alpha) and refined to R = 5.1%. The configuration of the thiophosphate group is Rp; conformational parameters are: glycosyl torsion angle anti, -151.9(5) degrees, sugar pucker C(3')-endo with P = 27.3 degrees, vmax = 45.5 degrees, six-membered cycle in chair form. The bond distances in the non-esterified P-S and P-O suggest that the negative charge is distributed between the groups. As illustrated in this and other studies, P-O has a much higher affinity for hydrogen bonds than P-S, indicated here by interactions with triethyl-ammonium N-H and O(2')-H as donors. One additional hydrogen bond N(3)-H---0(4) ties the bases which form a ribbon-like structure. 0(2) and S are not engaged in hydrogen bonds. The triethylammonium ion is two-fold disordered.     

Photomodulation of conformational states. III. Water-soluble bis-cysteinyl-peptides with (4-aminomethyl) phenylazobenzoic acid as backbone constituent

In previous studies we have shown that light-induced cis/trans isomerization of the azobenzene moiety in cyclo-[Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-AMPB] [AMPB: (4-aminomethyl)phenylazobenzoic acid] leads both in the monocyclic and in the oxidized bicyclic form to markedly differentiated conformational states in DMSO, a fact that lends itself for photomodulation of the redox potential of such bis-cysteinyl-peptides. For this purpose water-soluble systems are required, and this was achieved by replacing three residues outside the Cys-Ala-Thr-Cys active-site motif of thioredoxin reductase with lysines. The resulting cyclo-[Lys-Cys-Ala-Thr-Cys-Asp-Lys-Lys-AMPB] fully retains its photoresponsive properties in water as well assessed by uv and CD measurements. Paralleling results of the previously investigated azobenzene-containing cyclic peptides, the trans --> cis isomerization of the water-soluble monocyclic and oxidized bicyclic peptide is accompanied by a marked transition from a well-defined conformation to an ensemble of possible conformations. However, the conformational preferences are very dissimilar from those of the DMSO-soluble peptides. In fact, hydrogen bonds as well as secondary structure elements were found that change in the mono- and bicyclic peptide upon irradiation. The photo switch between different turn types and hydrogen bonding networks offers the structural rational for the significantly differentiated redox potentials, but also the possibility of monitoring by femtosecond uv-vis and ir spectroscopy fast and ultra fast backbone rearrangement processes following the electronic trans --> cis isomerization.     

High-pressure small-angle neutron scattering (SANS) study of 1,2-dielaidoyl-sn-glycero-3-phosphocholine bilayers

Small-angle neutron scattering of the trans-unsaturated DEPC has been investigated as a function of pressure at 12, 18.6 and 35 degrees C. A pressure-induced structural phase transition from a liquid-crystalline state to a gel state is observed at the temperatures studied. The critical pressure of this transition increases with increasing temperature with a delta P/delta T value of 51 bar/C degrees. The small-angle neutron scattering results indicate that the effect of the trans double bonds in DEPC is to enhance the conformational disorder in the hydrocarbon chains. In DEPC bilayers, a pressure-induced conformational ordering process is observed not only in the liquid-crystalline phase but also in the gel phase, which indicates that conformational disorder exists in the liquid-crystalline phase as well as in the gel phase.     

Effect of lengthening of peptide backbone by insertion of chiral beta-homo amino acid residues: conformational behavior of linear peptides containing alternating L-leucine and beta-homo L-leucine residues

The synthesis and the solution behavior of the linear peptides containing a beta-homo (beta-H) leucine residue-Boc-Leu-beta-HLeu-Leu-OMe, Boc-beta-HLeu-Leu-beta-HLeu-Leu-OMe, and Boc-Leu-beta-HLeu-Leu-beta-HLeu-Leu-OMe-as well as the solid structure of the tripeptide, are reported. The conformational behavior of the peptides was investigated in solution by two-dimensional nmr. Our data support the existence in solution with different families of conformers in rapid interchange. The crystals of the tripeptide are orthorhombic, space group P2(1)2(1)2, with a = 15.829(1) A, b = 29.659(1) A, c = 6.563(1) A, and Z = 4. The structure has been solved by direct methods and refined to final R1 and wR2 indexes of 0.0530 and 0.1436 for 2420 reflections with I > 2sigma(I). In the solid state, the tripeptide does not present intramolecular H bonds, and the peptide backbone of the two leucine residues adopts a quasi-extended conformation. For the beta-HLeu residue, the backbone conformation is specified by the torsion angles straight phi(2) = -120.9(4) degrees, mu(2) = 56.7(4) degrees, psi(3) = -133.2(4) degrees. The side chains of the three residues assume the same conformation (g(-), g(-), trans), and all peptide bonds, except the urethane group at the N-terminus, are in the trans conformation. Preliminary conformational energy calculations carried out on the Ac-NH-beta-HAla-NHMe underline that the conformations with mu angle equal to 180 degrees and 60 degrees assume lower energy with respect to the others. In addition, we found a larger conformational freedom for the psi angle with respect to the straight phi angle.     

Triplet Excitation Transfer between Carotenoids in the LH2 Complex from Photosynthetic Bacterium Rhodopseudomonas palustris

We have studied, by means of sub-microsecond time-resolved absorption spectroscopy, the triplet-excited state dynamics of carotenoids (Cars) in the intermediate-light adapted LH2 complex (ML-LH2) from Rhodopseudomonas palustris containing Cars with different numbers of conjugated double bonds. Following pulsed photo-excitation at 590 nm at room temperature, rapid spectral equilibration was observed either as a red shift of the isosbestic wavelength on a time scale of 0.6-1.0 mus, or as a fast decay in the shorter-wavelength side of the T(n)<--T(1) absorption of Cars with a time constant of 0.5-0.8 mus. Two major spectral components assignable to Cars with 11 and 12 conjugated double bonds were identified. The equilibration was not observed in the ML-LH2 at 77 K, or in the LH2 complex from Rhodobacter sphaeroides G1C containing a single type of Car. The unique spectral equilibration was ascribed to temperature-dependent triplet excitation transfer among different Car compositions. The results suggest that Cars of 11 and 12 conjugated bonds, both in close proximity of BChls, may coexist in an alpha,beta-subunit of the ML-LH2 complex.     

Two folded conformers of ubiquitin revealed by high-pressure NMR.

High-pressure 15N/1H two-dimensional NMR spectroscopy has been utilized to study conformational fluctuation of a 76-residue protein ubiquitin at pH 4.5 at 20 degrees C. The on-line variable pressure cell technique is used in conjunction with a high-field NMR spectrometer operating at 750 MHz for 1H in the pressure range between 30 and 3500 bar. Large, continuous and reversible pressure-induced 1H and 15N chemical shifts were observed for 68 backbone amide groups, including the 7.52 ppm 15N shift of Val70 at 3500 bar, indicating a large-scale conformational change of ubiquitin with pressure. On the basis of the analysis of sigmoid-shaped pressure shifts, we conclude that ubiquitin exists as an equilibrium mixture of two major folded conformers mutually converting at a rate exceeding approximately 10(4) s(-1) at 20 degrees C at 2000 bar. The second conformer exists at a population of approximately 15% (DeltaG(0) = 4.2 kJ/mol) and is characterized with a significantly smaller partial molar volume (DeltaV(0) = -24 mL/mol) than that of the well-known basic native conformer. The analysis of 1H and 15N pressure shifts of individual amide groups indicates that the second conformer has a loosened core structure with weakened hydrogen bonds in the five-stranded beta-sheet. Furthermore, hydrogen bonds of residues 67-72 belonging to beta5 are substantially weakened or partially broken, giving increased freedom of motion for the C-terminal segment. The latter is confirmed by the significant decrease in 15N[1H] nuclear Overhauser effect for residues beyond 70 at high pressure. Since the C-terminal carboxyl group constitutes the reactive site for producing a multi-ubiquitin structure, the finding of the second folded conformer with a substantially altered conformation and mobility in the C-terminal region will shed new light on the reaction mechanism of ubiquitin.     

Calorimetric and x-ray diffraction studies of rye glucocerebroside mesomorphism.

Glucocerebrosides (GlcCer) isolated from the leaves of winter rye (Secale cereale L. cv Puma) differ from the more commonly investigated natural and synthetic cerebrosides, in that greater than 95% of the fatty acids are saturated and monounsaturated hydroxy fatty acids. Isomers of the trihydroxy long chain base hydroxysphingenine (t1(8:18 cis or trans)) and isomers of sphingadienine (d18:2(4trans, 8 cis or trans)) comprise 77% and 17%, respectively, of the total long chain bases. The phase behavior of fully hydrated and dry rye leaf GlcCer was investigated using differential scanning calorimetry (DSC) and x-ray diffraction. On initial heating, aqueous dispersions of GlcCer exhibit a single endothermic transition at 56 degrees C and have an enthalpy (delta H) of 46 J/g. Cooling to 0 degrees C is accompanied by a small exothermic transition (delta H = -8 J/g) at 8 degrees C. On immediate reheating, a broad exothermic transition (delta H = -39 J/g) is observed between 10 and 20 degrees C in addition to a transition at 56 degrees C. These transitions are not reversible, and the exothermic transition rapidly diminishes when the sample is held at low temperature. Using x-ray diffraction, it was determined that the endotherm at 56 degrees C represents a transition from a highly ordered lamellar crystalline phase (Lc) with a d-spacing of 57 A and a series of wide-angle reflections in the 3-10 A range, to a lamellar liquid crystalline (L alpha) phase having a d-spacing of 55 A and a diffuse wide-angle scattering peak centered at 4.7 A. Cooling leads to the formation of a metastable gel phase (L beta) with a d-spacing of 64.0 A and a single broad reflection at 4.28 A. Subsequent warming to above 15 degrees C restores the original Lc phase. Thus, rye GlcCer in excess water exhibit a series of irreversible transitions and gel phase metastability. Dry GlcCer undergo an initial heating endothermic transition at 130 degrees C, which is ascribed to a transformation into the HII phase from a two phase state characterized by the coexistence of phases with disordered (alpha) and helical (delta) type chain conformations but of unknown lattice identity: An exotherm at 67.5 degrees C observed upon subsequent cooling is of unknown origin. Since an undercooled HII phase persists down to 19 degrees C, the exotherm may derive in part from an alpha-to-delta type chain packing conformational change especially under slow cooling conditions.(ABSTRACT TRUNCATED AT 400 WORDS)