Synthesis of delta(E)Phe-containing tripeptide via photoisomerization and its conformation in solution

A new synthetic route to (E)-beta-phenyl-alpha,beta-dehydroalanine (delta(E)Phe)-containing peptide was presented via photochemical isomerization of the corresponding (Z)-beta-phenyl-alpha,beta-dehydroalanine (delta(Z)Phe)-containing peptide. By applying this method to Boc-Ala-delta(Z)Phe-Val-OMe (Z-I: Boc, t-butoxycarbonyl; OMe, methoxy), Boc-Ala-delta(E)Phe-Val-OMe (E-I) was obtained. The identification of peptide E-I was evidenced by 1H-nmr, 13C-nmr, and uv absorption spectroscopy, elemental analysis, and hydrogenation. The conformation of peptide E-I in CDCl3 was investigated by 1H-nmr spectroscopy (solvent dependence of NH chemical shift and difference nuclear Overhauser effect). Interestingly, peptide E-I differed from peptide Z-I in the hydrogen-bonding mode. Namely, for peptide Z-I, only Val NH participates in intramolecular hydrogen bonding, which leads to a type II beta-turn conformation supported by hydrogen bonding between CO(Boc) and NH(Val). On the other hand, for peptide E-I, two NHs, delta(E)Phe NH and Val NH, participate in intramolecular hydrogen bonding. In both peptides, a remarkable NOE (approximately 11-13%) was observed for Ala C(alpha) H-deltaPhe NH pair. Based on the nmr data and conformational energy calculation, it should be concluded that peptide E-I takes two consecutive gamma-turn conformations supported by hydrogen bonding between CO(Boc) and NH(delta(E)Phe), and between CO(Ala) and NH(Val) as its plausible conformation.     

Thermal stability of the Z conformation of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG): evidence for a conformational transition before melting

The thermal stability of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG) has been studied at two nucleotide concentrations, in the presence of 1 M NaClO4. At low nucleotide concentration (7 X 10(-5) M), circular dichroism experiments show a conformational transition from the Z conformation to another conformation, named X, which is not the B conformation, as the temperature is increased from 0 to 35 degrees C. Between 40 and 65 degrees C, another transition is observed which corresponds to the melting of the X conformation. At higher nucleotide concentration (2 X 10(-3) M), circular dichroism and 31P nuclear magnetic resonance experiments show that at low temperature (br5dC-dG)3 adopts the Z conformation. There are associations between the oligonucleotides which progressively disappear as the temperature increases. In the range 35-60 degrees C a transition from the Z conformation to another conformation is observed. This new conformation is the X conformation detected at low nucleotide concentration.     

Temperature and ligand dependence of conformation and helical order in myosin filaments

Mammalian myosin filaments are helically ordered only at higher temperatures (>20 degrees C) and become progressively more disordered as the temperature is decreased. It had previously been suggested that this was a consequence of the dependence of the hydrolytic step of myosin ATPase on temperature and the requirement that hydrolysis products (e.g., ADP.P(i)) be bound at the active site. An alternative hypothesis is that temperature directly affects the conformation of the myosin heads and that they need to be in a particular conformation for helical order in the filament. To discriminate between these two hypotheses, we have studied the effect of temperature on the helical order of myosin heads in rabbit psoas muscle in the presence of nonhydrolyzable ligands. The muscle fibers were overstretched to nonoverlap such that myosin affinity for nucleotides was not influenced by the interaction of myosin with the thin filament. We show that with bound ADP.vanadate, which mimics the transition state between ATP and hydrolysis products, or with the ATP analogues AMP-PNP or ADP.BeF(x)() the myosin filaments are substantially ordered at higher temperatures but are reversibly disordered by cooling. These results reinforce recent studies in solution showing that temperature as well as ligand influence the equilibrium between multiple myosin conformations [Málnási-Csizmadia, A., Pearson, D. S., Kovács, M., Woolley, R. J., Geeves, M. A., and Bagshaw, C. R. (2001) Biochemistry 40, 12727-12737; Málnási-Csizmadia, A., Woolley, R. J., and Bagshaw, C. R. (2000) Biochemistry 39, 16135-16146; Urbanke, C., and Wray, J. (2001) Biochem. J. 358, 165-173] and indicate that helical order requires the myosin heads to be in the closed conformation. Our results suggest that most of the heads in the closed conformation are ordered, and that order is not produced in a separate step. Hence, helical order can be used as a signature of the closed conformation in relaxed muscle. Analysis of the dependence on temperature of helical order and myosin conformation shows that in the presence of these analogues one ordered (closed) conformation and two disordered conformations with distinct thermodynamic properties coexist. Low temperatures favor one disordered conformation, while high temperatures favor the ordered (closed) conformation together with a second disordered conformation.     

Conformational properties of the G.G mismatch in d(CGCGAATTGGCG)2 determined by NMR.

The conformational properties of the DNA duplex d(CGCGAATTGGCG)2, which contains two noncomplementary G.G base pairs, have been examined in aqueous solution by 1H and 31P NMR as a function of temperature. The G.G mismatch is highly destabilizing, with a Tm value 35 K below that observed for the native EcoRI dodecamer. The dodecamer appears symmetric in the NMR spectra and exists largely as an average B-type DNA conformation. However, the 1H and 31P NMR spectra give evidence of considerable conformational heterogeneity at the mismatched nucleotides and their nearest neighbors, which increases with increasing temperature. There is no evidence for a significant population of the syn purine conformation. The imino protons of the mispaired bases G4 and G9 are degenerate, resonate at high field, and exchange readily with solvent. These results indicate that the mispaired bases are only weakly hydrogen-bonded and are only partially stacked into the helix. On raising the temperature, the duplex shows increasing exchange between two or more conformations originating from the mismatch sites. However, these additional conformations maintain their Watson-Crick hydrogen bonding. The increase in chemical exchange is consistent with a quasimelting process for which the G.G sites provide local nuclei. Extensive modeling studies by dynamic annealing have confirmed that the G(anti).G(anti) conformation is favored and that the mispairs are poorly stacked within the helix. The results explain both the poor thermal stability and low hypochromicity of this duplex.     

A new bacterial polysaccharide (YAS34). I. Characterization of the conformations and conformational transition

This paper concerns the study of the conformational transition of a new exopolysaccharide (YAS34) using experimental techniques such as optical rotation, conductimetric and microcalorimetric measurements as a function of temperature. The behaviors of this polysaccharide in the acid or sodium salt form are compared; a deacetylated sample is also prepared to demonstrate the role of substituents. For the native structure (never heated), a conformational transition is observed but the deacetylated polysaccharide exhibits no ordered conformation. Multidetection size exclusion chromatography (SEC) analyses and conductimetric experiments allowed to determine the nature of each conformation and the molecular dimensions. From these results, it is suggested that the native conformation is a double helix which by heating over T(m) (temperature corresponding to half conformational transition) dissociates into disordered single chains. In the acid and sodium salt forms, by cooling below T(m), an ordered conformation is restored. This conformation seems to be an intramolecular double helix 'hairpin-like turn' (called renatured conformation). Nevertheless an irreversible denaturation is obtained progressively in the sodium salt form when the time of heating over T(m) increases. The conformation of the deacetylated polysaccharide corresponds to that of a single flexible chain (disordered conformation). The conformational transition for the native conformation was studied also in relation to the polyelectrolytic character of the polysaccharide: stability as a function of salt nature and salt and polymer concentrations was investigated for the polymer initially in the sodium and acid forms.     

Conformation of the antifreeze glycoprotein of polar fish

High-field proton and 13C NMR spectroscopy has been used to test and refine the recent proposal, based on vacuum uv circular dichroism results, of a threefold left-handed helical conformation for antifreeze glycoprotein (AFGP). Partial assignment of the protons of the glycotripeptide repeating unit has been made by comparison with spectra of model compounds, by selective decoupling, and by measurements of nuclear Overhauser effect (nOe). At 40 degrees C, AFGP fraction 8 (Mr 2600) shows 2-Hz linewidths which broaden at lower temperature. Neither 1H nor 13C chemical shifts depend strongly on temperature, suggesting no abrupt conformational transition. The nOe between alanine alpha and beta protons vary with temperature and with field strength, from small positive enhancements at 50 degrees C and 80 MHz to large negative effects at 3 degrees C and 300 MHz, indicating a substantial change of rotational correlation time with temperature. The higher-molecular-weight fraction 1-4 shows negative nOe at all temperatures. The CD spectra of fraction 1-4 show bands characteristic of the polyproline II structure at both 3 and 50 degrees C, while those bands in fraction 8 are weaker at 50 than 3 degrees C. The 1H nOe, the 13C T1, and CD data are interpreted as indicating that AFGP fraction 8 is an extended "rod-like" conformation at low temperature which becomes a flexible coil at high temperature, while fraction 1-4 is a flexible rod with sufficient segmental mobility to eliminate any long-range order.     

Trapping conformational intermediate states in the reaction center protein from photosynthetic bacteria

In protein, conformational changes are often crucial for function but not easy to observe. Two functionally relevant conformational intermediate states of photosynthetic reaction center protein (RCs) are trapped and characterized at low temperature. RCs frozen in the dark do not allow electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B). In contrast, RCs frozen under illumination in the product (P(+)Q(A)Q(B)(-)) state, with the oxidized electron donor, P(+), and reduced Q(B)(-), return to the ground state at cryogenic temperature in a conformation that allows a high yield of Q(B) reduction. Thus, RCs frozen under illumination are found to be trapped above the ground state in a conformation that allows product formation. When the temperature is raised above 120 K, the protein relaxes to an inactive conformation which is different from the RCs frozen in the dark. The activation energy for this change is 87 +/- 8 meV, and the active and inactive states differ in energy by only 16 +/- 3 meV. Thus, there are several conformational substates along the reaction coordinate with different transition temperatures. The ground state spectra of the RCs in active and inactive conformations report differences in the intraprotein electrostatic field, demonstrating that the dipole or charge distribution has changed. In addition, the electrochromic shift associated with the Q(A)(-) to Q(B) electron transfer at low temperature was characterized. The electron-transfer rate from Q(B)(-) to P(+) was measured at cryogenic temperature and is similar to the rate at room temperature, as expected for an exothermic, electron tunneling reaction in RCs.     

Temperature-dependent structure of the E x S complex of Bacillus stearothermophilus alcohol dehydrogenase

The catalytic chemistry of the thermophilic Bacillus stearothermophilus alcohol dehydrogenase (HtADH) closely resembles that of mesophilic horse liver alcohol dehydrogenase (HLADH). Molecular dynamics (MD) simulations of the htADH x NAD+ x EtO- complex at 298, 323, and 348 K show that the structure of the ligated Zn2+...EtO- complex varies slightly with change in temperature. The MD-created Boltzmann distribution of htADH x NAD+ x EtO- structures establishes the formation of multiple states which increase in number with a decrease in temperature. The motions of the cofactor domain are highly correlated with the motions of NAD+ at the optimal growth temperature (348 K), with NAD+ being pushed toward the substrate by Val260. With a decrease in temperature, the motion together of the cofactor and substrate is reversed, and at 298 K, the nicotinamide ring of the cofactor moves away from the substrate. Both the distance between and the angle of approach of C4 of NAD+ and HD of EtO- become distorted from those of the reactive conformation. The percentages of ground state present as the reactive conformation at different temperatures are approximately correlated with the kcat for the htADH enzymatic reaction. The rate constant for the htADH x NAD+ x EtOH --> htADH x NAD+ x EtO- proton dissociation, which is mediated by Thr40-OH, becomes slower at lower temperatures. The time-dependent distance between EtO- and Thr40-OH reveals that the Thr40 hydroxyl group sways between the substrate and NAD+ ribose 2'-hydroxyl group at the optimal enzyme growth temperature, and this movement is effectively frozen out as the temperature decreases. The temperature dependence of active site conformations is due to the change in both long-range and short-range motions of the E x S complex.     

Effect of temperature and protonation upon the conformation of 2'-o-methyladenosine. Correlation of conformational parameters in purine nucleosides.

Proton magnetic resonance studies of 2'-o-methyladenosine in 2H2O have been carried out at variable temperature and p2H. The chemical shifts and H-H coupling constants are discussed in terms of the molecular conformation. Comparison of the data with those of adenosine reveals that 2'-O-methylation has little influence on the conformation. At neutral p2H where the adenine base is not protonated, the molecules favor a 2' endo, gauche-gauche conformation. Protonation of the base at the N(1) position leads to a decrease in the 2' endo, gauche-gauche bias. The data for 2'-O-methyladenosine and adenosine, as well as for several other purine derivatives, reveal the presence of a correlation between the sugar pucker and the C(5')-C(4') conformer distribution which is the inverse of the correlation previously reported for pyrimidine derivatives.     

Thermal stability of the Z-conformation of the tetranucleoside triphosphate (m5dC-dG)2

The tetranucleoside triphosphate d(m5C-G)2 has been studied in solution by circular dichroism and 31P nuclear magnetic resonance as a function of temperature, in presence of 3 M NaClO4. It is shown that in such high ionic strength d(m5C-G)2 may adopt a Z-like conformation for temperatures lower than 5 degrees C. At these temperatures, another conformation, in slow equilibrium with the Z-like one, is also detected. Increasing the temperature leads to a transition from the Z-like conformation to intermediate forms before melting. It is demonstrated that these intermediates are not the B form.     

Circular dichroism evidence of the left-handed conformation adopted by cyclopropylpyrroloindole antitumor antibiotics in solution

The purpose of this work was to determine the conformation adopted in solution by adozelesin, carzelesin, and their derivatives obtained under basic or acidic conditions. Circular dichroism in the 270–330 nm wavelengths region was studied. In solution adozelesin can adopt two different conformations: a left-handed one which is thermodynamically favoured at low temperature and is mainly present at room temperature, and a right-handed one which is observed either at high temperature or at room temperature in the presence of DNA. This was ascertained by the presence of circular dichroism signals of the couplet type. The active form of carzelesin, i.e., U-76074, also exhibited a left-handed conformation in solution. Carzelesin and the derivatives obtained under acidic conditions that lack the cyclopropyl ring, and cyclopropylpyrroloindole, obtained under basic conditions, cannot adopt such a conformation. Chirality 8:585–589, 1996. © 1997 Wiley-Liss, Inc.     

Molecular assembly formation of cyclic hexa-beta-peptide composed of acetylated glycosamino acids

A novel cyclic hexamer of acetylated beta-glycosamino acid was synthesized and its conformation and molecular assembly formation was investigated. Variable temperature NMR study indicated that the cyclic hexapeptide took a C(3) symmetric conformation at room temperature, but at elevated temperatures a C(6) symmetric one, which was not due to averaging of the C(3) symmetric conformation, appeared. Computational geometry optimization showed that the C(6) symmetric conformation was a highly planar structure with amide groups orienting perpendicular to the ring plane. The cyclic hexa-beta-peptide formed rod-shaped crystals from an N,N-dimethyl formamide solution at elevated temperature. The optical microscopy observation with a sensitive tint plate under cross-nicol configuration and electron diffraction analysis of the crystals revealed that the cyclic hexa-beta-peptides were stacked one after the other to form a regular nanotube structure.     

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.     

Non-Watson-Crick structures in oligodeoxynucleotides: self-association of d(TpCpGpA) stabilized at acidic pH

The 1H NMR spectrum of the tetradeoxynucleotide d(TpCpGpA) was examined as a function of temperature, pH, and concentration. At pH 7 and above the solution conformation for this oligodeoxynucleotide appears to be a mixture of random coil and Watson-Crick duplex. At 25 degrees C, a pH titration of d(TpCpGpA) shows that distinct conformational changes occur as the pH is lowered below 7.0. These conformational changes are reversible upon readjusting the pH to neutrality, indicating the presence of a pH-dependent set of conformational equilibria. At 25 degrees C, the various conformational states in the mixture are in rapid exchange on the NMR time scale. Examination of the titration curve shows the presence of distinct conformational states at pH greater than 7, and between pH 4 and pH 5. At pH less than 4, a third conformational state is present. When the pH titration is repeated at 5 degrees C, the conformational equilibria are in slow exchange on the NMR time scale; distinct signals from each conformational state are observable. The stable conformational state present between pH 4 and pH 5 represents an ordered conformation of d(TpCpGpA) which dissociates to a less ordered structure upon raising the temperature. This ordered conformation does not result from an intramolecular rearrangement, as is shown by by spectra obtained by varying oligodeoxynucleotide concentration at constant pH. The ordered conformation differs from the Watson-Crick helix, as is shown from nuclear Overhauser enhancement experiments, as well as chemical shift data. An ordered conformation for d(TpCpGpA) was previously reported [Reid, D. G., Salisbury, S. A., Brown, T., & Williams, D. H. (1985) Biochemistry 24, 4325-4332].(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal in helix sense of copoly(beta-alkyl-L-aspartate-beta-benzyl-L-aspartate)

The conformation of copoly(beta-alkyl-L-aspartate-beta-benzyl-L-aspartate), in which the alkyl group is ethyl, propyl, butyl, hexyl, nonyl, dodecyl, or stearyl, was studied in solution and the solid state by optical rotatory dispersion and circular dichroism methods. The helix sense of the copolyaspartate studied here is transformed from a left-handed to right-handed alpha-helix as the degree of alkylation increases. Reversal in helix sense occurs, i.e., the left-handed alpha-helix based on the handedness of poly(beta-benzyl-L-aspartate) is transformed into a right-handed alpha-helix with increase in alkyl groups with right-handed nature. Reversal in helix sense is also observed for copolyaspartates with an intermediate or high degree of alkylation as temperature rises. Copolyaspartates with hexyl, nonyl, or dodecyl groups exhibit an induced circular dichroism around 230-238 nm and can form an ordered side chain structure which is broken down at high temperature. One has to consider the conformation of the omega-helix and beta-form of the copolyaspartates in the solid state in addition to the reversal in helix sense. Copolyaspartates with a low degree of alkylation are in the alpha-helical conformation over the low temperature range and adopt the omega-helical conformation in the high temperature range, indicative of a thermal alpha-omega transition. A small number of alkyl groups can be incorporated into the benzene ring stacking of the omega-helix, but not a large number. All the copolyaspartates can assume the beta-form at high temperatures. The helix conformation is not significantly affected by the formation of side chain crystals of the copolyaspartate with a large number of stearyl groups, in contrast to copolyglutamate.     

Regulation of tryptophan synthase by temperature, monovalent cations, and an allosteric ligand. Evidence from Arrhenius plots, absorption spectra, and primary kinetic isotope effects

To investigate the linkage between enzyme conformation and catalysis, we have determined the effects of temperature on catalytic properties of the tryptophan synthase alpha(2)beta(2) complex and beta(2) subunit in the absence or presence of different monovalent cations (Cs(+), Na(+), and GuH(+)) and of an allosteric ligand, alpha-glycerol 3-phosphate. Arrhenius plots of the activity data between 5 and 50 degrees C are nonlinear in the presence of certain ligands but not others. The conditions that yield nonlinear Arrhenius plots also yield temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. The results provide evidence that the nonlinear Arrhenius plots are caused by a temperature-dependent conformational change that precedes the rate-limiting step in catalysis. Thermodynamic analysis of the data associated with the conformational change shows that the activation energies are much higher at low temperatures than at high temperatures. We correlate the results with a model in which the enzyme is converted by increased temperature under certain conditions from a low-activity "open" conformation to a high-activity "closed" conformation. The allosteric ligand and different monovalent cations, including GuH(+), which also acts as a chaotropic agent, affect the equilibrium between the open and closed forms. The large positive entropy changes in the conformational conversion suggest that the closed conformation results from tightened hydrophobic interactions that exclude water from the active site of the beta subunit.     

Further evidence for the absence of polyproline II stretch in the XAO peptide

It has been suggested that the alanine-based peptide with sequence Ac-XX-[A](7)-OO-NH(2), termed XAO where X denotes diaminobutyric acid and O denotes ornithine, exists in a predominantly polyproline-helix (P(II)) conformation in aqueous solution. In our recent work, we demonstrated that this "polyproline conformation" should be regarded as a set of local conformational states rather than as the overall conformation of the molecule. In this work, we present further evidence to support this statement. Differential scanning calorimetry measurements showed only a very small peak in the heat capacity of an aqueous solution of XAO at 57 degrees C, whereas the suggested transition to the P(II) structure should occur at approximately 30 degrees C. We also demonstrate that the temperature dependence of the (3)J(HNHalpha) coupling constants of the alanine residues can be explained qualitatively in terms of Boltzmann averaging over all local conformational states; therefore, this temperature dependence proves that a conformational transition does not occur. Canonical MD simulations with the solvent represented by the generalized Born model, and with time-averaged NMR-derived restraints, demonstrate the presence of an ensemble of structures with a substantial amount of local P(II) conformational states but not with an overall P(II) conformation.     

Formation of the antiplanar-antiplanar phosphate conformation of dilauroylphosphatidylcholine bilayers

Infrared spectroscopy was used to investigate lipid conformational changes that occur in dilauroylphosphatidylcholine (diC12PC) bilayers with and without fatty-acid-amino-acids as guest molecules in the membrane. Incorporating 2.5 mole% N-decanoylglycine (decgly) into diC12PC liposomes caused formation of the antiplanar-antiplanar (ap-ap) phosphodiester conformation which was stable in room temperature IR spectra. Several other fatty-acid-amino-acids incorporated into diC12PC bilayers were found to also elicit the ap-ap phosphodiester conformation. Unlike these diC12PC/fatty-acid-amino-acid mixed bilayers, pure diC12PC bilayers would form the ap-ap phosphodiester conformation only under low temperature incubation conditions. Dry diC12PC films incubated at 5 degrees C for 0.5 h (brief incubation) or 16 h (prolonged incubation), and then rapidly hydrated (i.e., vortexed at 25 degrees C in D2O), caused the ap-ap phosphodiester conformation to persist in the diC12PC liposomes equilibrated to room temperature. Slow hydration for 16 h at 5 degrees C in both buffered and non-buffered D2O of diC12PC lipid films also produced the ap-ap phosphodiester conformation. In contrast, slow hydration for 16 h at 5 degrees C in PBS/D2O of diC12PC/decgly mixed films caused the greatest number of ap-ap phosphodiester conformers. Using pure diC12PC bilayers, infrared data indicate that incubation of diC12PC films causes the headgroup phosphodiester conformation to change from gauche-gauche (g-g) conformation to the ap-ap conformation. Under all liposome formation conditions examined, no changes in hydration of either the phosphate group or the carbonyl ester group were detected and in addition, no trans/gauche conformational changes in the acyl chain were observed.