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PubMed1991

Synthesis and NMR conformational analysis of a beta-turn mimic incorporated into gramicidin S. A general approach to evaluate beta-turn peptidomimetics.

The solution structure of a gramicidin S (GS) analog containing a beta-turn mimic [BTD4-5, Lys2.2']GS has been compared to that of native GS. The linear [BTD4-5, Lys2.2']GS was synthesized by solid phase methodology and the cyclized peptide was analyzed by NMR. In the peptide portion of [BTD4-5, Lys2.2']GS, the intramolecular hydrogen bonding pattern, inter-residue NOEs, including a transannular H alpha-H alpha NOE, and JN alpha coupling constants all describe a solution structure which is equivalent to that of native GS. These data confirm that the BTD group is a competent Type II' beta-turn mimic since it does not disrupt the native conformation of GS. It also supports the use of GS as a conformational model in which to test beta-turn mimics.

Amino Acid Sequence↗
PubMed1984

Conformational analysis of the single-helical DNA fragment d(T-A-A-T) in aqueous solution. The combined use of NMR proton chemical shifts and coupling constants obtained at 300 MHz and 500 MHz.

Proton NMR studies at 300 MHz and 500 MHz were carried out on the tetranucleoside trisphosphate d(T-A-A-T). The thermodynamics of the three stacking interactions, derived from chemical shift versus temperature profiles, were used to extrapolate the observed coupling constants, measured at a range of temperatures, to values appropriate to the fully stacked forms of the molecule. The data were interpreted in terms of N and S pseudorotational ranges [ Altona , C. and Sundaralingham , M. (1972) J. Am. Chem. Soc. 94, 8205-8212]. It is shown that the stacked state of the molecule cannot be described by one conformer, but consists of one major structure (60%) in which all sugar rings have S-type geometry and another structure (30%) in which residue dT(4) has an N-type sugar. The remainder of the stacked states consists of one or more conformers with two or three sugar residues in the N-type pseudorotational range. Detailed geometrical models are proposed for the major stacked conformers encountered in aqueous solution.

DNA, Single-Stranded↗
PubMed1992

Conformational analysis of a mitochondrial presequence derived from the F1-ATPase beta-subunit by CD and NMR spectroscopy.

Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood. We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the presequence for the beta-subunit of F1-ATPase, a mitochondrial matrix protein. Although this peptide is essentially unstructured in water, alpha-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE). In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and alpha-helical conformations, with a significant population of alpha-helix throughout the entire peptide. The helix is somewhat more stable in the N-terminal part of the presequence (residues 4-10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV. Addition of increasing amounts of TFE causes the alpha-helical content to increase even further, and the TFE titration data for the presequence peptide of the F1-ATPase beta-subunit are not consistent with a single, cooperative transition from random coil to alpha-helix. There is evidence that helix formation is initiated in two different regions of the peptide. This result helps to explain the redundancy of the targeting information contained in the presequence for the F1-ATPase beta-subunit.

Amino Acid Sequence↗
PubMed2005

Conformational analysis in solution of protein kinase C betaII V5-1 peptide.

One of protein kinase C (PKC) isozymes, PKC beta binds to receptor for activated C kinase 1 (RACK1), and their complex is suggested to be translocated to melanosomes. The binding site of PKC beta for RACK1 is considered one of its catalytic domains, V5 domain which consists of three motifs such as V5-1, V5-2, and V5-3. Among these, V5-1 region, extreme C-terminal residues of PKC beta showed the highest RACK1-binding affinity. PKC beta can be classified into PKC betaI and PKC betaII based on their different V5 domains. RACK1-binding affinity of PKC betaII is five times greater than that of PKC betaI. The structures of PKC betaI, PKC betaII, and RACK1 are not known. However, the conformational study on PKC betaII V5-1 region showing high RACK1-binding selectivity may help us in understanding the interaction between RACK1 and PKC betaII.

Catalytic Domain↗
PubMed2002

Structural and conformational analysis of the oxidase to dehydrogenase conversion of xanthine oxidoreductase.

Xanthine oxidoreductase (XOR) is a 300-kDa homodimer that can exist as an NAD+-dependent dehydrogenase (XD) or as an O2-dependent oxidase (XO) depending on the oxidation state of its cysteine thiols. Both XD and XO undergo limited cleavage by chymotrypsin and trypsin. Trypsin selectively cleaved both enzyme forms at Lys184, while chymotrypsin cleaved XD primarily at Met181 but cleaved XO at Met181 and at Phe560. Chymotrypsin, but not trypsin, cleavage also prevented the reductive conversion of XO to XD; thus the region surrounding Phe560 appears to be important in the interconversion of the two forms. Size exclusion chromatography showed that disulfide bond formation reduced the hydrodynamic volume of the enzyme, and two-dimensional gel electrophoresis of chymotrypsin-digested XO showed significant, disulfide bond-mediated, conformational heterogeneity in the N-terminal third of the enzyme but no evidence of disulfide bonds between the N-terminal and C-terminal regions or between XOR subunits. These results indicate that intrasubunit disulfide bond formation leads to a global conformational change in XOR that results in the exposure of the region surrounding Phe560. Conformational changes within this region in turn appear to play a critical role in the interconversion between the XD and XO forms of the enzyme.

Animals↗
PubMed1991

Hydrogen bonding and conformational analysis of (R)-norcarnitine monohydrate.

(R)-4-(N,N-Dimethylammonio)-3-hydroxybutanoate [(R)-norcarnitine] monohydrate, C6H13NO3.H2O, Mr = 165.2, triclinic, P1, a = 5.9081 (5), b = 6.0438 (4), c = 6.9084 (7) A, alpha = 65.584 (6), beta = 81.957 (8), gamma = 77.771 (6) degrees, V = 219.1 (1) A3, Z = 1, Dx = 1.252 g cm-3, lambda(Cu K alpha) = 1.54184 A, mu = 8.46 cm-1, F(000) = 90, T = 296 K, R = 0.035 for 1763 observations (of 1772 unique data). Intermolecular hydrogen bonding dominates the structure. The molecule of water contacts three different zwitterions, with O...O distances 2.7767 (13), 2.720 (2) and 2.722 (2) A. A hydrogen bond between carboxylate and dimethylammonio links the zwitterions in head-to-tail motif, N...O 2.672 (2) A. Comparison with two other 4-ammonio-3-hydroxybutanoates reveals that the title compound adopts a different conformation along the backbone, N(+)--C--C(OH)--C--CO2-, with N(+)--C--C(OH)--C anti and C--C(OH)--C--CO2- gauche-.

Carnitine↗
PubMed1998

Solid-phase synthesis, conformational analysis, and biological activity of AVR9 elicitor peptides of the fungal tomato pathogen Cladosporium fulvum.

The race-specific peptide elicitor AVR9 of the fungal pathogen Cladosporium fulvum specifically induces a hypersensitive response in tomato genotypes carrying the complementary resistance gene Cf-9. The total chemical syntheses of this 28-residue AVR9 peptide containing three disulfide bonds, and of three mutant peptides [R8K]AVR9, [F10A]AVR9 and [F21A]AVR9, have been accomplished. The syntheses were carried out using a stepwise solid-phase approach based on tBoc chemistry. The disulfide bridges were formed by air oxidation. The correctness of the chemical structure of all folded synthetic peptides was confirmed by combined NMR and MS analyses. The biological activity and a number of physicochemical properties of folded synthetic AVR9 are identical to those of native fungal 28-residue AVR9. The overall conformations of the folded synthetic mutant peptides were comparable to that of synthetic wild-type AVR9 as demonstrated by NMR spectroscopy. Mutant [R8K]AVR9 showed a threefold higher, and mutant [F10A]AVR9 a threefold lower necrosis-inducing activity when compared to synthetic wild-type AVR9. However, mutant [F21A]AVR9 showed hardly any necrosis-inducing activity. Affinity for polyclonal antibodies raised against native fungal AVR9 is positively correlated with the necrosis-inducing activity of the synthetic AVR9 peptides ([R8K]AVR9 > wild-type AVR9 > [F10A]AVR9 > [F21A]AVR9).

Blotting, Western↗
PubMed1997

Application of a genetic algorithm in the conformational analysis of methylene-acetal-linked thymine dimers in DNA: comparison with distance geometry calculations.

The three-dimensional spatial structure of a methylene-acetal-linked thymine dimer present in a 10 basepair (bp) sense-antisense DNA duplex was studied with a genetic algorithm designed to interpret NOE distance restraints. Trial solutions were represented by torsion angles. This means that bond angles for the dimer trial structures are kept fixed during the genetic algorithm optimization. Bond angle values were extracted from a 10 bp sense-antisense duplex model that was subjected to energy minimization by means of a modified AMBER force field. A set of 63 proton-proton distance restraints defining the methylene-acetal-linked thymine dimer was available. The genetic algorithm minimizes the difference between distances in the trial structures and distance restraints. A large conformational search space could be covered in the genetic algorithm optimization by allowing a wide range of torsion angles. The genetic algorithm optimization in all cases led to one family of structures. This family of the methylene-acetal-linked thymine dimer in the duplex differs from the family that was suggested from distance geometry calculations. It is demonstrated that the bond angle geometry around the methylene-acetal linkage plays an important role in the optimization.

Algorithms↗
PubMed1989

Conformational analysis of 9-substituted adenines in relation to their microsomal N1-oxidation.

Metabolic N-oxidation of adenine, 9-methyladenine, 9-benzyladenine, 9-benzhydryladenine and 9-trityladenine has been investigated using hepatic microsomes from hamster, guinea-pig, rabbit, mouse, rat, and dog. N1-Oxide formation occurs with 9-benzyladenine and 9-benzhydryladenine using liver preparations of all species examined, although to different extents. The N-oxidase activity was found, amongst rodents, in the order hamster greater than mouse greater than rabbit greater than rat greater than guinea-pig. Microsomal preparations from dog liver contained a small quantity of P-450 and yet produced a relatively large amount of the N-oxides, possibly indicating that other cytochromes in addition to P-450 may be involved in the N-oxidation of these compounds. The most favourable conformations of these 9-substituted analogues have been established using computer graphics modelling and 1H NMR techniques. Results obtained confirmed the importance of the stereochemical properties of these compounds in relation to N1-oxidation. These observations substantiate and extend our previous findings on the electronic, lipophilic, and stereochemical factors affecting the N-oxidation of adenine derivatives.

Adenine↗
PubMed1993

Synthesis, conformational analysis, and the glycosidic coupling reaction of substituted 2,7-dioxabicyclo[4.1.0]heptanes: 1,2-anhydro-3,4-di-O-benzyl-beta-L- and beta-D-rhamnopyranoses.

1,2-Anhydro-3,4-di-O-benzyl-alpha-L-rhamnopyranose was synthesized from L-rhamnose, while the D-enantiomer was synthesized from methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-mannopyranoside. For both of the syntheses, the key intermediates were 2-O-acetyl-3,4-di-O-benzyl-alpha-D- and -alpha-L-rhamnopyranosyl chlorides that were quantitatively prepared from the corresponding diacetates by chlorination. Ring closure of the chlorides was carried out readily with potassium tert-butoxide in oxolane, and crystalline 1,2-anhydro-3,4-di-O-benzyl-beta-D- and beta-L-rhamnopyranose were obtained in high yields. Conformational calculations, which were carried out using vicinal proton-proton coupling constants by the modified Karplus equation, suggested that the conformations of the pyranose rings of the title compounds were basically a half chair (4H5) with some flattening at C-4. Force-field calculations (MMP2) confirmed the experimental conformation with good agreement. The coupling reaction of the 1,2-anhydro-L-rhamnose ether with 1,2;3,4-di-O-isopropylidene-alpha-D-galactopyranose was effected in oxolane by catalysis by a Lewis acid, and only the alpha-linked disaccharide was obtained.

Antigens, Bacterial↗
PubMed1988

Homoallylic amines related to zimeldine. A comparative study on neuronal serotonin and norepinephrine reuptake based on conformational analysis.

A number of tertiary and secondary homoallylic amines, i.e. (Z)- and (E)-4-(4-bromophenyl)-4-(3-pyridyl)-3-buten-1-ylamines, were synthesized in diastereomerically pure forms. The compounds were evaluated as neuronal norepinephrine (NE) and serotonin (5-HT) uptake inhibitors under in vitro and ex vivo conditions and compared with the tricyclics amitriptyline and nortriptyline having homoallylic side chains and with the corresponding diastereomers in the zimeldine series having allylic side chains. The Z isomers of the new homoallylic derivatives (3Z, 4Z) were specific 5-HT uptake inhibitors in analogy with the corresponding allylic derivatives zimeldine (1Z) and norzimeldine (2Z). Likewise, the selectivity profile of the homoallylic (3E, 4E) and the allylic (1E, 2E) derivatives was comparable. In general, the homoallylic compounds were less potent inhibitors than their allylic counterparts. The similarities and discrepancies were evaluated in terms of conformational preferences determined by CAMSEQ molecular mechanics calculations. Homonorzimeldine (4Z) can accommodate energetically favored, but less populated, conformations having amino nitrogen atom to aromatic ring center distances comparable to those in norzimeldine. These facts correlate to retained 5-HT selectivity but diminished potency of 4Z compared to 2Z.

Animals↗
PubMed2006

Kinetic conformational analysis of human 8-oxoguanine-DNA glycosylase.

7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base followed by a much slower beta-elimination reaction at the 3'-side of the resulting abasic site. Many features of OGG1 mechanism, including its low beta-elimination activity and high specificity for a cytosine base opposite the lesion, remain poorly explained despite the availability of structural information. In this study, we analyzed the substrate specificity and the catalytic mechanism of OGG1 acting on various DNA substrates using stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine reporter fluorescence to follow DNA dynamics, we defined three pre-excision steps and assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii) insertion of several enzyme residues into DNA, and (iii) enzyme isomerization to the catalytically competent form. The individual rate constants were derived for all reaction stages. Of all conformational changes, we identified the insertion step as mostly responsible for the opposite base specificity of OGG1 toward 8-oxoG:C as compared with 8-oxoG:T, 8-oxoG:G, and 8-oxoG:A. We also investigated the kinetic mechanism of OGG1 stimulation by 8-bromoguanine and showed that this compound affects the rate of beta-elimination rather than pre-excision dynamics of DNA and the enzyme.

2-Aminopurine↗
PubMed1983

Carbon-13 NMR in conformational analysis of nucleic acid fragments. Heteronuclear chemical shift correlation spectroscopy of RNA constituents.

The assignment of the non-quaternary 13C resonances by means of two-dimensional heteronuclear chemical shift correlation spectroscopy is presented for several oligoribonucleotides: The dimers m6(2)AU, m6(2)Am6(2)A and mpUm6(2)A and the trimers m6(2)AUm6(2)A and m4(2)Cm4(2)Cm6(2)A. The temperature and concentration dependency of the 13C chemical shifts are studied with emphasis on the behaviour of the dimer m6(2)AU. The present study shows that in the 5-50 mM range the concentration-dependent chemical shift changes of the ribose carbons are negligible compared to chemical shift changes due to intramolecular events. All compounds studied show a surprising correlation between the chemical shifts of the carbon atoms of the ribose ring and the sugar conformational equilibrium as expressed by the percentage N or S conformer. Thus the chemical shift data can be used to obtain the thermodynamical parameters of the two-state N/S equilibrium. Parameters deduced for m6(2)AU are Tm = 306 K and delta S = -25 cal mol-1 K-1, which values are in satisfactory agreement with results obtained earlier from 1H NMR and from Circular Dichroism.

Dinucleoside Phosphates↗
PubMed1991

Primary structure and conformational analysis of peptide methionine-tyrosine, a peptide related to neuropeptide Y and peptide YY isolated from lamprey intestine.

A peptide belonging to the pancreatic-polypeptide-fold family of regulatory peptides has been isolated from the intestine of an Agnathan, the sea lamprey (Petromyzon marinus). The primary structure of the peptide (termed peptide methionine-tyrosine) was established as Met-Pro-Pro-Lys-Pro-Asp-Asn- Pro-Ser-Pro10-Asp-Ala-Ser-Pro-Glu-Leu-Ser-Lys-Tyr20-Met-Leu- Ala-Val-Arg-Asn- Tyr-Ile-Asn-Leu30-Ile-Thr-Arg-Gln-Arg-Tyr CONH2. This sequence shows stronger structural similarity with pig neuropeptide Y (64%), particularly in the COOH-terminal region, than with pig peptide tyrosine--tyrosine (61%) or with pig pancreatic polypeptide (42%). Molecular modelling and dynamic simulation, based upon sequence similarity with turkey pancreatic polypeptide, indicates that the conformations of the polyproline-helix-like region (residues 1-8) and the alpha-helical region (residues 15-30) in turkey pancreatic polypeptide are conserved in peptide methionine-tyrosine, and that non-bonded interactions between these domains have preserved the overall polypeptide fold in the molecule. The substitution of the otherwise totally conserved Gly9 residue by serine in lamprey peptide methionine-tyrosine, however, results in a preferred structure in which the conformation of the beta-turn between the two helical domains (residues 9-14) is appreciably different.

Amino Acid Sequence↗
PubMedDate not supplied

[Theoretical conformation analysis of MCD peptide].

The spatial structure of the MCD-peptide from bee venom has been calculated basing on the known sequence of 22 amino acid. The a priori calculations produce a system of two disulfide bonds, identical to that observed in the native structure. The calculated structure of MCD-peptide is close to that proposed earlier for the homologues peptide tertiapin and is confirmed by NMR and CD data.

Amino Acid Sequence↗
PubMedDate not supplied

Phase transfer catalysis in solid phase peptide synthesis. Preparation of cyclo[Xxx-Pro-Gly-Yyy-Pro-Gly] model peptides and their conformational analysis.

Relatively small cyclic peptides that contain functionalized side chains provide interesting model compounds for studying side chain-side chain interactions, peptide backbone flexibility (especially if X-Pro bonds are included), and as potential enzyme mimetics. In order to develop more efficient synthetic routes to compounds such as cyclo(Xxx-Pro-Gly-Yyy-Pro-Gly), using the Merrifield method, we have investigated several orthogonal solid phase synthesis strategies and contrasted the use of two solid phase peptide-resin cleavage techniques for preparing partially protected linear sequences. Phase transfer catalysis using tetrabutyl ammonium hydrogen sulfate in THF with saturated aqueous K2CO3 provides peptide acid salts in which most of the common protecting groups (Arg(NO2), Tyr(Bzl), Z-Lys, Lys(Boc), and Glu(tBu)) are not affected. Using 500 MHz proton NMR, peptides having a cyclo (L-L-Gly-L-L-Gly) sequence generally display two conformers in DMSO-d6 with the major isomer being the bis-cis conformer, while the minor form contains two beta turns. For peptides with a cyclo(D-L-Gly-L-L-Gly) sequence, the major conformer contains one cis and one trans X-Pro bond and one Type II beta turn, as previously predicted for related structure by Kopple and others.

Catalysis↗
PubMed1994

NMR studies and conformational analysis of a DNA four-way junction formed in a linear synthetic oligonucleotide.

A linear DNA oligomer (M(r) 14,000, 46 nucleotides) was especially designed, chemically synthesized, and studied by means of 1H NMR spectroscopy. The design of the oligomer was guided by the idea that incorporation of three short palindromic sequences, each interspersed by 5'-CTTG-3' motifs at predetermined positions in the oligomer, would give rise to the formation of three stable minihairpin loops [Ippel, J. H., et al. (1992) J. Biomol. Struct. Dyn. 9, 1-16], which in turn were expected to encourage further folding of the strand into a stable four-way junction containing three "hairpin" arms and an open-ended duplex stem as the fourth arm. Linear DNA four-way junctions constructed according to this concept can be more compact and are therefore expected to be more suitable as model compounds for conformational studies compared to junctions that are built from two or more separate strands. A stable cruciform conformation was substantiated for the 46-mer in aqueous solution in the presence of Mg2+. Complete sequential 1H NMR assignments of the nonexchangeable protons (except H4', H5', and H5") were obtained with the aid of NOESY and HOHAHA experiments. The NMR data gave evidence for the expected existence of minihairpin-loop structures at the three 5'-CTTG-3' motifs in the sequence. The complementary stem domains adopt a regular B-DNA form. Watson-Crick type base pairing is preserved for all residues in the stem domains, including the residues at the center of the junction. A systematic investigation of the interresidual NOEs observed between the protons of the eight central residues revealed the complete stacking pattern of the residues at the branch point.

Base Sequence↗