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PubMed1991

On the multiple-minima problem in the conformational analysis of polypeptides. V. Application of the self-consistent electrostatic field and the electrostatically driven Monte Carlo methods to bovine pancreatic trypsin inhibitor.

In connection with the accompanying paper to test various models for the hydration of polypeptides, we have explored a limited portion of the conformational energy hyperspace of the small protein bovine pancreatic trypsin inhibitor (BPTI) with the aid of two search methods developed in this laboratory. A series of low-energy conformations was obtained as a result of this study. These conformations constitute a set of local minima in the conformational energy space of the molecule as described by the ECEPP/2 (Empirical Conformational Energy Program for Peptides) potential energy function, without the inclusion of hydration. Five different initial conformations were used in this exploration: the first corresponds to an energy-refined structure based on the crystallographic coordinates (4PTI) provided by Deisenhofer and Steigemann and reported previously by Meirovitch and Scheraga. The remaining four initial conformations were obtained by using a Variable-Target-Function procedure, applied to the experimental Cartesian coordinates (5PTI) reported by Wlodawer et al. The self-consistent electrostatic field (SCEF) and the electrostatically driven Monte Carlo (EDMC) methods were used to search the conformational space. The SCEF and EDMC methodologies assume that a polypeptide or protein molecule is driven toward the native structure mainly by the action of the electrostatic interactions. Application of these methodologies led to a set of conformations (up to 50 kcal/mol lower than the starting ones) with ECEPP/2 energies lower than any of those that we had previously found. Application of both methods to the initial conformation generated from 4PTI led to a series of low-energy conformations exhibiting similar rms deviations with respect to the experimental data (4PTI) as did the starting conformation. However, statistical analysis of the runs that had started from the conformations generated by using the variable-target-function procedure (and applying the EDMC method) indicated that the rms deviations of the atomic positions of the new low-energy conformations tended to increase as the energy improved, when compared with the X-ray data from which the starting conformations had been generated. The structures with the lowest energies also had radii of gyration smaller than the experimentally observed one. These results indicated a need to include hydration in the potential function, and provided the conformations used in the accompanying paper to test various hydration models.

Animals↗
PubMed2002

Conformational analysis of the Galpha(s) protein C-terminal region.

The C-terminal domain of the heterotrimeric G protein a-subunits plays a key role in selective activation of G proteins by their cognate receptors. Several C-terminal fragments of Galpha(s) (from 11 to 21 residues) were recently synthesized. The ability of these peptides to stimulate agonist binding was found to be related to their size. Galpha(s)(380-394) is a 15-mer peptide of intermediate length among those synthesized and tested that displays a biological activity surprisingly weak compared with that of the corresponding 21-mer peptide, shown to be the most active. In the present investigation, Galpha(s)(380-394) was subjected to a conformational NMR analysis in a fluorinated isotropic environment. An NMR structure, calculated on the basis of the data derived from conventional 1D and 2D homonuclear experiments, shows that the C-terminal residues of Galpha(s)(380-394) are involved in a helical arrangement whose length is comparable to that of the most active 21 -mer peptide. A comparative structural refinement of the NMR structures of Galpha(s)(380-394) and Galpha(s)(374-394)C379A was performed using molecular dynamics calculations. The results give structural elements to interpret the role played by both the backbone conformation and the side chain arrangement in determining the activity of the G protein C-terminal fragments. The orientation of the side chains allows the peptides to assume contacts crucial for the G protein/receptor interaction. In the 15-mer peptide the lack as well as the disorder of some N-terminal residues could explain the low biological activity observed.

Amino Acid Sequence↗
PubMed1992

Identification of factor IX mutations in haemophilia B: application of polymerase chain reaction and single strand conformation analysis.

The molecular characterization of two haemophilia B defects, Calgary 1 and Calgary 2, was carried out using polymerase chain reaction (PCR) amplification and direct dideoxy sequencing. It had been previously shown that the Calgary 1 mutation affects the 5' TaqI restriction site of exon VIII, whereas Calgary 2 involves the loss of the 3' TaqI site of exon VIII of the factor IX gene. Sequencing data has now revealed that each of these alterations involves a C-to-T transition within a CpG dinucleotide. In each instance an arginine residue is replaced by a stop codon. These cases represent the recurrence of each particular alteration, both of which are predicted to result in the production of a truncated protein lacking a significant part of the catalytic region. A recently developed technique that reveals base substitutions as single-strand conformation polymorphisms (SSCP) was adapted for modelling in the detection of point mutations. Referred to here as single-strand conformation (SSC) analysis, this procedure, used in association with PCR, provided a reliable and sensitive system for molecular diagnosis in each of the cases presented. Computer-generated secondary structure predictions demonstrated a strong correlation with experimental results and the technique was used to screen 11 additional patients in the same region. A change detected by SSC analysis in one patient was localized to 55 base pairs, sequenced, and identified as a conservative amino acid substitution. This patient is now referred to as Calgary 3.

Base Sequence↗
PubMed2000

Multimerization-cyclization of DNA fragments as a method of conformational analysis.

Ligation of short DNA fragments results in the formation of linear and circular multimers of various lengths. The distribution of products in such a reaction is often used to evaluate fragment bending caused by specific chemical modification, by bound ligands or by the presence of irregular structural elements. We have developed a more rigorous quantitative approach to the analysis of such experimental data based on determination of j-factors for different multimers from the distribution of the reaction products. j-Factors define the effective concentration of one end of a linear chain in the vicinity of the other end. To extract j-factors we assumed that kinetics of the reaction is described by a system of differential equations where j-factors appear as coefficients. The assumption was confirmed by comparison with experimental data obtained here for DNA fragments containing A-tracts. At the second step of the analysis j-factors are used to determine conformational parameters of DNA fragments: the equilibrium bend angle, the bending rigidity of the fragment axis, and the total twist of the fragments. This procedure is based on empirical equations that connect the conformational parameters with the set of j-factors. To obtain the equations, we computed j-factors for a large array of conformational parameters that describe model fragments. The approach was tested on both simulated and actual experimental data for DNA fragments containing A-tracts. A-tract DNA bend angle determined here is in good agreement with previously published data. We have established a set of experimental conditions necessary for the data analysis to be successful.

Base Sequence↗
PubMed2004

Conformational analysis of canonical 2-deoxyribonucleotides. 2. Purine nucleotides.

The molecular structure of different conformers of isolated canonical purine 2'-deoxyribonucleotides 2-deoxyadenosine-5'-phosphate (pdA) and 2'-deoxyguanosine-5'-phosphate (pdG) was optimized using the B3LYP/6-31G(d) method. The results of the calculations reveal that the geometrical parameters and relative stability of the conformers significantly depend on the nature of the nucleobase, its orientation, the conformation of the furanose ring, the charge of the phosphate group and the character of the intramolecular hydrogen bonds. Analysis of the electron density distribution in purine nucleotides reveals the existence of a number of intramolecular hydrogen bonds. In general, the south conformer has a lower energy at the anti orientation of the base, and both conformers occur as the most stable for the syn orientation of the nucleobases. The results of the calculations reveal that the geometry and relative energy of the conformers of purine DNTs may be easily tuned by the charge of the phosphate group.

Anions↗
PubMed1997

Rapid and enhanced detection of mitochondrial DNA variation using single-strand conformation analysis of superposed restriction enzyme fragments from polymerase chain reaction-amplified products.

A strategy is described for detecting mitochondrial (mt) DNA variation which permits rapid and straightforward screening for forensic purposes. The method is based on the selection of fragments with adequate length for performing single strand conformation polymorphism (SSCP) analysis selecting a set of restriction enzymes (RE) which yield fragments with prefixed lengths. After digestion of mtDNA by the appropriate enzyme or set of enzymes, SSCP analysis is performed in a semiautomatic electrophoretic system using a silver staining detection method. The conformational changes due to single mutations were therefore found not to change the electrophoretic protocol but to change the relative position of the mutations within the fragment. The discrimination power of this method is estimated to be 90% when two restriction enzymes (MspI and HinfI) are used, but it is considerably higher when other enzymes are added.

DNA Mutational Analysis↗
PubMed1999

Configurative correlation and conformational analysis of strictosidine and vincoside derivatives

On the basis of the configuration of C-15 of the secologanin unit, using detailed NMR analysis, the configuration of C-3, the solution conformation around C-14, and the glucosidic bridge, as well as those of the dihydropyran and tetrahydropyridine rings, were determined in the vincosamide and strictosamide derivatives 4b and 5b. The stereochemical analysis was extended by chemical correlation to the 4-benzylated strictosidine and vincoside derivatives 3c and 3d. Experimental proof was presented for the interpretation of the "anomalous" chemical shift of acetylated strictosamide derivatives.

Journal Article↗
PubMed2005

Conformational analysis of a tetrasaccharide based on NMR spectroscopy and molecular dynamics simulations.

The conformational preference of the human milk oligosaccharide lacto-N-neotetraose, beta-d-Galp-(1 --> 4)-beta-d-GlcpNAc-(1 --> 3)-beta-d-Galp-(1 --> 4)-d-Glcp, has been analyzed using (1)H,(1)H T-ROESY and (1)H,(13)C trans-glycosidic J coupling experiments in isotropic solution and (1)H,(13)C residual dipolar couplings (RDCs) obtained in lyotropic liquid crystalline media. Molecular dynamics simulations of the tetrasaccharide with explicit water as the solvent revealed that two conformational states are significantly populated at the psi glycosidic torsion angle, defined by C(anomeric)-O-C-H, of the (1 --> 3)-linkage. Calculation of order parameters, related to the molecular shape, were based on the inertia tensor and fitting of experimental RDCs to different conformational states showed that psi(+) > 0 degrees is the major and psi(-) < 0 degrees is the minor conformation in solution, in complete agreement with a two-state analysis based on the T-ROESY data. Attention was also given to the effect of salt (200 mM NaCl) in the anisotropic medium, which was a ternary mixture of n-octyl-penta(ethylene glycol), n-octanol, and D(2)O.

Carbohydrate Sequence↗
PubMed1997

Conformational analysis of the thrombin receptor agonist peptides SFLLR and SFLLR-NH2 by NMR: evidence for a cyclic bioactive conformation.

The conformational properties of the pentapeptide Ser-Phe-Leu-Leu-Arg (P5), a human thrombin receptor-derived sequence forming part of a tethered ligand which activates the thrombin receptor, and its more active amide derivative Ser-Phe-Leu-Leu-Arg-NH2 (P5-NH2), have been studied by proton NMR spectroscopy in dimethylsulfoxide. Measurements of nuclear Overhauser effects, performed using two-dimensional rotating frame nuclear Overhauser (ROESY) and one-dimensional nuclear Overhauser enhancement (NOE) spectroscopy, revealed that P5 exists mainly in an extended conformation. However, proton-proton 1D-NOEs between Phe C alpha H and Ser C alpha H Leu3 C alpha H and Leu3 NH, and Leu4 C alpha H and Leu4 NH, as well as between the Ser and Arg sidechains, also implicated a minor conformer for P5 having a curved backbone and a near-cyclic structure. In contrast to P5, measurements of NOEs and ROEs for P5-NH2 revealed a more stabilized cyclic structure which may account for its higher biological potency. Thus strong interresidue sequential NH (i)-NH (i + 1) interactions, as well as C-terminal carboxamide to N-terminal side-chain interactions, i.e., Arg CONH2 to Phe ring and Arg CONH2 to Ser C alpha/C beta beta', observed at lower levels of the ROESY spectrum, supported a curved backbone structure for SFLLR-NH2. Since the higher potency P5-NH2 analogue adopts predominantly a cyclic structure, a cyclic bioactive conformation for thrombin receptor agonist peptides is suggested.

Amino Acid Sequence↗
PubMed1994

The transformation of nascent disc shaped high density lipoproteins into mature spherical high density lipoproteins: conformational analysis of apolipoprotein A-I in model intermediate particles.

The conformation of apolipoprotein A-I (apo A-I) was studied in reassembled lipoprotein particles containing various proportions of apo A-I, phospholipids and cholesteryl esters or triglycerides, using both theoretical and experimental approaches. The proportions of the components were chosen to permit the formation of swollen discs mimicking the intermediate particles that must result from the lecithin:cholesterol acyl transferase (LCAT)-mediated transformation of nascent discoidal high-density lipoprotein (HDL) into mature spherical HDL. Three groups of particles were defined by the number of helices per apo A-I molecule that could fit the edge of the swollen discs. Groups I, II and III had six, seven and eight helices per apo A-I, respectively. The conformation of apo A-I was studied by quenching of tryptophan fluorescence in the presence of KI. In all groups the tryptophan (Trp) residues belonging to the helices located on the edge of the particles were very sparingly hydrated. In contrast, the degree of hydration of Trp residues in external position was different in each group. The structural organization of phospholipids was assessed by measurements of the fluorescence anisotropy of 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) probe. In group I, which contained the less swollen particles, a segment of apo A-I of about 46 amino acids contributed to the area of the faces of the particles, resulting in a highly condensed and ordered phospholipid matrix. In groups II and III, the external segment of apo A-I did not interact with phospholipids.

Apolipoprotein A-I↗
PubMed1991

N.m.r. and conformational analysis of some 2,3-disubstituted methyl alpha-L-rhamnopyranosides.

Conformational studies of the branched trisaccharide glycosides X-(1----2)[Y-(1----3)]-alpha-L-Rha-OMe (where X and Y are residues of alpha-L-, beta-L-, alpha-D-, and beta-D-hexopyranoses) were based on 1H- and 13C-n.m.r. data (n.O.e.'s, 13C chemical shifts) and theoretical calculations. In the majority of the trisaccharide glycosides, there is insignificant restriction of rotation around the glycosidic linkages in the disaccharide units as compared to the corresponding disaccharide glycosides X-(1----2)-alpha-L-Rha-OMe and Y-(1----3)-alpha-L-Rha-OMe. Differences in the conformations observed for several compounds resulted in changes of the n.O.e. patterns and in deviations from additivity of glycosylation effects in the 13C-n.m.r. spectra.

Carbohydrate Conformation↗
PubMed1991

Molecular recognition. Conformational analysis of limited proteolytic sites and serine proteinase protein inhibitors.

The conformations of known tryptic limited proteolytic sites have been analysed and compared to the structures of the binding regions of serine proteinase inhibitors, as they are found when complexed to a serine proteinase. Conformational parameters studied include main-chain torsion angles, root-mean-square fits, accessibility, mobility and protrusion indices. As observed before, the inhibitors share a common main-chain conformation at the binding loop from P3-P'3 (Schechter & Berger notation), which is maintained throughout all the serine proteinase inhibitor families for which X-ray data is available, despite lack of similarity in the rest of the protein. This canonical structure is not found amongst the limited proteolytic sites (or nicksites), which differ markedly from the inhibitor binding loop conformation, and also amongst themselves. The experimentally determined nicksites are in general both accessible and protruding; as are the inhibitor binding loops, as well as being typically flexible regions of structure, as denoted by elevated temperature factors from crystallographic determinations. For cleavage by serine proteinases these loops must radically alter their local conformations and a large motion of the loop relative to the structure, in some cases, would be required to orientate these sites for cleavage.

Amino Acid Sequence↗
PubMed1989

Conformational analysis and molecular modelling of the branching point of amylopectin.

The conformational properties of 6(2) alpha-D-glucosylmaltotriose have been studied using energy calculations that include van der Waals interactions, hydrogen bond stabilization, exo-anometric effect and torsional potential contributions. The calculations focused mainly on the conformational properties displayed at the alpha(1----6) linkage within the tetrasaccharide for which the conformational space is reported. The tetrasaccharide molecule was then considered as a model compound of the branching point in amylopectin. From molecular modelling, some basic structural features associated with branching were clearly established. It was found that, among the low energy arrangements, the side chain would fold back onto the main backbone, thereby producing dense three-dimensional structures in which a 'parallel' arrangement is achieved. The branching between two strands of the double helix, as found in the crystalline moiety of A and B starches, was further investigated. It was found that one particular set of conformations about the glycosidic linkages in the two different strands, could result in an arrangement such that strands could be connected through an alpha(1----6) glycosidic linkage, with a minimum of distortion. The three-dimensional features derived from the molecular modelling agree with the physical properties and mode of biogenesis within the starch granule; they are in accord with a 'cluster' type of structure.

Amylopectin↗
PubMedDate not supplied

Conformational analysis of the 20 naturally occurring amino acid residues using ECEPP.

Conformational energy calculations using ECEPP (Empirical Conformational Energy Program for Peptides) were carried out on the N-acetyl-N'-methylamides of the 20 naturally occurring amino acids. Minimum-energy conformations were located, and the relative conformational energy, librational entropy, and free energy each minimum were calculated. The effects of intrinsic torsional potentials, intramolecular hydrogen bonds, and librational entropy on relative conformational energies and locations of minima are discussed. The results are categorized most easily by use of a new conformational letter code that is introduced here.

Amino Acids↗
PubMed1999

Conformational analysis of the phosphorylated epidermal growth factor receptor.

Phosphorylation-induced conformational changes have been well documented with different receptor tyrosine kinases. However. the susceptible epitopes and the tyrosine residue(s) involved in particular structural alteration mostly remain to be determined. Using a conformation-specific anti-peptide antibody, we have not only identified one such domain in the C-terminal tail of the EGF receptor but also identified the phosphate acceptor sites that are involved in the conformational change.

3T3 Cells↗
PubMedDate not supplied

[Conformational analysis of complexes of RNA polymerase from Escherichia coli with DNA].

Conformational behaviour of T2 DNA during its complex formation with E.coli RNA polymerase was studied by spin label technique. T2 DNA was selectively modified at its readily melting AT-rich sites by bromoacetooxypiperidine-1-oxyl-radical. Specific conformational changes are induced in DNA structure by RNA polymerase attachment. The changes are observed under the conditions when open transcriptionally competent complexes are formed. The readily melting sites of T2 DNA were shown to be involved in the formation of functionally active promoters.

Bacterial Proteins↗
PubMed1992

[Conformational analysis of biologically active thyroliberin analogs by two-dimensional NMR spectroscopy].

Preferable conformations of thyrotropin-releasing hormone (TRH, Glp-His-Pro-NH2) and its analogues Glp-Glu(R)-Pro-NH2 (R = NHCH(CH3)CH2Ar), Glp-Gln-Abu-NH2, Dho-Gln-Abu-NH2 in DMSO solution are determined using two-dimensional 1H NMR spectroscopy (delta-J-correlated, COSY and NOESY). Torsion angles psi i and chi i for every amino acid were calculated on the basis of the spin-spin coupling constants 3JNH-C alpha H and 3JC alpha H-C beta H values. The NOESY data were used for selecting the peptide conformations realized in solution. Distances between protons interacting by the dipole mechanism (d-contacts) were calculated using NOE values. These experiments allow one to estimate the torsion angles psi (between C alpha H-CO). TRH has an intramolecular H-bond between NH2-protons and His carbonyl with the torsion angles omega 3 = 180 degrees and psi 3 = 0 degrees. It is formation of this H-bond that apparently promotes the domination of the trans configuration of the His-Pro peptide bond. An intramolecular NH2-C alpha CO (Glp) H-bonding is revealed in other investigated compounds. It is known that a similar conformation of the TRH is realized in the course of its interaction with receptor.

Amino Acid Sequence↗
PubMed2005

Conformational analysis and stereoelectronic effects in trans-1,2-dihalocyclohexanes: 1H NMR and theoretical investigation.

The conformational equilibrium of trans-1,2-difluoro- (1), trans-1,2-dichloro- (2) and trans-1,2-dibromo-cyclohexane (3) was studied through a combined method of NMR, theoretical calculations and solvation theory. The solvent dependence of the 3JH1,H2 NMR coupling constants together with theoretical calculations allow the direct determination of the conformational equilibria without recourse to model compounds. The coupling constants were obtained with the aid of spectrum simulation, since these symmetric molecules present complex coupling systems. The observed couplings, when analysed by solvation theory and utilising DFT geometries (B3LYP/6-311+G**), gave energy values of E(ee) -E(aa) of 0.10, 0.95 and 1.40 kcalmol(-1) in the vapour phase for 1, 2 and 3, respectively, decreasing to -0.63, 0.36 and 0.93 kcalmol(-1) in CCl4 and to -1.91, -0.80 and -0.05 kcalmol(-1) in DMSO solution. The diaxial preference for all compounds is explained by natural bond orbital (NBO) analysis, which shows important hyperconjugative effects in this conformation. The "gauche effect" for compounds with more electronegative substituents, which are in gauche arrangement in the ee conformation, also plays a relevant role in more polar solvents.

Cyclohexanes↗