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PubMed1990

Enkephalin analogues containing beta-naphthylalanine at the fourth position.

To examine the importance of the aromatic side chains of enkephalin on opiate activity, we report the synthesis and conformational analysis of a series of analogues related to enkephalin with beta-naphthylalanine in place of phenylalanine at the fourth position. Three linear analogues (Tyr-D-Ala-Gly-(L and D)-beta Nal(1)-Leu-NH2 and Tyr-D-Ala-Gly-beta Nal(2)-Leu-NH2) were initially synthesized to examine the effect of the substitution on biological activity. The increased activity of these peptides at the mu-opiate receptor, compared to native Leu-enkephalin, prompted us to examine the more conformational constrained analogues, Tyr-c[D-A2bu-Gly-(L and D)-beta Nal(1)-Leu], incorporating a alpha, gamma-diaminobutyric acid at the second position and cyclization to the carboxylic end of the leucine. These two cyclic analogues provide insight into the necessity for the L chirality of the aromatic residue at position 4. The Tyr-c[D-A2bu-Gly-L-beta Nal(1)-Leu] analogue is highly potent and displays a slight preference for the mu receptor. The conformational analysis indicates that despite the high flexibility of the tyrosine side chain, the aromatic rings of the tyrosine and naphthylalanine are relatively distant from each other. The presence of two intramolecular hydrogen bonds help maintain the conformation of the 14-membered backbone ring that keeps the side chains directed away from each other. These findings are in agreement with our model of an extended structure required for mu selectivity and a folded form with close aromatic ring placement for delta selectivity.

Alanine↗
PubMed1984

Interactions of aminoglycoside antibiotics with negatively charged lipid layers. Biochemical and conformational studies.

Previous studies [Laurent et al., Biochem. Pharmac. 31, 3861 (1982)] have demonstrated that aminoglycoside antibiotics bind to negatively charged phospholipid bilayers and inhibit the activity of lysosomal phospholipases. This inhibition also occurs in vivo in animal and man. It is considered to be an early and significant step in the development of aminoglycoside-induced nephrotoxicity. The binding of 6 aminoglycosides in current clinical use (dibekacin, gentamicin, tobramycin, kanamycin A, amikacin and streptomycin) to phosphatidylinositol has been studied by gel filtration technique and by conformational analysis. Variation of the phosphatidylinositol content from 0 to 27% of total phospholipids causes a cooperative increase in aminoglycoside binding. At fixed phosphatidylinositol concentration, the binding of the different aminoglycosides is related to the number of aminogroups carried by the drug (viz., gentamicin greater than kanamycin A greater than streptomycin) and is largely, but not entirely dependent upon electrostatic interactions. Conformational analysis of the interaction of aminoglycosides with phosphatidylinositol monolayers was made by a step-wise computation approach. We first have taken into account the Vander Waals, torsional and electrostatic energies and we have calculated the hydrophobic and hydrophilic centers of each molecule. Assembly was then computed by successive association of one molecule of drug and up to 4 molecules of phosphatidylinositol. The calculated interaction energies varied from -8.5 kcal/mol (gentamicin) to -4.9 kcal/mol (amikacin) and -3.9 kcal/mol (streptomycin).(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoglycosides↗
PubMed1999

Fourier transform infrared linked analysis of conformational changes in annexin V upon membrane binding.

Annexins are ubiquitous cellular proteins of unknown primary function that bind to anionic phospholipid membranes in a calcium-dependent manner. Correlative studies involving X-ray crystallography and electron microscopy suggest that annexins undergo a structural change upon binding to supported lipid monolayer membranes. In this investigation, novel spectroscopic and analytical techniques have been applied to verify and characterize this change. Soluble annexin V was examined with ordinary transmission infrared spectroscopy, while membrane-bound annexin V was examined with both transmission and internal reflection infrared spectroscopy. Spectra were processed by linked analysis, whereby multiple spectra are fit simultaneously with component bands that are constrained to share common fitting parameters. This approach is shown to enhance the sensitivity and accuracy of the bandfitting procedure. Our results are consistent with the general mode of membrane binding inferred from electron microscopy studies, and they provide independent support for the conclusion that annexin V undergoes a conformational change upon binding to lipid monolayer membranes. Most likely, this change involves the formation of new beta structure in which interstrand hydrogen bonds orient parallel to the membrane surface.

Amides↗
PubMed1985

Interaction of streptomycin and streptomycylamine derivatives with negatively charged lipid layers. Correlation between binding, conformation of complexes and inhibition of lysosomal phospholipase activities.

Aminoglycoside antibiotics induce a lysosomal phospholipidosis in kidney proximal tubules after conventional therapy in animals and man. We have previously demonstrated that these drugs bind to negatively charged phospholipid bilayers at acid pH and inhibit the activity of lysosomal acid phospholipases in vitro and in vivo. A combined biochemical and conformational study [Brasseur et al., Biochem. Pharmac. 33, 629 (1984)] showed major and consistent differences between 6 aminoglycosides in current clinical use with respect to the stability of the complexes they form with phosphatidylinositol, their inhibitory potency towards the activity of lysosomal phospholipases and their current toxicity ranking (e.g. gentamicin greater than amikacin greater than streptomycin). In the present study we have extended this approach to experimental derivatives of streptomycin. The derivatives examined were: dihydrostreptomycin, dideguanyldihydrostreptomycin, streptomycylamine, dideguanylstreptomycylamine, N-butyl- and N-benzyl-dideguanylstreptomycylamine. These compounds were examined for (i) their binding to negatively charged liposomes, measured by gel permeation on Sepharose 4B; (ii) their interactions with phosphatidylinositol assessed by semi-empirical conformational analysis and (iii) their inhibitory effect on the activities of lysosomal phospholipases towards phosphatidylcholine present in negatively charged liposomes. Streptomycin and gentamicin were also used as reference compounds with low and high affinity (and inhibitory potency), respectively. Our observations can be summarized as follows: (i) the replacement of the aldehyde in the streptose ring by a methylamino group strikingly changes the conformation of the molecule, allowing a better interaction with phosphatidylinositol. Thus, streptomycylamine binds much more tightly to phospholipid bilayers and shows a higher inhibitory potency towards phospholipase activity, as compared to streptomycin. The conformational analysis shows, however, that this effect is only partially due to the additional cationic charge carried by streptomycylamine. Other modifications of the streptomycin molecule, such as the replacement of the guanidinium groups by aminogroups or the addition of hydrophobic moieties (butyl or benzyl groups) to the streptose do not markedly further strengthen the interactions of the molecule with phosphatidylinositol. (ii) Even though some derivatives (e.g. dideguanylstreptomycylamine) bind as tightly to phospholipids as gentamicin, they remain much less inhibitory towards lysosomal phospholipases.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoglycosides↗
PubMed1988

Analysis of conformational parameters in nucleic acid fragments. II. Co-crystal complexes of nucleic acid bases.

Studies have been made of conformational parameters in co-crystal complexes and compounds of nucleic acid bases in which there is the possibility of formation of hetero-base-pairs. Using published data extracted from the Cambridge structural database, a total of 37 base-pairs were found, of which 25 were hetero-pairs and 12 homo-pairs. These base-pairs were subject to analysis to reveal hydrogen bond parameters, propeller twist, buckle and C1'-C1' separation (or a similar parameter if C1' atoms were not present). Hetero-pairs were found to show larger twists than homo-pairs, the magnitude of twist being unrelated to hydrogen bond parameters or buckle value. The propeller twisting is less pronounced in these nucleic acid bases than in nucleosides, but still has a significant magnitude. Propeller twisting in hetero-pairs is found to be larger than in homo-pairs. Hetero-pairs appear to be formed preferentially in competitive situations.

Crystallization↗
PubMed1979

The conformations of the Lewis blood group determinants, sucrose and kanamycin A.

The main purpose is to illustrate the potential, in terms of structure and conformational analysis in the general area of carbohydrate chemistry, presented by the new generation of high frequency FT nuclear magnetic resonance spectrometers based in the highly stable magnetic fields generated by superconducting solenoids. In the case of the LEWIS human blood group antigenic determinants, specific interunit deshielding effects on 1H-chemical shifts and specific interunit nuclear OVERHAUSER enhancements of 1H-signals allowed specific assignments of overall conformations for these branched oligosaccharides. Furthermore, these conformations are shown to be well anticipated by way of simple hard-sphere calculations but taking into consideration important contributions by the exo-anomeric effect in the assignment of conformational preferences at the glycosidic bonds. The presence of an intramolecular hydrogen bond between OH-2g and OH-1f for sucrose dissolved in dimethylsulfoxide-d6(DMSO-d6) is demonstrated by examining the spectrum of a solution which has near 50% of the OH groups in the OD form. The 1H-signals in the structural unit Clf-O-H comes from O(C2g)-H comes from DMSO-d6 are shown to be deuterium shifted by 0.005-0.007 ppm when in the forms Clf-O-H comes from O(C2g)-D comes from DMSO-d6 (deshielded) and Clf-O-D comes from O(C2g)-H comes from DMSO-d6 (shielded). These small shifts were readily discerned at 400 MHz. The (1H)(400 MHz) and 13C (100 MHz) spectra of kanamycin A both as the free amine and the sulfate salt are presented to illustrate how modern nuclear magnetic resonance spectroscopy together with interpretations based in conformational analysis may allow the elucidation of the structures (except for the absolute configuration) of such substances. It is demonstrated that the effect on 13C chemical shifts of the protonation of the amino groups can provide useful clues as to their relative dispositions in three-dimensional space.

Chemical Phenomena↗
PubMed1983

[Conformation NMR analysis of the spatial structure of Buthus eupeus insectotoxin I5A].

1H NMR spectroscopy has been used to collect data related to the spatial structure of insectotoxin I5A Buthus eupeus: pH-dependence of the chemical shifts, deuterium exchange rates of individual amide hydrogens, spin-spin coupling of the H-N-C alpha-H and H-C alpha-C beta-H protons, and nuclear Overhauser effect between distinct protons belonging to amino acid residues remote in the sequence. Molecular conformation in the regions from Asp9 to Cys19 (beta-turn 9-12 and right-hand alpha-helix 12-19) and from Asn23 to Asn34 (antiparallel beta-sheet with the beta-turn 27-30) directly follows from the observed parameters. Pseudoatomic approach of distance geometry algorithm was used to solve the overall folding of the molecule and propose the most probable set of disulfide bridges: Cys2-Cys19, Cys5-Cys31, Cys16-Cys26 and Cys20-Cys33. The spatial structure of insectotoxin I5A B. eupeus demonstrates remarkable similarity with that of a "long" type scorpion neurotoxin V-3 Centruroides sculpturatus.

Amino Acid Sequence↗
PubMed2000

Lisch corneal dystrophy is genetically distinct from Meesmann corneal dystrophy and maps to xp22.3.

PURPOSE: There is an ongoing discussion whether Lisch corneal dystrophy (band-shaped and whorled microcystic dystrophy of the corneal epithelium) represents a disorder that is different from Meesmann corneal dystrophy. The purpose of this study was to evaluate at the molecular level if Lisch and Meesmann corneal dystrophies are genetically distinct. METHODS: We examined at the slit lamp a total of 48 members of a family with an aggregation of Lisch corneal dystrophy. Genomic DNA was extracted from leukocytes of the peripheral blood of seven affected and six unaffected members of this family. Mutational hotspots in the cornea-specific keratin genes K3 and K12 were scanned for mutations by single-strand conformation analysis. To test for linkage to the keratin K3 or K12 loci or for X-chromosomal inheritance, six (K3) and four (K12) microsatellite markers each flanking the keratin loci as well as 22 microsatellite markers covering the X-chromosome were typed. Linkage was analyzed using the MLINK and FASTMAP procedures. RESULTS: A total of 19 trait carriers were identified in six generations of the family. No hereditary transmission from father to son was observed. Linkage was excluded for the keratin K3 and K12 genes. Furthermore, single-strand conformation analysis detected no mutations in these genes. Multipoint linkage analysis revealed linkage with a maximum likelihood of the odds (LOD) score of 2.93 at Xp22.3. Linkage was excluded for Xp22.2 to Xqter. CONCLUSIONS: Lisch corneal dystrophy is genetically different from Meesmann corneal dystrophy. Evidence was found for linkage of the gene for Lisch corneal dystrophy to Xp22.3.

Adolescent↗
PubMed1999

Effect of conformation on the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH to its cyclic imide degradation product.

The objective of this study was to explain the increased propensity for the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH (1), a vitronectin-selective inhibitor, to its cyclic imide counterpart cyclo-(1,7)-Gly-Arg-Gly-Asu-Ser-Pro-Asp-Gly-OH (2). Therefore, we present the conformational analysis of peptides 1 and 2 by NMR and molecular dynamic simulations (MD). Several different NMR experiments, including COSY, COSY-Relay, HOHAHA, NOESY, ROESY, DQF-COSY and HMQC, were used to: (a) identify each proton in the peptides; (b) determine the sequential assignments; (c) determine the cis-trans isomerization of X-Pro peptide bond; and (d) measure the NH-HCalpha coupling constants. NOE- or ROE-constraints were used in the MD simulations and energy minimizations to determine the preferred conformations of cyclic peptides 1 and 2. Both cyclic peptides 1 and 2 have a stable solution conformation; MD simulations suggest that cyclic peptide 1 has a distorted type I beta-turn at Arg2-Gly3-Asp4-Ser5 and cyclic peptide 2 has a pseudo-type I beta-turn at Ser5-Pro6-Asp7-Gly1. A shift in position of the type I beta-turn at Arg2-Gly3-Asp4-Ser5 in peptide 1 to Ser5-Pro6-Asp7-Gly1 in peptide 2 occurs upon formation of the cyclic imide at the Asp4 residue. Although the secondary structure of cyclic peptide 1 is not conducive to succinimide formation, the reaction proceeds via neighbouring group catalysis by the Ser5 side chain. This mechanism is also supported by the intramolecular hydrogen bond network between the hydroxyl side chain and the backbone nitrogen of Ser5. Based on these results, the stability of Asp-containing peptides cannot be predicted by conformational analysis alone; the influence of anchimeric assistance by surrounding residues must also be considered.

Chromatography, High Pressure Liquid↗
PubMed1996

Low incidence of androgen receptor gene mutations in human prostatic tumors using single strand conformation polymorphism analysis.

It is possible that structural changes of the androgen receptor (AR) contribute to the insensitivity of prostatic carcinomas to endocrine therapy. We have isolated DNA from 58 human prostate tumor specimens (31 carcinomas pretreatment, 13 carcinomas after relapse to hormonal therapy, and 14 benign prostatic hyperplasia), three established human prostate carcinoma cell lines and two transplantable human prostatic carcinoma xenografts. Twelve pairs of oligonucleotide primers were used to amplify the majority of the coding region of the AR gene and the products screened for mutations using single-strand conformation polymorphism (SSCP) techniques. In one tumor sample a cytosine to guanine transition in exon F which leads to substitution of glutamic acid for the wild type glutamine at position 798 of the ligand binding domain was detected. The same mutation was also found in the patient's genomic DNA and as been described in a patient with partial androgen insensitivity syndrome. Intronic mutations were detected in two of the benign prostatic hyperplasia samples, and a silent mutation at nucleotide 995 was found to be present in eight poorly differentiated carcinomas, one BPH specimen, as well as in the cell line DU145 (18% of the samples studied). In agreement with most of the literature, these studies indicate that AR mutations are rare both prior to therapy and even in androgen relapsed tumors.

Humans↗
PubMed2005

Polymerase chain reaction-single strand conformational polymorphism analysis of rearranged during transfection proto-oncogene in Chinese familial Hirschsprung's disease.

AIM: To investigate the relationship between mutations of rearranged during transfection (RET) proto-oncogene and Chinese patients with Hirschsprung's disease (HD), and to elucidate the genetic mechanism of familial HD patient at the molecular level. METHODS: Genomic DNA was extracted from venous blood of probands and their relatives in two genealogies. Polymerase chain reaction (PCR) products, which were amplified using specific primers (RET, exons 11, 13, 15 and 17), were electrophoresed to analyze the single-strand conformational polymorphism (SSCP) patterns. The positive amplified products were sequenced. Forty-eight sporadic HD patients and 30 normal children were screened for mutations of RET proto-oncogene simultaneously. RESULTS: Three cases with HD in one family were found to have a G heterozygous insertion at nucleotide 18,974 in exon 13 of RET cDNA (18,974insG), which resulted in a frameshift mutation. In another family, a heterozygosity for T to G transition at nucleotide 18,888 in the same exon which resulted in a synonymous mutation of Leu at codon 745 was detected in the proband and his father. Eight RET mutations were confirmed in 48 sporadic HD patients. CONCLUSION: Mutations of RET proto-oncogene may play an important role in the pathogenesis of Chinese patients with HD. Detection of mutated RET proto-oncogene carriers may be used for genetic counseling of potential risk for HD in the affected families.

Base Sequence↗
PubMed2003

Structural and rheological characterization of Scleroglucan/borax hydrogel for drug delivery.

The polysaccharide Scleroglucan, one of the most rigid polymers found in nature, can form a chemical/physical gel, in the presence of borax. The obtained hydrogel was loaded with three different model molecules (Theophylline, Vitamin B12 and Myoglobin) and then, after freeze-drying, was used as a matrix for tablets. The release profiles of the substances from the dosage forms were evaluated; the matrix appeared capable to modulate the diffusion of the chosen molecules, and different diffusion rates were observed, according to the different radii of the tested molecules. Interestingly, in the dissolution medium the matrix undergoes an anisotropic swelling taking place only in the axial direction, while a negligible radial variation occurs. The water uptake of the matrix occurs according to a Fickian process. Samples at two different polymer concentrations (0.7 and 2.3%, w/v) were characterized in terms of rheological and mechanical parameters and the properties were interpreted in terms of the molecular structure obtained by conformational analysis. The flow curves acquired in the viscoelasticity interval, show the effect of the borate ion in improving the resistance of the gel in comparison to the polymer alone. The evaluation of the moduli indicates that the system is viscoelastic, with an appreciable liquid component that increases as the polymer concentration decreases. Also the cohesion of the gel is higher in comparison to the Scleroglucan and is strongly dependent on temperature. The combination of experimental and theoretical conformational analysis approaches, allowed us to propose a model for the structure of the macromolecular network and to give an explanation to the anomalous swelling that was observed. It came out that the polymer can built up a channel structure, mediated via borax ion interaction, that can accommodate guest molecules of different size.

Borates↗
PubMed2003

Detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis strains by single-strand conformation polymorphism analysis and restriction fragment length polymorphism.

Anti-Mycobacterium tuberculosis drug-resistance, mainly multi-drug resistance (MDR-TB), represents an important public health problem in several countries. Aim of our study is to identify the presence of these mutations in M. tuberculosis isoniazid- and rifampin-resistant strains isolated in our Institute; to evaluate linkage between type of mutation and level of resistance; to determine the usefulness of easy molecular techniques for rapid detection of such mutations on body specimens. Isoniazid- and rifampin-resistance was tested on 67 M. tuberculosis strains by Single-Strand Conformation Polymorphism (SSCP) and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assays, using HaeIII, PstuI, BsteII, BstuI enzymes. Drug-resistance of control strains was determined by cultural techniques (fluorimetry- BACTEC 9120). Cultural assay showed isoniazid- and rifampin-resistance in 6.12 and 2%, respectively (data confirmed by SSCP assay). Mutation of katG, linked to isoniazid resistance, was detected using BstuI enzyme, and mutation of rpoB, expression of reduced sensitivity to rifampin, using HaeIII. 15 body specimens, M. tuberculosis-positive to conventional assays, were tested by SSCP technique. Epidemiologic reports of numerous cases of tuberculosis due to MDR strains induce to detect quickly both Mycobacteria and drug-resistance, in order to start prompt effective therapy. On this basis, molecular assays are useful for a rapid therapeutic decision.

Antitubercular Agents↗
PubMed1986

Plastocyanin conformation. An analysis of its near ultraviolet absorption and circular dichroic spectra.

The near-ultraviolet absorption and circular dichroic spectra of plastocyanin are dependent upon the redox state, solution pH, and ammonium sulfate concentration. This dependency was observed in plastocyanin isolated from spinach, poplar, and lettuce. Removal of the copper atom also perturbed the near-ultraviolet spectra. Upon reduction there are increases in both extinction and ellipticity at 252 nm. Further increases at 252 nm were observed upon formation of apo plastocyanin eliminating charge transfer transitions as the cause. The spectral changes in the near-ultraviolet imply a flexible tertiary conformation for plastocyanin. There are at least two charge transfer transitions at approximately 295-340 nm. One of these transitions is sensitive to low pH's and is attributed to the His 87 copper ligand. The redox state dependent changes observed in the near-ultraviolet spectra of plastocyanin are attenuated either by decreasing the pH to 5 or by increasing the ammonium sulfate concentration to 2.7 M. This attenuation cannot be easily explained by simple charge screening. Hydrophobic interactions probably play an important role in this phenomenon. The pH and redox state dependent conformational changes may play an important role in regulating electron transport.

Circular Dichroism↗
PubMed2006

Synthesis and biological evaluation of bicyclic nucleosides as inhibitors of M. tuberculosis thymidylate kinase.

Herein we describe the synthesis and conformational analysis of a series of bicyclic thymidine derivatives and their evaluation as inhibitors of thymidine monophosphate kinase from Mycobacterium tuberculosis (TMPKmt), based on previously discovered bicyclic sugar nucleosides. With a K(i) value of 2.3 microm, 1-[3-aminomethyl-3,5-dideoxy-2-O,6-N-(thiocarbonyl)-beta-D-ribofuranosyl]thymine emerged as the most potent TMPK inhibitor of this series. Moreover, this promising compound displays inhibitory potency against Mycobacteria cultures with an IC(99) value of 100 microg mL(-1), thus promoting TMPKmt for the first time as a validated target for further inhibitory design. Attempts to rationalise the observed structure-activity relationship (SAR) involving molecular modelling and conformational analysis are described.

Binding Sites↗
PubMed1991

Conformational energy analysis of the substitution of Val for Gly 233 in a functional region of platelet GPIb alpha in platelet-type von Willebrand disease.

Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder in which patient platelets exhibit an abnormally increased binding of circulating von Willebrand factor (vWF). We have recently shown that this abnormality is associated with a point mutation resulting in substitution of Val for Gly 233 in platelet membrane glycoprotein Ib alpha (GPIb alpha), a major component of the platelet GPIb/IX receptor for vWF. To investigate the effect of this substitution on the three-dimensional structure of this region of the protein, we have generated the allowed (low energy) conformations of the region of the GPI alpha protein containing residues 228-238 (with 5 residues on either side of the critical residue 233) with Gly 233 (wild type) and Val 233 (PT-vWD) using the computer program ECEPP (Empirical Conformational Energies of Peptides Program). The wild-type sequence is Tyr-Val-Trp-Lys-Gln-Gly-Val-Asp-Val-Lys-Ala. We find that the Gly 233-containing peptide can exist in two low energy conformers. The lowest energy conformer is a structure containing a beta-turn at Gln 232-Gly 233 while the alternative conformation is an amphipathic helical structure. Only the amphipathic helical structure is allowed for the Val 233-containing peptide which contains a hydrophobic 'face' consisting of Val 229, Val 233 and Val 236 and another hydrophilic surface composed of such residues as Lys 231 and Asp 235. No such surfaces exist for the lowest energy bend conformer for the Gly 233-containing peptide, but do exist in the higher energy helical structure. The amphipathic surfaces in the 228-238 region of the Val 233-containing GPIb alpha protein may associate strongly with complementary surfaces during vWF binding to the GPIb/IX receptor complex and may help explain heightened association of vWF with this receptor in PT-vWD.

Amino Acid Sequence↗