Theoretical conformational analysis and synthesis of analogues of the heptapeptide antibiotic K-582 A.

A detailed theoretical conformational analysis of the linear heptapeptide antibiotic [Arg2]K-582 A (Arg-Arg-D-Orn-Thr-D-Orn-Lys-D-Tyr) was carried out. The results of the computer simulation suggest that the linear peptide has a high propensity to fold in solution into a quasi-cyclic conformation in equilibrium with pi(L-D) helices. The synthesis of two inactive analogues with an L-Lys in place of D-Orn3 or D-Orn5 confirms the importance of the proposed folding pattern for the occurrence of the antimicrobial activity of K-582 A.

Evolutionary relationships among bacterial carbamoyltransferases.

An immunological approach was used for the study of ornithine carbamoyltransferase (OTCase) evolution in bacteria. Antisera were prepared against the anabolic and catabolic OTCases of Pseudomonas aeruginosa and Aeromonas formicans as well as against OTCase and putrescine carbamoyltransferases from Streptococcus faecalis; these antisera were then tested against the unpurified OTCases, either anabolic or catabolic, of 34 bacterial strains. Extensive cross-reactions were observed between the antisera to catabolic OTCases from P. aeruginosa, A. formicans and S. faecalis and the catabolic enzymes from other species or genera. These antisera cross-reacted also with the anabolic OTCases of strains of the Enterobacteriaceae but not with the anabolic OTCases of the same species or of other species or genera. The cross-reaction measured between the antisera against P. aeruginosa anabolic OTCase and the anabolic OTCases of other Pseudomonas were largely in agreement with the phylogenic subdivision of Pseudomonas proposed by N. J. Palleroni. The correlation was also significantly higher with the anabolic enzyme of an archaeobacterium, Methanobacterium thermoaceticum, than with the catabolic or anabolic OTCases from other genera in the eubacterial line. The antiserum raised against A. formicans anabolic OTCase was quite specific for its antigen and appeared to be raised against the heaviest of the various oligomeric structures of the enzyme.

Inhibition of steroid-protein interactions by dicyclohexane derivatives.

Sixteen dicyclohexane derivatives including the parent compound d,1-3,4-bis (4-oxocyclohexyl)-hexane (PRDX) have been synthesized and studied for putative interference with androgen binding to transport proteins, metabolizing enzymes, and receptors from rat tissues. Several of these analogues inhibited competitively the binding of dihydrotestosterone to ABP, the epididymal androgen transport protein. One compound had an affinity for ABP as high as Kd = 70 nM. Some dicyclohexanes also inhibited the aromatase enzyme which catalyses conversion of androgens into estrogens, as well as the NADPH-dependent, particulate form of 3 alpha(beta)-hydroxysteroid dehydrogenase, the enzyme that converts dihydrotestosterone into 5 alpha-androstanediol. For both enzymes the inhibition potency Ki of PRDX was about equal to the Km of the substrate. All of these interactions were specific in that they were modulated by single substitutions on the dicyclohexane molecule and they did not occur with other steroid binding proteins such as 5 alpha-reductase and the intracellular androgen receptor. A conformational study showed that dicyclohexanes can assume a 'steroidoid' conformation that differs from the crystal structure and which could account for the specific interactions with the steroid binding sites described here.

Proposed folding pattern for apolipoprotein A-II based on a structural analogy with uteroglobin.

The tertiary structure observed in the crystalline state for uteroglobin, a small steroid binding protein, is used as a template to build an approximated model for apolipoprotein A-II. The presence of four proline residues and four hydrophobic clusters located at similar positions in apolipoprotein A-II and uteroglobin is taken as the major source of stability in such tertiary structures. A brief description of plausible specific binding sites appearing on the model of apolipoprotein A-II is given. It is suggested that the internal cavity and the four surface pockets observed for uteroglobin and postulated for apolipoprotein A-II might be used to insure specific binding of triglycerides, phospholipids, or cholesterol.

Modification of human hemoglobin by glutathione. III. Perturbations of hemoglobin conformation analyzed by computer modeling.

The perturbations of the conformation of human deoxyhemoglobin induced by the covalent attachment of glutathione at cysteine beta 93 have been investigated by computer simulation in conjunction with molecular graphics. In the first phase of the analysis, a systematic search was carried out of the conformational space of glutathione attached to deoxyhemoglobin. In this search, the conformation of the hemoglobin molecule was held constant, while the relative energies of a series of 186,624 glutathione conformations involving systematic variation of six dihedral angels were calculated. From this search, the most favorable conformation was selected as the starting conformation for energy minimization of the glutathionyl hemoglobin molecule as a function of all Cartesian coordinates. In order to provide a reference state, an independent minimization by the same procedures was carried out for deoxyhemoglobin in the absence of glutathione. Comparison of the minimized structures with and without glutathione attached revealed a number of significant differences. The most conspicuous difference in the protein moiety concerned the salt bridge between aspartate beta 94 and histidine beta 146 which is destabilized upon minimization of the glutathionyl-hemoglobin complex due to interactions of the aspartate residue with the glycyl NH group of glutathione. Other observed differences in the minimized structures are located at the alpha 1-beta 2 interface and include displacement of the carboxyl group of aspartate beta 99. In the minimized complex, the glutathione portion assumes a quasi-cyclic conformation stabilized through interactions between the free (gamma-glutamyl) amino and (glycyl) carboxyl ends of the tripeptide and between this carboxyl end and the epsilon amino group of lysine alpha 40. In a parallel conformational study of glutathione alone, a similar structure was found as the lowest energy form. These quasi-cyclic conformations contrast with the extended structures reported by Wright (Wright, W.B. (1955) Acta Crystallogr. 11, 632-642) for crystals of glutathione where interactions between molecules play a major role. The conclusions of our analysis are in agreement with the experimental investigations reported in the two preceding papers and permit, moreover, a coherent interpretation of the observed functional and structural changes in deoxyhemoglobin induced by glutathione.

Production and characterization of a rat monoclonal antibody against leu5 enkephalin.

A rat X mouse hybridoma line producing a monoclonal antibody against leu5 enkephalin has been obtained. The monoclonal antibody belongs to the IgG2b class and has an affinity constant of 8.0 X 10(8) M-1 at 4 degrees C. This antibody exhibits approximately 40% cross-reactivity with 1-6 dynorphin but very weak cross-reactivities with met5 enkephalin (1.4%), 1-13 dynorphin (1.3%) and beta-endorphin (0.0045%). The detailed study, by competitive assay, of the interaction between this antibody and various enkephalin derivatives shows that the carboxy-terminal part of the molecule and, particularly, the leu side chain constitutes the immunodominant group. Nevertheless, the tyrosyl residue also contribute considerably to the binding, probably via a peculiar conformation effect, although we can not exclude the tyrosyl residue acting as a secondary contact residue. This monoclonal antibody has been used in a radioimmunoassay to determine leu5 enkephalin like immunoreactive material present in rat brain and hypothalamus.

Conformational properties of the pentapeptide fragment [32-36] of the thymic hormone thymopoietin.

The conformational energy for the pentapeptide Arg-Lys-Asp-Val-Tyr (TP5) is calculated using empirical potential functions. Calculation of the local interactions for each independent residue gives a local energy term for which the probabilities as a function of phi, psi are plotted on Ramachandran-type maps. The interaction energy between residues is calculated only for these points in the maps with maximum probability. The most probable conformation for TP5 is found to have an extended backbone arrangement having the Arg and Tyr sidechains folded over the backbone. 13C n.m.r. spin lattice relaxation time measurements show no increase in T1 of the alpha-carbons at the first and terminal amino acids. The increase in T1 along the sidechain as found for Lys does not occur for Arg and Tyr. These signs of reduced mobility are consistent with a set of folded conformations in which the Arg and Tyr sidechains have hindered internal rotations. The vicinal NH-C alpha H couplings agree well with those calculated for the most probable conformer. This is not so for the C alpha H-C beta H couplings. These data are consistent with previous n.m.r. and structure activity studies.

Tertiary structure of H-Pro-Leu-Gly-NH2, the factor that inhibits release of melanocyte stimulating hormone, derived by conformational energy calculations.

Conformational energy calculations were carried out on H-Pro-Leu-Gly-NH(2), the factor that inhibits the release of melanocyte stimulating hormone, and its biologically active analog, H-Pro-Ala-Gly-NH(2). Both peptides were found to be relatively compact molecules that retain, however, some degree of flexibility. After structure refinement, H-Pro-Leu-Gly-NH(2) possesses at least three preferred compact conformations. Two of these conformations occupy rather broad and flat energy troughs, while a third occupies a narrow and deep potential energy well. This third structure, which consists of a 10-membered beta-turn closed by a (4 --> 1) hydrogen bond between the proton of the trans carboxamide of Gly and the C=O of Pro, is the one that was proposed for H-Pro-Leu-Gly-NH(2) in dimethylsulfoxide and was also found by x-ray analysis.