Approach to the study of natural peptides from structure to functions. III. Structural organization of an adrenorphin molecule and its synthetic analogs

Spatial structure of the peptide hormone adrenorphin was investigated by the theoretical conformational method. A solution of the "reverse conformational problem" for adrenorphin made it possible to predict a series of the modified synthetic analogues, which may assume one of the low-energy conformations of the native hormone.     

Characterization of the conformational domains of bradykinin by computational methods

The AMBER 4.0 force field was used to perform a characterization of the conformational profile of the nonapeptide bradykinin. A thorough conformational search was carried out using molecular dynamics as sampling technique, by computing cycles of high (900 K) and low (300 K) temperature trajectories. A total of 2400 minima were generated and subsequently clustered using the root-mean-square of the backbone dihedral angles as criterium. After the use of a tolerance value of 20 degrees, the conformations were clustered in 233 unique conformations with energies up to 40 kcal/mol above the lowest minimum. The analysis of the low-energy conformations indicate that the peptide exhibits a high tendency to adopt a beta-turn at the C-terminus and a propensity to adopt a bent structure at the N-terminus. These results are in agreement with the experimental evidence reported in the literature and provide detailed information necessary to understand the conformational preferences of the peptide.     

Spontaneous transients of [Ca2+]i depend on external calcium and the activation of L-type voltage-gated calcium channels in a clonal pituitary cell line (AtT-20) of cultured mouse corticotropes

Based upon the message-address concept, this molecular modeling study used the delta-selective agonist spiroindanyloxymorphone (SIOM) as a molecular template for a conformational search and analysis of delta-selective opioid peptides. It was assumed that the tyramine moiety plays the same role for delta-opioid receptor recognition in both peptide and non-peptide ligands. Using 20 reported low-energy conformations of Tyr-cyclo[D-Cys-D-Pen]-OH (JOM-13) for comparison, the geometrical relationship of the two aromatic rings present in SIOM was used for the identification of potential active conformations of JOM-13, from which two delta-receptor-binding models (I and II) were constructed. Models I and II differ from each other in the arrangement of the peptide backbones. To evaluate the two models, a conformational search of two other known delta-selective ligands, [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Pen2,L-Pen5]enkephalin (DPLPE) was performed, using the geometrical relationship of the two aromatic rings defined in the two receptor-binding models as a molecular template. Among the conformations generated from the molecular simulation, low-energy conformers of DPDPE and DPLPE conforming to models I and II were identified. Unlike model I, conformers of DPDPE and DPLPE that fit model II contain a cis amide bond in the Gly3 residue.     

Conformational analysis and hemolytic activity of immunoglobulin G fragment 285-292 and its analogs

Theoretical conformational analysis was carried out for the 285-292 fragment of human immunoglobulin G (His-Asn-Ala-Lys-Thr-Lys-Pro-Arg) and its analogues containing Arg, Glu, Gly, Lys, or Trp residue instead of the His residue in position 1. Spectropolarimetic investigation of these peptides showed the analogues to have different activities in the C1q-mediated erythrocytes hemolysis assay. Comparison of the low-energy structures sets of the compounds tested allowed to suggest a model of the "biological active" conformation for the peptide molecule in the course of the C1q complement component binding.     

2D 1H-NMR conformational study of phosphatidylserine diluted in perdeuterated dodecylphosphocholine micelles. Evidence for a pH-induced conformational transition

The conformation of phosphatidylserine (DMPS) diluted in perdeuterated dodecylphosphocholine micelles (DPC) has been investigated by 1D and 2D proton NMR spectroscopy. Chemical shift pH dependence showed that the pK relative to the serine carboxyl titration (3.4 +/- 0.05) was nearly identical to that measured in bilayers. Chemical shift and NOE data revealed that the phosphatidylserine molecule undergoes a conformational transition upon titration of the serine carboxyl group. The NOE network observed between the different parts of the molecule was sufficiently abundant to allow, in combination with molecular modeling methods, an assessment of the conformational changes. The conformational changes mainly involve the glycerol backbone, which is parallel to the whole molecule, that is, to the layer normal, at low pH and becomes perpendicular to the whole molecule at neutral pH. In both cases, the conformations are remarkably close to those observed for the crystal forms of zwitterionic and negatively charged phospholipids. Two-dimensional proton NMR study of phospholipids, diluted in perdeuterated DPC micelles, appears to be a simple and relevant method to obtain complete and direct information on their conformations in a model membrane-solution interface.     

Characterization of the bioactive form and molecular determinants of recognition of cyclic enkephalin peptides at the delta-opioid receptor

An extensive and systematic search strategy to determine the conformational profile of 12 cyclic disulfide-bridged opioid peptides with varying affinities at the delta receptor has been carried out to identify the structure that is recognized by the delta receptor for each analogue. The methods and procedures used here for the conformational search have already been validated for [D-Pen2,D-Pen5] enkephalin (DPDPE), one member of this family. Use of these methods led to a low-energy solution conformation of DPDPE in excellent agreement with all the geometric properties deduced from its solution nmr spectra. Each of the analogue was subjected to the same procedure, involving a combination of molecular dynamics simulations at high and low temperature. The study was repeated in two environmental conditions, an apolar environment, simulated by using a distance-dependent dielectric constant, and a polar environment by embedding the peptides in a high constant dielectric (epsilon = 80). An automated comparison of the different conformers based on their backbone rms and average distance between the key aromatic moieties was followed by graphic analysis using maximum structural overlap. The cross-comparison of the conformations for each analogue revealed a unique conformer that may be recognized by the delta receptor for each high-affinity analogue that permitted maintaining the critical elements required for recognition in a simple spatial orientation, while maximizing similarity in other regions.     

Search for low-energy conformations of a neurotoxic protein by means of predictive rules, tests for hard-sphere overlaps, and energy minimization

A method to obtain models for the three-dimensional structure of the neurotoxin alpha from Naja nigricollis from its amino acid sequence is explored here. Empirical predictive rules were used to estimate the positions of helices, extended structures and bends; advantage was taken of the availability of 14 homologous sequences for the neurotoxins in an attempt to increase the reliability of these predictions. Unassigned residues were allowed to take up several possible conformations determined from the frequencies of occurrence of each type of conformation of that residue in x-ray structures of many proteins. The conformational space of the molecule was explored initially by testing for hard-sphere overlaps and approximate closure of disulfide loops with the aid of a computer; this procedure yielded a limited number of conformations, whose conformational energies were then determined and minimized by optimizing the backbone and side-chain dihedral angles of each residue. Five compact conformations with low energy were found for this neurotoxin. The procedure used here provides an illustration as to how empirical protein algorithms may be used to limit the conformational space, in which energy minimization has to be carried out.     

Conformational changes due to vicinal glycosylation: the branched alpha-L-Rhap(1-2)[beta-D-Galp(1-3)]-beta-D-Glc1-OMe trisaccharide compared with its parent disaccharides

Conformations of the alpha-L-Rhap(1-2)-beta-D-Glc1-OMe and beta-D-Galp(1-3)-beta-D-Glc1-OMe disaccharides and the branched title trisaccharide were examined in DMSO-d6 solution by 1H-nmr. The distance mapping procedure was based on rotating frame nuclear Overhauser effect (NOE) constraints involving C- and O-linked protons, and hydrogen-bond constraints manifested by the splitting of the OH nmr signals for partially deuteriated samples. An "isotopomer-selected NOE" method for the unequivocal identification of mutually hydrogen-bonded hydroxyl groups was suggested. The length of hydrogen bonds thus detected is considered the only one motionally nonaveraged nmr-derived constraint. Molecular mechanics and molecular dynamics methods were used to model the conformational properties of the studied oligosaccharides. Complex conformational search, relying on a regular phi, psi-grid based scanning of the conformational space of the selected glycosidic linkage, combined with simultaneous modeling of different allowed orientations of the pendant groups and the third, neighboring sugar residue, has been carried out. Energy minimizations were performed for each member of the phi, psi grid generated set of conformations. Conformational clustering has been done to group the minimized conformations into families with similar values of glycosidic torsion angles. Several stable syn and anti conformations were found for the 1-->2 and 1-->3 bonds in the studied disaccharides. Vicinal glycosylation affected strongly the occupancy of conformational states in both branches of the title trisaccharide. The preferred conformational family of the trisaccharide (with average phi, psi values of 38 degrees, 17 degrees for the 1-->2 and 48 degrees, 1 degree for the 1-->3 bond, respectively) was shown by nmr to be stabilized by intramolecular hydrogen bonding between the nonbonded Rha and Gal residues.     

Does conformational free energy distinguish loop conformations in proteins?

Limitations in protein homology modeling often arise from the inability to adequately model loops. In this paper we focus on the selection of loop conformations. We present a complete computational treatment that allows the screening of loop conformations to identify those that best fit a molecular model. The stability of a loop in a protein is evaluated via computations of conformational free energies in solution, i.e., the free energy difference between the reference structure and the modeled one. A thermodynamic cycle is used for calculation of the conformational free energy, in which the total free energy of the reference state (i.e., gas phase) is the CHARMm potential energy. The electrostatic contribution of the solvation free energy is obtained from solving the finite-difference Poisson-Boltzmann equation. The nonpolar contribution is based on a surface area-based expression. We applied this computational scheme to a simple but well-characterized system, the antibody hypervariable loop (complementarity-determining region, CDR). Instead of creating loop conformations, we generated a database of loops extracted from high-resolution crystal structures of proteins, which display geometrical similarities with antibody CDRs. We inserted loops from our database into a framework of an antibody; then we calculated the conformational free energies of each loop. Results show that we successfully identified loops with a "reference-like" CDR geometry, with the lowest conformational free energy in gas phase only. Surprisingly, the solvation energy term plays a confusing role, sometimes discriminating "reference-like" CDR geometry and many times allowing "non-reference-like" conformations to have the lowest conformational free energies (for short loops). Most "reference-like" loop conformations are separated from others by a gap in the gas phase conformational free energy scale. Naturally, loops from antibody molecules are found to be the best models for long CDRs (> or = 6 residues), mainly because of a better packing of backbone atoms into the framework of the antibody model.     

Molecular structures and conformational studies of triarylcyclopropyl and related nonsteroidal antiestrogens

Molecular structures and conformational characteristics of a series of 1,1-dichloro-2,2,3-triarylcyclopropanes (DTACs), which were reported previously to be distinctly antiestrogenic and inhibitors of the estrogen-receptor-positive MCF-7 human breast cancer cells in culture, are reported. In addition, structural and conformational features of the DTACs were compared to the first-known nonsteroidal antiestrogen, MER25, and the clinically useful antiestrogen Tamoxifen. The molecular structures of four DTAC compounds were determined by X-ray diffraction. Crystallographic structures show that the DTAC molecules have nearly the same relative conformation for the three aryl rings which is designated as a "nonpropeller" conformation in contrast to the observed "propeller" conformation for the three rings in all known triarylethylenes. Systematic conformational searches were performed to find the conformational preferences of DTACs, MER25, and Tamoxifen using idealized model compounds built from their respective crystal structure. Energy-minimization and conformational-search studies demonstrated that all DTAC molecules have a common, single global minimum energy conformer for their central core containing the dichlorotriarylcyclopropyl system, which is similar to that found in their crystal structures. Conformational search of MER25 showed that the molecule can assume a number of low-energy conformers of which two, one anti (A1) and one gauche (G1A), have about the same energy. The anti conformation is similar to the one observed in its crystal structure and resembles the estrogenic E-isomer of Tamoxifen, while the lowest energy gauche conformer of MER25 resembles more closely the antiestrogenic Z-isomer of Tamoxifen. NMR spectroscopic analysis of MER25 showed that the molecule exists predominantly in the anti conformation in solution. A comparative review of the structural features and bioactivities of Tamoxifen, DTACs, and MER25 provides a possible explanation for their low estrogen receptor binding affinity which is common to these compounds together with their antiestrogenic activity.     

Role of hydrophobicity and solvent-mediated charge-charge interactions in stabilizing alpha-helices

A theoretical study to identify the conformational preferences of lysine-based oligopeptides has been carried out. The solvation free energy and free energy of ionization of the oligopeptides have been calculated by using a fast multigrid boundary element method that considers the coupling between the conformation of the molecule and the ionization equilibria explicitly, at a given pH value. It has been found experimentally that isolated alanine and lysine residues have somewhat small intrinsic helix-forming tendencies; however, results from these simulations indicate that conformations containing right-handed alpha-helical turns are energetically favorable at low values of pH for lysine-based oligopeptides. Also, unusual patterns of interactions among lysine side chains with large hydrophobic contacts and close proximity (5-6 A) between charged NH3+ groups are observed. Similar arrangements of charged groups have been seen for lysine and arginine residues in experimentally determined structures of proteins available from the Protein Data Bank. The lowest-free-energy conformation of the sequence Ac-(LYS)6-NMe from these simulations showed large pKalpha shifts for some of the NH3+ groups of the lysine residues. Such large effects are not observed in the lowest-energy conformations of oligopeptide sequences with two, three, or four lysine residues. Calculations on the sequence Ac-LYS-(ALA)4-LYS-NMe also reveal low-energy alpha-helical conformations with interactions of one of the LYS side chains with the helix backbone in an arrangement quite similar to the one described recently by (Proc. Natl. Acad. Sci. U.S.A. 93:4025-4029). The results of this study provide a sound basis with which to discuss the nature of the interactions, such as hydrophobicity, charge-charge interaction, and solvent polarization effects, that stabilize right-handed alpha-helical conformations.     

Contact interactions method: a new algorithm for protein folding simulations

Computer simulations of simple exact lattice models are an aid in the study of protein folding process; they have sometimes resulted in predictions experimentally proved. The contact interactions (CI) method is here proposed as a new algorithm for the conformational search in the low-energy regions of protein chains modeled as copolymers of hydrophobic and polar monomers configured as self-avoiding walks on square or cubic lattices. It may be regarded as an extension of the standard Monte Carlo method improved by the concept of cooperativity deriving from nonlocal contact interactions. A major difference with respect to other algorithms is that criteria for the acceptance of new conformations generated during the simulations are not based on the energy of the entire molecule, but cooling factors associated with each residue define regions of the model protein with higher or lower mobility. Nine sequences of length ranging from 20 to 64 residues were used on the square lattice and 15 sequences of length ranging from 46 to 136 residues were used on the cubic lattice. The CI algorithm proved very efficient both in two and three dimensions, and allowed us to localize energy minima not localized by other searching algorithms described in the literature. Use of this algorithm is not limited to the conformational search, because it allows the exploration of thermodynamic and kinetic behavior of model protein chains.     

Structure-functional organization of the angiotensin II molecule. III. Polyfunctional nature

Conformational properties of the molecules of angiotensin II and some of its analogs are analysed with regard to the quantitative characteristics of their biological activities. Connections between low-energy conformations of angiotensin II and its biological functions are established. The hormone's spatial structures plausible in terms of interactions with the specific receptors located in smooth muscles and adrenal cortex tissues have been identified.     

The determinants of fat intake in a multi-ethnic New Zealand population. Fletcher Challenge--University of Auckland Heart and Health Study Management Committee

The problem of a variety of denatured forms of the protein molecule under equilibrium conditions is considered. The experimental conditions are described at which the protein molecule can exist in various non-native states. The history of the discovery of a universal intermediate molten globule state and the current status of research in this field are briefly outlined. Particular emphasis is placed on the fact that the molten globule state is a thermodynamic state of the protein molecule that is separated from both the native and the completely unfolded state by "all-or-none" transitions, i.e., intramolecular analogs of the 1st-order phase transitions. It is also shown that the molten globule state is not the only intermediate state observed for a particular protein under equilibrium conditions. The main structural features of the protein molecule in various denatured conformations are described. How many molten globule states there exist? A molten globule, a precursor of the molten globule, a highly structured molten globule: are these particular conformational states or different forms of the unique intermediate state? Or different forms of the native protein molecule with different degrees of disorder? Or differently structured forms of the unfolded polypeptide chain? This review is an attempt to answer these questions.     

Activation of protein kinase C induces cortical granule exocytosis in a Ca(2+)-independent manner, but not the resumption of cell cycle in porcine eggs

N-Acetylneuraminic acid (1) is a common sugar in many biological recognition processes. Neuraminidase enzymes recognize and cleave terminal sialic acids from cell surfaces. Viral entry into host cells requires neuraminidase activity, thus inhibition of neuraminidase is a useful strategy for development of drugs for viral infections. A recent crystal structure for influenza viral neuraminidase with sialic acid bound shows that the sialic acid is in a boat conformation [Prot Struct Funct Genet 14: 327 (1992)]. Our studies seek to determine if structural pre-organization can be achieved through the use of sialyllactones. Determination of whether siallylactones are pre-organized in a binding conformation requires conformational analysis. Our inability to find a systematic study comparing the results obtained by various computational methods for carbohydrate modeling led us to compare two different conformational analysis techniques, four different force fields, and three different solvent models. The computational models were compared based on their ability to reproduce experimental coupling constants for sialic acid, sialyl-1,4-lactone, and sialyl-1,7-lactone derivatives. This study has shown that the MM3 forcefield using the implicit solvent model for water implemented in Macromodel best reproduces the experimental coupling constants. The low-energy conformations generated by this combination of computational methods are pre-organized toward conformations which fit well into the active site of neuraminidase.     

Circadian rhythm of intraocular pressure in the rat

In order to investigate the relationship between the bioactive conformation of a peptide and its set of thermodynamically accessible structures in solution, the conformational profile of the tetrapeptide Ac-Pro-Ala-Pro-Tyr-OH was characterized by computational methods. Search of the conformational space was performed within the molecular mechanics frame-work using the AMBER4.0 force field with an effective dielectric constant of 80. Unique structures of the peptide were compared with its bioactive conformation for the protein Streptomyces griseus Protease A, as taken from the crystal structure of the enzyme-peptide complex. The results show that the bound conformation is close to one of the unique conformations characterized in the conformational search of the isolated peptide. Moreover, the lowest energy minimum characterized in the conformational search exhibits large deviations when compared to the bound conformation of the crystal structure.     

Investigation of conformational specificity at GPIIb/IIIa: evaluation of conformationally constrained RGD peptides

RGD-containing proteins and peptides are known to bind to the platelet GPIIb/IIIa receptor and inhibit platelet aggregation. That a conformational component to the specificity exists is suggested by significantly lower activity of linear RGD analogs relative to closely related cyclic peptides and small proteins containing the RGD sequence. Recently, conformations for a suite of RGD containing cyclic peptides have been defined by NMR-based methods and, for one molecule, by X-ray diffraction. We report here the NMR-based conformational analysis of an additional cyclic peptide, cyclo(Pro-Arg-Gly-Asp-D-Pro-Gly), and compare the conformational variations in the suite of peptides and related analogs. Biological activity data for these peptides shows a preference of the platelet GPIIb/IIIa receptor for one conformation of the RGD sequence, but suggests its ability to bind a second, distinct conformation.     

Conformational analysis of two cyclic analogs of angiotensin: implications for the biologically active conformation

Conformations of two cyclic analogs of angiotensin (Asp1-Arg2-Val3-Tyr4-Val/Ile5-His6-Pro7-Phe8, AT), cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT, were studied, independently employing two complementary techniques, energy calculations and NMR measurements in DMSO solution. NMR data were indicative of well-defined solution conformations for the cyclic moieties of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT, including the phi values for the Cys3/HCys3 and Tyr4 residues, as well as the chi 1 value for the Tyr4 residue. Solution conformations for the exocyclic linear parts of both molecules cannot be described by the NMR data with the same precision. At the same time, independent energy calculations revealed the same conformations of cyclic moieties of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT among low-energy conformers for both peptides. Moreover, the same conformations are compatible with the model of AT receptor-bound conformation (Nikiforovich & Marshall, 1993), which assumes the particular spatial arrangement of aromatic moieties of Tyr4, His6, and Phe8 residues and the C-terminal carboxyl. These conformers of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT contain "an open turn" in the backbone of the Tyr4-Val5 residues, instead of the earlier proposed beta-like reversal, thus confirming the suggestion that the conformation(s) ensuring binding of AT analogs with specific receptors should not be described in terms of a unique backbone conformer.