Anti-insulin antibody structure and conformation. II. Molecular dynamics with explicit solvent.

Molecular dynamics at 300 K was used as a conformation searching tool to analyze a knowledge-based structure prediction of an anti-insulin antibody. Solvation effects were modeled by packing water molecules around the antigen binding loops. Some loops underwent backbone and side-chain conformational changes during the 95-ps equilibration, and most of these new, lower potential energy conformations were stable during the subsequent 200-ps simulation. Alterations to the model include changes in the intraloop, main-chain hydrogen bonding network of loop H3, and adjustments of Tyr and Lys side chains of H3 induced by hydrogen bonding to water molecules. The structures observed during molecular dynamics support the conclusion of the previous paper that hydrogen bonding will play the dominant role in antibody-insulin recognition. Determination of the structure of the antibody by x-ray crystallography is currently being pursued to provide an experimental test of these results. The simulation appears to improve the model, but longer simulations at higher temperatures should be performed.     

Dogfish insulin. Primary structure, conformation and biological properties of an elasmobranchial insulin

Insulin from an elasmobranch, the spiny dogfish (Squalus acanthias) has been purified to near homogeneity by means of acid-ethanol extraction and salt precipitation. The amino acid sequences of the performic-acid-oxidised A and B chains have been determined and exhibit some unusual features. The A chain contains a total of 22 amino acids; only the insulin from coypu (a member of the Rodentia suborder, Hystricomorpha), has previously been reported to contain an extension past the A21 asparagine. The B10 histidine, which is involved in the formation of the insulin hexamers in higher vertebrates through the co-ordination of zinc, is present in this elasmobranch insulin. Several substitutions relative to bovine insulin occur in the proposed receptor binding region (A5Gln leads to His, B21Glu leads to Pro, B22Arg leads to Lys, B25Phe leads to Tyr). In spite of these substitutions, the maximal response in the rat epididymal fat cell assay is the same for bovine and dogfish insulins; the concentration required to produce the half-maximal response is, however, approximately threefold greater for dogfish insulin than that of bovine insulin. The use of interactive computer graphics model-building predicts that the dogfish insulin can attain a three-dimensional structure very similar to that of bovine insulin; circular dichroic spectra are presented which support the model-building studies.     

Molecular mechanics modeling of azobenzene-based photoswitches

We present an extension of the generalized amber force field to allow the modeling of azobenzenes by means of classical molecular mechanics. TD-DFT calculations were employed to derive different interaction models for 4-hydroxy-4'-methyl-azobenzene, including the ground (S(0)) and S(1) excited state. For both states, partial charges and the -N = N- torsion potentials were characterized. On this basis, we pave the way to large-scale model simulations involving azobenzene molecular switches. Using the example of an isolated molecule, the mechanics of cyclic switching processes are demonstrated by classical molecular dynamics simulations.     

Coypu insulin. Primary structure, conformation and biological properties of a hystricomorph rodent insulin.

Insulin from a hystricomorph rodent, coypu (Myocaster coypus), was isolated and purified to near homogeneity. Like the other insulins that have been characterized in this Suborder of Rodentia, coypu insulin also exhibits a very low (3%) biological potency, relative to pig insulin, on lipogenesis in isolated rat fat-cells. The receptor-binding affinity is significantly higher (5-8%) in rat fat-cells, in rat liver plasma membranes and in pig liver cells, indicating that the efficacy of coypu insulin on receptors is about 2-fold lower than that of pig insulin. The primary structures of the oxidized A- and B-chains were determined, and our sequence analysis confirms a previous report [Smith (1972) Diabetes 21, Suppl. 2, 457-460] that the C-terminus of the A-chain is extended by a single residue (i.e. aspartate-A22), in contrast with most other insulin sequences, which terminate at residue A21. In spite of a large number of amino acid substitutions (relative to mammalian insulins), computer-graphics model-building studies suggest a similar spatial arrangement for coypu insulin to that for pig insulin. The substitution of the zinc-co-ordinating site (B10-His----Gln) along with various substitutions on the intermolecular surfaces involved in the formation of higher aggregates are consistent with the observation that this insulin is predominantly 'monomeric' in nature. The c.d. spectrum of coypu insulin is relatively similar to those of casiragua insulin and of bovine insulin at low concentration.     

A porous-medium approach for modeling heart mechanics. I. theory

A method is developed for evaluating the distribution of the blood pressure, stresses and strains of the muscle fibers, and motion of the cardiac wall due to the cyclic contractions of the heart. The cardiac system is subdivided into two media: the chambers and the wall; the latter is enclosed by two impermeable surfaces (with one interface separating the two media and the other confining the wall). The momentum balance equation for the blood in the (two) cardiac ventricles is averaged, yielding a modification of Forchheimer's law, namely inclusion of the time derivative of the flux. The contracting muscle provides the driving force for the blood flow, and the endocardial velocity is thus taken as identical to that of the blood in the cavity next to the wall. Translation of the endocardium is governed by the blood pressure gradients in the ventricles. The blood pressure and stress-strain pattern in the myocardium are analyzed by applying concepts of the theory of mixtures to the blood and to the saturated solid matrix. With the blood pressure simulated by a modified Darcy law with relative fluid-solid velocity, fiber stresses and strains can be assessed with the aid of appropriate constitutive and compatibility laws.     

Inactive conformation of an insulin despite its wild-type sequence.

The peptide group between residues B24 and B25 of insulin was replaced by an ester bond. This modification only in the backbone was meant to eliminate a structurally important H-bond between the amide proton of B25 and the carbonyl oxygen of A19, and consequently to enhance detachment of the C-terminal B-chain from the body of the molecule, exposing the underlying A-chain. According to a model derived from the effects of side-chain substitutions, main-chain shortening, and crosslinking, this conformational change is prerequisite for receptor binding. Contrary to the expectation that increased flexibility would increase receptor binding and activity, depsi-insulin ([B24-B25 CO-O]insulin) has turned out be only 3-4% potent. In search of an explanation for this observation, the solution structure of depsi-insulin was determined by two-dimensional 1H-NMR spectroscopy. It was found that the loss of the B25-A19 H-bond does not entail detachment of the C-terminal B-chain. On the contrary, it is overcompensated by a gain in hydrophobic interaction achieved by insertion of the Phe B25 side chain into the molecule's core. This is possible because of increased rotational freedom in the backbone owing to the ester bond. Distortion of the B20-B23 turn and an altered direction of the distal B-chain are consequences that also affect self-association. The exceptional position of the B25 side chain is thus the key feature of the depsi-insulin structure. Being buried in the interior, it is not available for guiding the interaction with the receptor, a crucial role attributed to it by the model. This seems to be the main reason why the structure of depsi-insulin represents an inactive conformation.     

Molecular mechanics studies of dinucleoside methylphosphonates: influence of methylphosphonate and its chirality on the phosphodiester conformation.

Molecular mechanics studies are performed on single stranded as well as base paired forms of dinucleoside methylphosphonates comprising different base sequences for both the S- and R-isomers of methylphosphonate (MP). S-MP produces noticeable distortions in the geometry, locally at the phosphate center, and this enables the stereochemical feasibility of compact g- g- phosphodiester. Besides, it tends to perturb the conformations around the P-O3' and glycosyl bonds, causing minor variations in stacking interactions. In single stranded dinucleosides, the gain in adjacent base stacking interaction energies seems to be sufficient to overcome the barrier to P-O3' bond rotation arising due to S-MP...sugar interaction, and this results in transition to a compact phosphodiester (BI-type) from an initial extended phosphodiester (BII-type) conformation. Such a thing seems rather difficult in base pair constrained duplexes. Dinucleosides with R-MP behave analogous to normal phosphate duplexes as the methyl group is away from the sugar. It is found that dinucleoside methylphosphonates are energetically less favoured than the corresponding dinucleoside phosphates mainly due to the depletion of contributions from electrostatic attractive interactions involving the base and sugar with the methylphosphonate consequent to the nonionic nature of the latter. Neither S-MP nor R-MP seem to significantly alter the stereochemistry of duplex structure.     

Molecular modeling of an antigenic complex between a viral peptide and a class I major histocompatibility glycoprotein.

Computer simulation of the conformations of short antigenic peptides (5-10 residues) either free or bound to their receptor, the major histocompatibility complex (MHC)-encoded glycoprotein H-2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern-recognition technique, several energy-minimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall alpha-helical conformation through regular i,i + 4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i + 3 hydrogen bonds at their N-terminal end. Experimental data documented that the C-terminal residue is critical for interaction of the peptide with H-2 Ld. This finding could be satisfactorily explained by a 3-D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3-D model is proposed for the complex between a high-affinity nonapeptide and the H-2 Ld receptor. First, the H-2 Ld molecule was built from X-ray coordinates of two homologous proteins: HLA-A2 and HLA-Aw68, energy-minimized and studied by MD simulations. With HLA-A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X-ray conformation. Water simulation of the H-2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template-derived optimal parameters. The bound peptide retains mainly its alpha-helical conformation and binds to hydrophobic residues of H-2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor-binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts alpha-helical conformation for the bound peptide.     

Synthesis and properties of chemotactic peptide analogs. I. Crystal structure and molecular conformation of HCO-Met-leu-Ain-OMe

HCO-Met-Leu-Ain-OMe (2), an analog of the chemotactic peptide HCO-Met-Leu-Phe-OH, containing the conformationally blocked residue of the 2-aminoindane-2-carboxylic acid (Ain) has been synthesized and its crystal and molecular conformation has been determined. Crystals of 2 are monoclinic, space group P2(1), with a = 15.059(7), b = 18.548(7), c = 9.600(4) A; beta = 85.04(3) degrees. The structure has been solved by direct methods and refined to R = 0.069 for 2813 independent reflections with I greater than 2.5 sigma (I). Two independent molecules A and B have been found in the asymmetric unit of the crystal of 2. Their conformation can be described as extended at the Met and Leu residues, but folded at the C-terminal Ain residue. The helical folding is left- and right-handed in the A and B molecule, respectively. The crystal packing is characterized by ribbons of intermolecular hydrogen bonded molecules extended along the c direction. The constrained analog 2 is highly active in the superoxide production, thus indicating that a stabilization of a helical folding at the C-terminal region of chemotactic tripeptides maintains the activity. The orientation of the aromatic ring, with respect to its adjacent backbone atoms, does not seem critical for the activity.     

Molecular conformation of achatin-I, an endogenous neuropeptide containing D-amino acid residue. X-ray crystal structure of its neutral form

The molecular conformation of achatin-I neutral form (H-Gly-D-Phe-Ala-Asp-OH), an endogenous neuropeptide, was elucidated by X-ray crystal analysis. The molecule has a type II' beta-turn structure with the D-Phe-Ala residues at the corner of the bend, which is further stabilized by two NH(Gly)...C gamma = O sigma(Asp) and NH(Asp)...C gamma = O sigma(Asp) intramolecular hydrogen bonds. This turn conformation may be an important feature of achatin-I related to its neuroexcitatory activity.     

Molecular conformation of thyroid hormones: structure and binding interactions of thyroxine

In an accurately determined X-ray diffraction study of the thyroid hormone thyroxine (T4), the two independent conformations in the crystal lattice show significant differences in the outer phenyl ring geometry when compared with that of 3,5,3'-triiodothyronine (T3). The major differences between the T4 and T3 structures are a shortened C4'-O4' bond, contraction of the C3'-C4'-C5' angle and an increase in the C3' and C5' angles of T4. These changes can be correlated with the difference in acidity of the 4'-OH of T4 and T3 and help to explain binding affinity differences among thyroactive compounds. The hydrogen bond directionality observed in T4 and other thyroid structures offers an insight into the molecular details of the hormone-receptor site. The conformation of one T4 molecule is cisoid, that of the other transoid, the first such instance of different overall conformations to be found in the same crystal lattice. One T4 molecule has the side chain nearly coplanar with the inner ring, an unusual conformation among thyroid structures.     

Steroid structure and function. Molecular conformation of 4-hydroxyestradiol and its relation to other catechol estrogens

Hydroxylation of estrogens at C(2) or C(4) effects differentially their binding affinity to and dissociation rate from the estrogen receptor. The X-ray crystal structure of 4-hydroxyestradiol (4-OH-E2) is reported here and compared with that of 2-hydroxyestradiol (2-OH-E2), the 2- and 4-hydroxylated derivatives of estrone (E1) and with that of the parent estrogens, E1 and E2. The overall molecular shape and hydrogen bonding patterns of each were examined for their possible relevance to their binding to the estrogen receptor and their biological activity. A shift in the B-ring conformation away from the symmetrical 7 alpha,8 beta-half-chair form toward the 8 beta-sofa form is induced by both 2- and 4-hydroxy substitution. This shift appears to be larger in the case of E2 than E1 derivatives and to be correlated with an observed change in the hydrogen bonding potential of the C(3) hydroxyl. In 4-OH-E2, as in E2 and 4-OH-E1, the C(3) hydroxyl functions both as a hydrogen bond donor and acceptor. In contrast in 2-OH-E2 the hydroxyl functions only as a donor. The markedly reduced affinity of 2-hydroxylated estrogens for the estrogen receptor could be due to a combination of steric interactions, competition between O(2) and O(3) for hydrogen bonds for a common site on the receptor, and to general interference with hydrogen bond formation of O(3). The C(4) hydroxyl participates in the formation of a chain of hydrogen bonds in the solid state that is similar to a chain seen in single crystals of E2. The presence of a similar chain of hydrogen bonds involving O(3) in the receptor site could account for the decreased dissociation rate of the 4-OH-E2 receptor complex.     

Anti-insulin receptor serum mimics the developmental role of insulin in mouse mammary explants

A number of growth factors can maintain hormonally-responsive epithelium in murine mammary explants as well as insulin, but only insulin can promote the synthesis of casein and alpha-lactalbumin, in the presence of glucocorticoid and prolactin. Anti-insulin receptor serum can elicit these effects of insulin on milk protein gene expression. The anti-serum is unique in its ability to mimic the developmental role of insulin in murine mammary epithelium.     

Solution conformation of an oligonucleotide containing a G.G mismatch determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three dimensional solution structure of the non-selfcomplementary oligonucleotide, d(GAGGAGGCACG). d(CGTGCGTCCTC) in which the central base pair is G.G. This is the first structural determination of a G.G mismatch in a oligonucleotide. Two dimensional nuclear magnetic resonance spectra show that the bases of the mismatched pair are stacked into the helix and that the helix adopts a classical B-DNA form. Spectra of the exchangeable protons show that the two guanosines are base paired via their imino protons. For the non-exchangeable protons and for some of the exchangeable protons nuclear Overhauser enhancement build up curves at short mixing times have been measured. These give 84 proton-proton distances which are sensitive to the helix conformation. One of the guanosines adopts a normal anti conformation while the other is syn or close to syn. All non-terminal sugars are C2' endo. These data sets were incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. The G.G mismatch shows a symmetrical base pairing structure. Although the mismatch is very bulky many of its features are close to that of normal B-DNA. The mismatch induces a small lateral shift in the helix axis and the sum of the helical twist above and below the mismatch is close to that of B-DNA.