1H nuclear-magnetic-resonance studies of the porcine-pancreatic secretory trypsin inhibitor at 270 MHz

The pancreatic secretory trypsin inhibitor from porcine pancreas has been investigated by high-resolution 1H nuclear magnetic resonance (NMR) at 270 MHz. The presence of a number of slowly exchanging labile protons indicates that the protein is highly globular. Of the two tyrosyl rings, one is free-rotating and solvent-exposed while the other one is hindered in its mobility and buried in the interior of the protein. A lineshape analysis of the temperature dependence of aromatic resonances gave the dynamic parameters for activation of ring mobility. The inhibitor exhibits at least three well-resolved high-field ring-current-shifted methyl resonances. Form II of the inhibitor, that lacks the first four residues, has been compared with the intact form I. No detectable differences were found between the spectra of I and II, which indicates that the presence of the N-terminal tetrapeptide does not appreciably affect the overall conformation of the protein.     

A slow fluctuation in the molecular conformation of a soya bean trypsin inhibitor

The kinetics of the hydrogen-deuterium exchange reaction in a trypsin inhibitor (Kunitz) from soya bean have been followed by infrared absorption measurements in aqueous solutions at various temperatures and pH values. It was found that, in every case, 49% of the total peptide hydrogen atoms exchange relatively slowly. This amount corresponds to 83 peptide groups per molecule, and this is considered to be equal to the number of peptide NH groups involved in hydrogen bonds with the carbonyls of other peptide groups in the protein molecule in its native form. Each rate constant (k) determined at pH 2.75 for this category of the NH groups is in good agreement with the value expected from an idea that the breaking of the peptide-peptide hydrogen bonds takes place very slowly, and that this is the rate-determining process in the hydrogen-deuterium exchange reaction. Thus, by ultraviolet absorption measurements at 297 nm, the equilibrium constant of the native and denatured forms has been determined in the temperature range from 42 to 53.5 °C, as well as the reaction rate of reaching equilibrium from an off-equilibrium state. From these data the rate constant (k₁) of the denaturation reaction is determined, and the k₁ value is found to be practically equal to the hydrogen exchange rate constant (k). The Arrhenius plot of this rate constant (k) gives a straight line in the 25 to 55 °C region, and this gives a value of 48.6 kcal/mol for the activation energy of the denaturation reaction. The rate of this reaction is found to be very low at 25 °C; its half-life is about eleven days. Infrared absorption spectra observed in the amide I region suggest that the very slow denaturation of this protein is accompanied by a conformation change from an α-helix to a β-form. The number of the peptide groups involved in this α → β change is estimated to be 9 ± 3.     

Structural stability of disulfide mutants of basic pancreatic trypsin inhibitor: A molecular dynamics study

The structure and folding of basic pancreatic trypsin inhibitor (BPTI) has been studied extensively by experimental means. We report a computer simulation study of the structural stability of various disulfide mutants of BPTI, involving eight 250-psec molecular dynamics simulations of the proteins in water, with and without a phosphate counterion. The presence of the latter alters the relative stability of the single disulfide species [5–55] and [30–51]. This conclusion can explain results of mutational studies and the conservation of residues in homologues of BPTI, and suggests a possible role of ions in stabilizing one intermediate over another in unfolding or folding processes. © 1996 Wiley-Liss, Inc.     

Bovine pancreatic trypsin inhibitor crystals with different morphologies: a molecular dynamics simulation study

A molecular dynamics simulation study is reported for three polymorphic protein crystals (4PTI, 5PTI and 6PTI) of bovine pancreatic trypsin inhibitor (BPTI). The simulated lattice constants are in good agreement with experimental data, indicating the reliability of force field used. The fluctuation patterns of peptide chains in the three crystals are similar, and the protein structures are fairly well maintained during simulation. We observe that water forms a pronounced hydration layer near the protein surface. The diffusion coefficients of water in the three crystals are smaller than in bulk phase, and thus, the activation energies are higher. The porosity, fluctuation of peptide chains and solvent-accessible surface area as well as the diffusion coefficients of water and counterion in 5PTI are the largest among the three crystals. The diffusion of water and counterion is anisotropic, and the degree of anisotropy increases in the order of 4PTI < 5PTI < 6PTI. Despite a slight difference, the structural and diffusion properties in the three BPTI crystals are generally close. This simulation study reveals that crystal polymorphism does not significantly affect microscopic properties in the BPTI crystals with different morphologies.     

Structure and internal dynamics of the bovine pancreatic trypsin inhibitor in aqueous solution from long-time molecular dynamics simulations

Structural and dynamic properties of bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution are investigated using two molecular dynamics (MD) simulations: one of 1.4 ns length and one of 0.8 ns length in which atom-atom distance bounds derived from NMR spectroscopy are included in the potential energy function to make the trajectory satisfy these experimental data more closely. The simulated properties of BPTI are compared with crystal and solution structures of BPTI, and found to be in agreement with the available experimental data. The best agreement with experiment was obtained when atom-atom distance restraints were applied in a time-averaged manner in the simulation. The polypeptide segments found to be most flexible in the MD simulations coincide closely with those showing differences between the crystal and solution structures of BPTI. © 1995 Wiley-Liss, Inc.     

Solution conformation of a cyclic neurokinin antagonist: A nmr and molecular dynamics study

The solution structure of a hexapeptide, cyclo(Gln-Trp-Phe-Gly-Leu-Met), which is a selective NK-2 antagonist, has been studied by a combination of two-dimensional nmr and molecular dynamics (MD) techniques. The simulation based on nmr and MD data resulted in the convergence to a family of structures. Free molecular dynamics for 50 ps in the presence of DMSO solvent molecules shows that the structure is energetically stable. One intramolecular hydrogen bond between the amide proton of Gin and the carbonyl oxygen of Gly was revealed. This result is consistent with the results from the measurement of the temperature coefficient of the amide protons. The extent of intermolecular hydrogen bonding between the amide protons of the peptide and DMSO was also revealed by the free MD simulation. The resulting structure of the cyclic peptide contains a variation type I′ β-turn in the Gly-Leu-Met-Gln segment. Comparison of the structure of this peptide with that of other NK-2 antagonist cyclic hexapeptides was made, and the activity of cyclic antagonists appears to be inversely related to the conformational rigidity of the cyclic peptides. © 1994 John Wiley & Sons, Inc.     

Solution conformation of human neuropeptide Y by 1H nuclear magnetic resonance and restrained molecular dynamics.

The solution structure of human neuropeptide Y has been solved by conventional two-dimensional NMR techniques followed by distance-geometry and molecular-dynamics methods. The conformation obtained is composed of two short contiguous alpha-helices comprising residues 15-26 and 28-35, linked by a hinge inducing a 100 degree angle. The first helix (15-26) is connected to a polyproline stretch (residues 1-10) by a tight hairpin (residues 11-14). The helices and the polyproline stretch are packed together by hydrophobic interactions. This structure is related to that of the homologous avian pancreatic polypeptide and bovine pancreatic polypeptide. The C- and N-terminii, known to be involved in the biological activity for respectively the receptor binding and activation, are close together in space. The side chains of residues Arg33, Arg35 and Tyr36 on the one hand, and Tyr1 and Pro2 on the other, form a continuous solvent-exposed surface of 4.9 mm2 which is supposed to interact with the receptor for neuropeptide Y.     

Raman studies of the conformation of the basic pancreatic trypsin inhibitor.

The vibrational Raman spectra of the basic pancreatic trypsin inhibitor in aqueous solution, as lyophilized powder and in a single crystal and presented. The thermal stability of this protein is demonstrated by the fact that minor alterations in the spectrum, mainly in the amide III band near 1260 cm-1, occur in the solution spectrum only at temperatures above 75 degrees C. No significant spectral changes appear when the pH value of the solution is varied in the range from 1.5 to 8.7. The distinct differences of the powder spectrum compared to that of the solution, show that lyophilization causes appreciable conformational changes both in the main-chain and in the side-chains. A difference in main chain conformation of the basic pancreatic trypsin inhibitor in single crystal and in solution is suggested by different amide III frequencies.     

On the dependence of molecular conformation on the type of solvent environment: a molecular dynamics study of cyclosporin A

The dependence of the conformation of cyclosporin A (CPA), a cyclic undecapeptide with potent immunosuppressive activity, on the type of solvent environment is examined using the computer simulation method of molecular dynamics (MD). Conformational and dynamic properties of CPA in aqueous solution are obtained from MD simulations of a CPA molecule dissolved in a box with water molecules. Corresponding properties of CPA in apolar solution are obtained from MD simulations of CPA in a box with carbontetrachloride. The results of these simulations in H2O and in CCl4 are compared to each other and to those of previous simulations of crystalline CPA and of an isolated CPA molecule. The conformation of the backbone of the cyclic polypeptide is basically independent of the type of solvent. In aqueous solution the beta-pleated sheet is slightly weaker and the gamma-turn is a bit less pronounced than in apolar solution. Side chains may adopt different conformations in different solvents. In apolar solution the hydrophobic side chain of the MeBmt residue is in an extended conformation with its hydroxyl group hydrogen bonded to the backbone carbonyl group. In aqueous solution this hydrophobic side chain folds over the core of the molecule and the mentioned hydrogen bond is broken in favor of hydrogen bonding to water molecules. The conformation obtained from the MD simulation in CCl4 nicely agrees with experimental atom-atom distance data as obtained from nmr experiments in chloroform. In aqueous solution the relaxation of atomic motion tends to be slower than in apolar solution.     

Design of fluorinated sialic acid analog inhibitor to H5 hemagglutinin of H5N1 influenza virus through molecular dynamics simulation study

Abstract

Influenza epidemics and pandemics are caused by influenza A virus. The cell surface protein of hemagglutinin and neuraminidase is responsible for viral infection and release of progeny virus on the host cell membrane. Now 18 hemagglutinin and 11 neuraminidase subtypes are identified. The avian influenza virus of H5N1 is an emergent threat to public health issues. To control the influenza viral infection it is necessary to develop antiviral inhibitors and vaccination. In the present investigation we carried out 50 ns Molecular Dynamics simulation on H5 hemagglutinin of Influenza A virus H5N1 complexed with fluorinated sialic acid by substituting fluorine atoms at any two hydroxyls of sialic acid by considering combinatorial combination. The binding affinity between the protein–ligand complex system is investigated by calculating pair interaction energy and MM-PBSA binding free energy. All the complex structures are stabilized by hydrogen bonding interactions between the H5 protein and the ligand fluorinated sialic acid. It is concluded from all the analyses that the fluorinated complexes enhance the inhibiting potency against H5 hemagglutinin and the order of inhibiting potency is SIA-F9 ? SIA-F2 ≈ SIA-F7 ≈ SIA-F2F4 ≈ SIA-F2F9 ≈ SIA-F7F9 > SIA-F7F8 ≈ SIA-F2F8 ≈ SIA-F8F9 > SIA-F4 ≈ SIA-F4F7 ≈ SIA-F4F8 ≈ SIA-F8 ≈ SIA-F2F7 ≈ SIA > SIA-F4F9. This study suggests that one can design the inhibitor by using the mono fluorinated models SIA-F9, SIA-F2 and SIA-F7 and difluorinated models SIA-F2F4, SIA-F2F9 and SIA-F7F9 to inhibit H5 of H5N1 to avoid Influenza A viral infection.

Communicated by Ramaswamy H. Sarma     

The molecular stoichiometry of trypsin inhibition by human alpha-1-proteinase inhibitor

The stoichiometry of interaction of human alpha-1-proteinase inhibitor with porcine trypsin has been determined using a highly purified preparation of inhibitor. In contrast to the reports of others, one mole of alpha-1-proteinase inhibitor was found to inhibit two moles of trypsin. Disc gel electrophoresis indicates that the 2:1 complex is preferentially formed even when free alpha-1-proteinase inhibitor is still present.     

Determination and comparative analysis of the conformation of bovine pancreatic trypsin inhibitor and trypsin inhibitors E and K from the data of two-dimensional 1H-NMR spectroscopy

On the basis of joint consideration of distance dependences between amide proton NH and protons C alpha H, NH, C beta H of the preceding in amino acid sequence residue from the torsion angles phi psi, chi 1, the correlation diagram of these proton-proton distances with the regions of sterically allowed conformational space (phi, psi) is presented and the method for the determination of the L-amino acid residues backbone conformations is proposed. The diagram was used for the determination of backbone conformations of bovine pancreatic trypsin inhibitor and trypsin inhibitors E and K from Dendroaspis polylepis using the data from two-dimensional 1H-NMR spectroscopy. The analysis of backbone conformations was carried out. The individual elements of these protein molecules secondary structure were characterized and their high conformational homology was shown. The inference about qualitative coincidence of three protein molecules conformation in solution, preservation of secondary structure basic elements and their similarity with bovine pancreatic trypsin inhibitor crystalline structure was made.     

A molecular orbital study of the conformation of formycin

Semiempirical quantum mechanical calculations, using the iterative extended Huckel theory, are carried out for the evaluation of conformational energies, dipole moment and net atomic charges as a function of the rotation about the glycosidic bond. Torsion about the C(4′)-C(5′) bond has also been considered. The energy diagrams for either the gg or gt rotamers of formycin predict that neither the first or the second energy minimum fall in the classical anti or syn regions. The predicted energy difference between the two most preferred conformations is rather large (3 kcal/mole). In contrast adenosine is predicted to favor the anti conformation by less than 1 kcal/mole. Barriers to internal counter-clockwise rotation about the glycosidic bond are higher for adenosine.     

A molecular orbital study of the conformation of barbiturates

The all-valence electrons molecular orbital method PCILO (Perturbative Configuration Interaction using Localized Orbitals) is applied to the study of the conformational and electronic properties of barbiturates. The results indicate the preference for specific conformations which correspond to a tendency for at least a partial folding of the aliphatic substituents towards the barbituric ring, and the eclipsing by the cyclic substituents of the bonds ending at C₅. The theoretical results agree with available experimental data from X-ray crystallography. On the other hand, the substituents at C₅ exert a negligible influence on the charge distribution in the barbituric ring. This situation agrees with the hypothesis that the factors responsible for the pharmacological activity of these drugs reside probably essentially in the electronic properties of the barbituric ring system (perhaps in its ability to hydrogen-bond with adenine), the role of the substituents at C₅ being mainly to favor the transfer of the drug to its receptors and the establishment of an appropriate contact with it.     

A highly strained nuclear conformation of the exportin Cse1p revealed by molecular dynamics simulations

To investigate the stability of the open nuclear state of the exportin Cse1p and its closing mechanism at the atomic level, we have performed multiple molecular dynamics simulations. The simulations revealed a strikingly fast transition of Cse1p from the open conformation to the closed cytoplasmic form, consistent with the proposal that Cse1p represents a "spring-loaded molecule." The structure of the ring-shaped state obtained in the simulations is remarkably close to the crystal structure of the cytoplasmic state, though the open nuclear structure was used as the only input. The conformational change is initially driven by release of strain due to RanGTP/importin-alpha binding. Subsequently, a stable closed state is formed, driven by attraction of electrostatically complementary interfaces. These results are consistent with and extend previous proposals. Reverse-charge and neutral mutants remained in an open state. The simulations predict a detailed reaction pathway and resolve the role of suggested hinge regions.     

A study of the lysyl residues in the basic pancreatic trypsin inhibitor using 1H nuclear magnetic resonance at 360 Mhz.

Fourier transform 1H nuclear magnetic resonance (NMR) experiments at 360 MHz using convolution difference techniques to improve the spectral resolution were employed to investigate the resonances of the lysyl residues in bovine pancreatic trypsin inhibitor. The observations in both native protein and in chemically modified protein containing Nepsilon-dimethyllsysine show that three of the four lysines extend predominantly freely into the solvent, whereas lysine-41 is involved in an intramolecular interaction with tyrosine-10. Since in the single crystal structure tyrosine-10 is involved in an intermolecular interaction with arginine-42 of the neighboring protein molecule, the NMR data thus reveal a local conformation difference for bovine pancreatic trypsin inhibitor in solution and in the crystalline form which appears to result primarily from intermolecular interaction in the crystal lattice.