Molecular Dynamics Simulations of β-turn Forming Tetra- and Hexapeptides

Abstract

It was previously shown that the structural ensemble of model peptides DDKG and GKDG (H. Ishii et al. Biopolymers 24, 2045–2056, 1985), DEKS (A. Otter et al. J. Biomol. Struct. Dyn. 7, 455–476, 1989) NPGQ (F. R. Carbone et al. Int. J. Pept. Protein. Res. 26, 498–508, 1985), SALN (H. Santa et al. J. Biomol. Struct. Dyn. 16, 1033–1041, 1999), SYPFDV and SYPYDV (J. Yao et al. J. Mol. Biol. 243, 736–753, 1994), VP^DAH and VP^DSH (B. Imperiali et al. J. Am. Chem. Soc. 114, 3182–3188, 1992) in solution contains a significant—or in some cases dominant—proportion of β-turn conformation. In this study, a protein database was searched for the above, unprotected sequences which incorporate only L-amino acid residues. Simulated annealing and 25 ns MD simulations of structures were also performed. The DSSP and STRIDE secondary structure-assigning algorithms and clustering were used to analyze trajectories and i, i+3 hydrogen bonds were also sought. The DSSP analysis showed a fluctuation between β-turn and random meander structure, although bend structures were not detected because of the insufficient length of peptide chains. This alternating trend was confirmed when the STRIDE algorithm was used to analyze trajectories, but STRIDE assigned more turn structures. The population of the strongest clusters was above 40% and the middle structures adopted β-turn structure for most sequences. These results are in good agreement with previous experimental results and support the idea of the ultra-marginal stability of turns in the absence of stabilizing long-range interactions of the neighboring segments of a polypeptide chain. However, interactions between the side-chains in tetrapeptides could also contribute to turn stability and result in unusual stability in some cases. Our observations suggest that such interactions are the consequence rather than the driving force of turn formation.     

The auto-orientation in high magnetic fields of oxidized cytochrome b562 as source of constraints for solution structure determination

¹⁵N-¹H ¹J couplings were measured at 500 MHz and 800 MHz for ¹⁵N enriched oxidized cytochrome b ₅₆₂ from E. coli. The magnetic field dependence of 70 ¹J values, which could be measured without signal overlap, shows that there is a molecular magnetic anisotropy which provides partial molecular orientation in the magnetic field and, consequently, residual dipolar couplings (rdc). The rdc were used as further constraints to improve the existing structure [Arnesano et al. (1999) Biochemistry, 38, 8657–8670] with a protocol which uses the rhombic anisotropy [Banci et al. (1998) J. Am. Chem. Soc., 120, 12903–12909]. The overall large molecular magnetic anisotropy has been found to be determined by both the low spin iron (III) and the four helix bundle structure magnetic susceptibility anisotropy contributions.     

Nuclear Overhauser Data and Stereochemical Considerations Suggest that Netropsin Binds Symmetrically Within the Minor Groove of Poly(dA)·Poly(dT), Forming Hydrogen Bonds with Both Strands of the Double Helix

Abstract

Sarma et al. (J. Biomol. Str. and Dynam. 2, 1085 (1985) have proposed, on the basis of nuclear magnetic resonance experiments on the complex of netropsin with poly(dA)·poly(dT), that the drug molecule lies asymmetrically along the dA side of the minor groove and makes hydrogen bonds only with the dA strand. If the crystal structure analyses of B-DNA (Fratini et al., J. Biol. Chem. 257, 14686 (1982)) and of its complex with netropsin (Kopka et al., J. Mol. Biol. 183, 553 (1985)) are any guide, this off-center, wide-groove model is stereochemically unlikely. More to the point, the off-center model is unnecessary to explain the observed nmr data. All of the nuclear Overhauser and other observations are fully explained by the structure seen in the x-ray crystal analysis, in which netropsin sits squarely centered within the minor groove, making bifurcated hydrogen bonds with both strands.     

Two- and three-dimensional 31P-driven NMR procedures for complete assignment of backbone resonances in oligodeoxyribonucleotides

Summary We describe a strategy for sequential assignment of ³¹P and deoxyribose ¹H NMR resonances in oligodeoxyribonucleotides. The approach is based on ³¹P–¹H J-cross-polarization (hetero TOCSY) experiments, recently demonstrated for the assignment of resonances in RNA [Kellogg, G.W. (1992) J. Magn. Reson., 98, 176; Kellogg, G.W. et al. (1992) J. Am. Chem. Soc., 114, 2727]. Two-dimensional hetero TOCSY and hetero TOCSY-NOESY experiments are used to connect proton spin systems from adjacent nucleotides in the dodecamer d(CGCGAATTCGCG)₂ entirely on the basis of through-bond scalar connectivities. All phosphorus resonances of the dodecamer are assigned by this method, and many deoxyribose ¹H resonances can be assigned as well. A new three-dimensional hetero TOCSY-NOESY experiment is used for backbone proton 4, 5 and 5 resonance assignments, completing assignments begun on this molecule in 1983 [Hare, D.R. et al. (1983) J. Mol. Biol., 171, 319]. Numerical simulations of the time dependence of coherence transfer aid in the interpretation of hetero TOCSY spectra of oligonucleotides and address the dependence of hetero TOCSY and related spectra on structural features of nucleic acids. The possibility of a generalized backbone-driven ¹H and ³¹P resonance-assignment strategy for oligonucleotides is discussed.To whom correspondence should be addressed.     

A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients

Summary General pulse sequence elements that achieve sensitivity-enhanced coherence transfer from a heteronucleus to protons of arbitrary multiplicity are introduced. The building blocks are derived from the sensitivity-enhancement scheme introduced by Cavanagh et al. ((1991) J. Magn. Reson., 91, 429–436), which was used in conjunction with gradient coherence selection by Kay et al. ((1992) J. Am. Chem. Soc., 114, 10663–10665), as well as from a multiple-pulse sequence effecting a heteronuclear planar coupling Hamiltonian. The building blocks are incorporated into heteronuclear correlation experiments, in conjunction with coherence selection by the formation of a heteronuclear gradient echo. This allows for efficient water suppression without the need for water presaturation. The methods are demonstrated in HSQC-type experiments on a sample of a decapeptide in H₂O. The novel pulse sequence elements can be incorporated into multidimensional experiments.     

Structure of Lennard–Jones fluid near large spherical particles: further test of the “universality” of adjustable parameter in perturbation density functional theory

Grand canonical Monte Carlo simulation is used to study the density profiles of Lennard–Jones (LJ) fluid next to a large hard sphere (mimicking a colloidal particle) of various sizes. The LJ fluid in the inhomogeneous system thus maintains equilibrium with the bulk LJ fluid. The chosen density and potential parameters for the bulk fluid correspond to the conditions situated at “dangerous” regions of the phase diagram, i.e. near the critical temperature or close to the gas–liquid coexistence curve. The aim of present extensive simulations is to provide exact data for the broad range of the bulk parameters against which the “universality” of adjustable parameter associated with a perturbation density functional approximation (DFA) can be tested. Here the term “universality” means independence of this parameter on the particular external field responsible for the generation of a non-uniform density profile of the fluid. It is shown that the “universality” of this parameter associated with a third order+second order perturbation DFA holds also in the present case of a large spherical particle as a source of external potential, similarly as established in previous studies dealing with other interaction potential and other external fields [J. Chem. Phys., 122, 064503 (2005); J. Chem. Phys., 123 124708 (2005)]. This DFA can be used as input into a recently proposed framework for the calculation of interparticle potential of mean force (PMF).     

Homologous IRCM-Serine Protease 1 from pituitary,heart atrium and ventricle: A common pro-hormone maturation enzyme?

IRCM-Serine Protease 1 (IRCM-SP1) has recently been isolated and characterized from porcine pituitary anterior and neurointermediate lobes (Cromlishet al., 1986a,J. Biol. Chem. 261:10850–10858; Cromlishet al., 1986b,J. Biol. Chem. 261:10859–10870). This pituitary serine protease was shown to selectively cleave human proopiomelanocortin (POMC)-derived peptides at both pairs of basic residues and C-terminal to specific Arg residues, all known to be cleavedin vivo. Here, a similar enzyme was isolated from rat heart atria and ventricles. Rat IRCM-SP1 was shown to be highly specific for the same cleavage sites in POMC, as the porcine pituitary homologue. Furthermore, the rat and the porcine enzymes cleave rat pro-Atrial Natriuretic Factor (pro-ANF 1–126) to yield ANF 103–126, 102–126 and 99–126 in that order of preference. This suggests thatin vitro the cleavage sites preferred in pro-ANF resemble those found in brain and hypothalamus. The enzyme is nine times more abundant in atria versus ventricles/mg protein. It is concluded that IRCM-SP1, could well represent a common pro-hormone maturation enzyme for POMC and Pro-ANF and possibly many other pro-hormones.     

An improved method for large-scale purification of recombinant human glucagon

Glucagon was expressed inEscherichia coli as a fusion protein including the glucagon sequence [Ishizakiet al. (1992),Appl. Microbiol. Biotechnol.36, 483–486]. The high-level expression of a protein inE. coli often results in an insoluble aggregate called an inclusion body containing a fusion protein. In our previous report [Yoshikawaet al. (1992),J. Protein Chem. 11, 517–525], we solubilized this inclusion body by using guanidinium chloride. However, the existence of denaturant caused problems such as a low proteolytic activity for transforming the fusion protein into glucagon and complicated purification methods. We tried to improve the method to enable large-scale purification. At alkaline pH, the inclusion body could be solubilized to a high concentration and cleaved by amino acid-specific endopeptidases. By utilizing isoelectric precipitations as a new economical purification method for glucagon from intermediates, the glucagon obtained was shown to be over 99.5% pure by analytical RP-HPLC. The yield was almost equal that of our previous method, and the glucagon produced was chemically and biochemically equivalent to natural glucagon.     

Site-Directed Mutagenesis in Basic Amino Acid Residues of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V _max/K _mADP. These results indicate that Arg76 is not involved in CO₂ binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413–418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207–210], does not perform an essential function for the enzyme activity.     

Reading the three-dimensional structure of lattice model-designed proteins from their amino acid sequence.

While all the information required for the folding of a protein is contained in its amino acid sequence, one has not yet learned how to extract this information to predict the detailed, biological active, three-dimensional structure of a protein whose sequence is known. Using insight obtained from lattice model simulations of the folding of small proteins (fewer than 100 residues), in particular of the fact that this phenomenon is essentially controlled by conserved contacts (Mirny et al., Proc Natl Acad Sci USA 1995;92:1282) among (few) strongly interacting ("hot") amino acids (Tiana et al., J Chem Phys 1998;108:757-761), which also stabilize local elementary structures formed early in the folding process and leading to the (postcritical) folding core when they assemble together (Broglia et al., Proc Natl Acad Sci USA 1998;95:12930, Broglia & Tiana, J Chem Phys 2001;114:7267), we have worked out a successful strategy for reading the three-dimensional structure of lattice model-designed proteins from the knowledge of only their amino acid sequence and of the contact energies among the amino acids.     

Prediction of the stability of coiled coils using molecular dynamics simulations

We have performed 40–80 ns-long molecular dynamics (MD) simulations of the GCN4 leucine zipper and synthetic coiled coils using the GROMOS96 (43a2) and OPLS-AA force fields, with the aim of predicting coiled coil stability. Starting with an initial configuration of two peptides placed in an ideal coiled coil configuration, we find that changing the amino acid sequence modestly or decreasing peptide length can lead to a decrease in the final α-helicity of coiled coils, although for peptides as long or longer than 16 residues, the values of helicity do not decrease to the low values seen in the experimental results of Lumb et al. (Biochemistry. 1994, 33, 7361–7367) or of Su et al. (Biochemistry. 1994, 33, 15501–15510), presumably because the simulations are not long enough. We find, however, that helicity correlates positively with the number of close hydrophobic interactions between the two peptides, showing that stable coiled coils in the simulations are tightly packed. The minimum interhelical distances are 0.50–0.66 nm for charged groups, indicating that favorable charge interactions are also important for the stability of the coiled coil.     

The heme environment of mouse neuroglobin: histidine imidazole plane orientations obtained from solution NMR and EPR spectroscopy as compared with X-ray crystallography

The ¹H NMR chemical shifts of the heme methyl groups of the ferriheme complex of metneuroglobin (Du et al. in J. Am. Chem. Soc. 125:8080–8081, 2003) predict orientations of the axial histidine ligands (Shokhirev and Walker in J. Biol. Inorg. Chem. 3:581–594, 1998) that are not consistent with the X-ray data (Vallone et al. in Proteins Struct. Funct. Bioinf. 56:85–94, 2004), and the EPR spectrum (Vinck et al. in J. Am. Chem. Soc. 126:4516–4517, 2004) is only marginally consistent with these data. The reasons for these inconsistencies appear to be rooted in the high degree of aqueous solution exposure of the heme group and the fact that there are no strong hydrogen-bond acceptors for the histidine imidazole N–H protons provided by the protein. Similar inconsistencies may exist for other water-soluble heme proteins, and ¹H NMR spectroscopy provides a simple means to verify whether the solution structure of the heme center is the same as or different from that in the crystalline state.     

Analysis of chemotactic bacterial distributions in population migration assays using a mathematical model applicable to steep or shallow attractant gradients

The mathematical model developed by Riveroet al. (1989,Chem. Engng Sci. 44, 2881–2897) is applied to literature data measuring chemotactic bacterial population distributions in response to steep as well as shallow attractant gradients. This model is based on a fundamental picture of the sensing and response mechanisms of individual bacterial cells, and thus relates individual cell properties such as swimming speed and tumbling frequency to population parameters such as the random motility coefficient and the chemotactic sensitivity coefficient. Numerical solution of the model equations generates predicted bacterial density and attractant concentration profiles for any given experimental assay. We have previously validated the mathematical model from experimental work involving a step-change in the attractant gradient (Fordet al., 1991Biotechnol. Bioengng.37, 647–660; For and Lauffenburger, 1991,Biotechnol. Bioengng,37, 661–672). Within the context of this experimental assay, effects of attractant diffusion and consumption, random motility, and chemotactic sensitivity on the shape of the profiles are explored to enhance our understanding of this complex phenomenon. We have applied this model to various other types of gradients with successful intepretation of data reported by Dalquistet al. (1972,Nature New Biol. 236, 120–123) forSalmonella typhimurum validating the mathematical model and supportin the involvement of high and low affinity receptors for serine chemotaxis by these cells.     

Structural Features of Glycera dibranchiata Monomer Hemoglobins. Primary Sequences of Monomer Hemoglobin Components II and III

Primary sequences for the remaining two members (GMH2, GMH3) of the group of three major monomeric hemoglobins from the marine annelid Glycera dibranchiata have been obtained. Full sequences of each 147-amino acid globin were achieved with a high degree of confidence using standard Edman technology in combination with molecular mass determinations of the intact globins and of the cyanogen bromide cleavage fragments using electrospray ionization mass spectrometry. When minor assumptions concerning Q/E identities are made these new results indicate the likely correspondence of GMG2 with the protein represented by the first Glycera dibranchiata monomer hemoglobin complete sequence [Imamura et al., (1972), J. Biol. Chem. 247, 2785–2797]. When these new sequences are combined with the previously determined primary sequence for the third major monomer hemoglobin, GMH4 [Alam et al., J. Protein Chem. (1994), 13, 151–164], it becomes clear that these three (GMG2–4) are truly distinct proteins, contrary to previous suggestions. Surprisingly, our results show that none of these three primary sequences is identical to the published sequence of the refined monomer hemoglobin crystal structure protein; however, there is a strong correspondence to the GMG2 sequence. The present sequencing results, in combination with the published GMH4 sequence, confirm the presence of a distal Leu in place of the more commonly encountered distal His in all three of the major monomer hemoglobins isolated in this laboratory and indicate that the unusual B10 Phe occurs only in GMH4. Analysis of the sequences presented here, along with comparison of amino acid content for Glycera dibranchiata monomer hemoglobins isolated from three different laboratories, and comparison of NMR results from two laboratories suggest further correspondences which unify disparate published isolations.     

Molecular Dynamics Simulations of Rhodopsin Point Mutants at the Cytoplasmic Side of Helices 3 and 6

Abstract

The present work reports on a structural analysis carried out through different computer simulations of a set of rhodopsin mutants with differential functional features in regard to the wild type. Most of these mutants, whose experimental features had previously been reported [Ramon et al. J Biol Chem 282, 14272–14282 (2007)], were designed to perturb a network of electrostatic interactions located at the cytoplasmic sides of transmembrane helices 3 and 6. Geometric and energetic features derived from the detailed analysis of a series of molecular dynamics simulations of the different rhodopsin mutants, involving positions 134(3.49), 247(6.30), and 251(6.34), suggest that the protein structure is sensitive to these mutations through the local changes induced that extend further to the secondary structure of neighboring helices and, ultimately, to the packing of the helical bundle. Overall, the results obtained highlight the complexity of the analyzed network of electrostatic interactions where the effect of each mutation on protein structure can produce rather specific features.     

Transient phase kinetics of activation of human plasminogen

Equations for the time-dependent concentrations of all species involved in the general mechanism of human plasminogen activation proposed by Wohlet al. (J. biol. Chem. 255, 2005–2013, 1980) have been derived. These equations are valid for the whole course of the reaction: for both the transient phase and the steady state. In addition, we compare our results with the ones obtained by the above-mentioned authors for the steady state assuming rapid equilibrium conditions. Finally, we propose a method for the determination of all velocity constants.     

Peroxynitrite decay in the presence of hydrogen peroxide,mannitol and ethanol: A reappraisal

We have reported previously that the apparent rate of peroxynitrite (ONOO^-) decay, as followed from its absorbance at 302 nm, decreases in the presence of hydrogen peroxide, mannitol and ethanol (Alvarez et al., 1995, Chem. Res. Toxicol. 8:859-864; Alvarez et al., 1998, Free Radic. Biol. Med. 24:1331–1337). Recently, two papers confirmed the observation and proposed that this slowing effect was due to the formation of absorbing peroxynitrate (O₂NOO^-) as intermediate (Goldstein and Czapski, 1998, J. Am. Chem. Soc. 120:3458–3463; Hodges and Ingold, 1999, J. Am. Chem. Soc. 121:10695–10701). Peroxynitrate would be formed from the reaction of peroxynitrite-derived nitrogen dioxide with superoxide. Superoxide, in turn, would arise from the one-electron oxidation of hydrogen peroxide, or from the reaction of reductive radicals derived from mannitol and ethanol with dioxygen. In agreement with this concept, we show herein that under the conditions of our previous work, the slowing effect is prevented by superoxide dismutase and, in the case of mannitol and ethanol, by reducing the dioxygen concentration of the reaction solutions. Thus, superoxide formation is necessary for the decrease in the rate of absorbance decay. In addition, by simulations using known rate constants and absorption coefficients, we show that the slowing effect can be quantitatively accounted for by the formation of peroxynitrate.     

Comparison of ion current noise predicted from different models of the sodium channel gating mechanism in nerve membrane

Summary The theoretical power density spectrumS(f) of ion current noise is calculated from several models of the sodium channel gating mechanism in nerve membrane. Sodium ion noise experimental data from the frog node of Ranvier [Conti, F.,et al. (1976),J. Physiol. (London) 262:699] is used as a test of the theoretical results. The motivation for recent modeling has been evidence for a coupling between sodium activation and inactivation from voltage clamp data. The two processes are independent of one another in the Hodgkin and Huxley (HH) model [Hodgkin, A.L., Huxley, A.F. (1952),J. Physiol. (London) 117:500] The noise data is consistent with HH, as noted by Contiet al. (1976). The theoretical results given here appear to indicate that only one case of coupling models is also consistent with the noise data.     

Clean SEA-HSQC: a method to map solvent exposed amides in large non-deuterated proteins with gradient-enhanced HSQC

The recent introduction of the SEA-TROSY experiment (Pellecchia et al. (2001) J. Am. Chem. Soc., 123, 4633–4634) can alleviate the problem of resonance overlap in ¹⁵N/²H labeled proteins. This method selectively observes solvent exposed amide protons with a SEA element. However, SEA-TROSY spectra may be contaminated with exchange-relayed NOE contributions from fast exchanging hydroxyl or amine protons and longitudinal relaxation contributions. Furthermore, for non-deuterated proteins or protein-ligand complexes, SEA-TROSY spectra may contain NOE contributions from aliphatic protons. In this communication, a modified version of the SEA element, a Clean SEA element, is introduced to eliminate these artifacts.