A sequence of seventy-three nucleotides from the coliphage R17 genome

1. A sequence of 73 nucleotides of the RNA genome from coliphage R17 was determined. It can be read through in only one translational frame. The fragment is not part of the coatprotein cistron (Min Jou et al., 1972), nor does it come from the untranslated sequences described previously (Steitz, 1969; Nichols, 1970; Cory et al., 1970; de Wachter et al., 1971; Contreras et al., 1971; Cory et al., 1972). It contains two sequences of 23 and 24 nucleotides, 22 of which are identical. This kind of reiteration is the first one found in bacteriophage nucleic acid. 2. Improved conditions were found and tested for blocking oligonucleotides with carbodi-imide and cleaving by ribonuclease A at cytidylate residues. 3. A synthetic medium is described which allows labelling in vivo with (32)P to give specific radioactivities higher than those obtained in the procedures used previously.     

Homology models of mu-opioid receptor with organic and inorganic cations at conserved aspartates in the second and third transmembrane domains

Metal ions affect ligand binding to G-protein-coupled receptors by as yet unknown mechanisms. In particular, Na(+) increases the affinity for antagonists but decreases it for agonists. We had modeled the mu-opioid receptor (muR) based on the low-resolution structure of rhodopsin by G. F. X. Schertler, C. Villa, and R. Henderson (1993, Nature 362, 770-772) and proposed that metal ions may be directly involved in the binding of ligands and receptor activation (B. S. Zhorov and V. S. Ananthanarayanan, 1998, J. Biomol. Struct. Dyn. 15, 631-637). Developing this concept further, we present here homology models of muR using as templates the structure of rhodopsin elaborated by I. D. Pogozheva, A. L. Lomize, and H. I. Mosberg (1997, Biophys. J. 70, 1963-1985) and J. M. Baldwin, G. F. X. Schertler, and V. M. Unger (1997, J. Mol. Biol., 272, 144-164). Using the Monte Carlo minimization (MCM) method, we docked the Na(+)-bound forms of muR ligands: naloxone, bremazocine, and carfentanyl. The resultant low-energy complexes showed that the two positive charges in the protonated metal-bound ligands interact with the two negative charges at Asp(3.32) and Asp(2.50) (for notations, see J. A. Ballesteros and H. Weinstein, 1995, Methods Neurosci. 25, 366-426). MCM computation on morphine docked inside the model of muR by I. D. Pogozheva, A. L. Lomize, and H. I. Mosberg (1998, Biophys. J. 75, 612-634) yielded two binding modes with the ligand's ammonium group salt-bridged either to Asp(3.32) (generally regarded as the ligand recognition site) or to Asp(2.50). The latter is the presumed site for Na(+) ion, which is known to modulate ligand binding. Assuming that in the low-dielectric transmembrane region of muR, organic and inorganic cations would compete for Asp(3.32) and Asp(2.50), we propose that ligand binding, as visualized in the above models, would first displace Na(+) from Asp(3.32). A subsequent progress of the ligand toward Asp(2.50) would result in either the retention of Na(+) at Asp(2.50) in the case of antagonists or the displacement of Na(+) from Asp(2.50) in the case of agonists. The displaced Na(+) would move toward the salt-bridged Asp(3.49)-Arg(3.50) and disengage the salt bridge. This, in turn, would result in conformational changes at the cytoplasmic face of the receptor that facilitate the interaction with the G-protein.     

Identification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage

Fenton chemistry [Fenton (1894) J. Chem. Soc. 65, 899-910] techniques were employed to identify the residues involved in metal binding located at the active sites of restriction endonucleases. This process uses transition metals to catalytically oxidize the peptide linkage that is in close proximity to the amino acid residues involved in metal ligation. Fe2+ was used as the redox-active transition metal. It was expected that Fe2+ would bind to the endonucleases at the Mg2+-binding site [Liaw et al. (1993) Biochemistry 32, 7999-4003; Ermácora et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387; Soundar and Colman (1993) J. Biol. Chem. 268, 5264-5271; Wei et al. (1994) Biochemistry 33, 7931-7936; Ettner et al. (1995) Biochemistry 34, 22-31; Hlavaty and Nowak (1997) Biochemistry 36, 15515-15525). Fe2+-mediated oxidation was successfully performed on TaqI endonulease, suggesting that this approach could be applied to a wide array of endonucleases [Cao and Barany (1998) J. Biol. Chem. 273, 33002-33010]. The restriction endonucleases BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, BsoBI, EcoRI, EcoRV, MspI, and HinP1I were subjected to oxidizing conditions in the presence of Fe2+ and ascorbate. All proteins were inactivated upon treatment with Fe2+ and ascorbate. BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, and BsoBI were specifically cleaved upon treatment with Fe2+/ascorbate. The site of Fe2+/ascorbate-induced protein cleavage for each enzyme was determined. The Fe2+-mediated oxidative cleavage of BamHI occurs between residues Glu77 and Lys78. Glu77 has been shown by structural and mutational studies to be involved in both metal ligation and catalysis [Newman et al. (1995) Science 269, 656-663; Viadiu and Aggarwal (1998) Nat. Struct. Biol. 5, 910-916; Xu and Schildkraut (1991) J. Biol. Chem. 266, 4425-4429]. The sites of Fe2+/ascorbate-induced cleavage for PvuII, FokI, BglI, and BsoBI agree with the metal-binding sites identified in their corresponding three-dimensional structures or from mutational studies [Cheng et al. (1994) EMBO J. 13, 3297-3935; Wah et al. (1997) Nature 388, 97-100; Newman et al. (1998) EMBO J. 17, 5466-5476; Ruan et al. (1997) Gene 188, 35-39]. The metal-binding residues of BglII, SfiI, and BssSI are proposed based on amino acid sequencing of their Fe2+/ascorbate-generated cleavage fragments. These results suggest that Fenton chemistry may be a useful methodology in identifying amino acids involved in metal binding in endonucleases.     

A 1-D model to explore the effects of tissue loading and tissue concentration gradients in the revised Starling principle

The recent experiments in Hu et al. (Am J Physiol Heart Circ Physiol 279: H1724-H1736, 2000) and Adamson et al. (J Physiol 557: 889-907, 2004) in frog and rat mesentery microvessels have provided strong evidence supporting the Michel-Weinbaum hypothesis for a revised asymmetric Starling principle in which the Starling force balance is applied locally across the endothelial glycocalyx layer rather than between lumen and tissue. These experiments were interpreted by a three-dimensional (3-D) mathematical model (Hu et al.; Microvasc Res 58: 281-304, 1999) to describe the coupled water and albumin fluxes in the glycocalyx layer, the cleft with its tight junction strand, and the surrounding tissue. This numerical 3-D model converges if the tissue is at uniform concentration or has significant tissue gradients due to tissue loading. However, for most physiological conditions, tissue gradients are two to three orders of magnitude smaller than the albumin gradients in the cleft, and the numerical model does not converge. A simpler multilayer one-dimensional (1-D) analytical model has been developed to describe these conditions. This model is used to extend Michel and Phillips's original 1-D analysis of the matrix layer (J Physiol 388: 421-435, 1987) to include a cleft with a tight junction strand, to explain the observation of Levick (Exp Physiol 76: 825-857, 1991) that most tissues have an equilibrium tissue concentration that is close to 0.4 lumen concentration, and to explore the role of vesicular transport in achieving this equilibrium. The model predicts the surprising finding that one can have steady-state reabsorption at low pressures, in contrast to the experiments in Michel and Phillips, if a backward-standing gradient is established in the cleft that prevents the concentration from rising behind the glycocalyx.     

A model for the modulation of microvessel permeability by junction strands

To investigate the effect of junction strands on microvessel permeability, we extend the previous analytical model developed by Fu et al. (1994, J. Biomech. Eng., 116, pp. 502-513), for the interendothelial cleft to include multiple junction strands in the cleft and an interface between the surface glycocalyx layer and the cleft entrance. Based on the electron microscopic observations by Adamson et al. (1998, Am. J. Physiol., 274(43), pp. H1885-H1894), that elevation of intracellular cAMP levels would increase number of tight junction strands, this two-junction-strand and two-pore model can successfully account for the experimental data for the decreased permeability to water, small and intermediate-sized solutes by cAMP.     

Molecular modeling studies on CNG channel from bovine retinal rod: a structural model of the cyclic nucleotide-binding domain

A dimeric model of the cyclic nucleotide-binding domain of the all-alpha homomeric cyclic nucleotide-gated channel from bovine retinal rod is constructed. The model, based on the structure of the fairly homologous catabolite gene activator protein (Weber and Steitz, J Mol Biol 1987;198:311-326), is obtained by use of comparative modeling and molecular dynamics simulations. Our model provides a structural basis for the experimentally measured difference in activity between cAMP and cGMP, as well as the different solvent accessibilities of GLY597 in the complex with cGMP, with cAMP and in the protein in free state. In addition, it provides support for the rearrangement of the domain C helix on ligand binding and releasing proposed by Matulef et al. (Neuron 1999;24:443-452).     

Role of divalent cations in the 3',5'-exonuclease reaction of DNA polymerase I.

X-ray studies of the proofreading 3',5'-exonuclease site of the large (Klenow) fragment of DNA polymerase I have detected a binuclear metal complex consisting of a pentacoordinate metal (site A) which shares a ligand, Asp-355, with an octahedral metal (site B) [Freemont, P. S., Friedman, J. M., Beese, L. S., Sanderson, M. R., & Steitz, T. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8924-8928; Beese, L. S., & Steitz, T. A. (1991) EMBO J. 10, 25-33]. Kinetic studies of the activation of the 3',5'-exonuclease reaction by Co2+, Mn2+, or Mg2+, at low concentrations of DNA, reveal sigmoidal activation curves for the three metal ions with Hill coefficients of 2.3-2.4 and K0.5 values of 16.6 microM, 4.2 microM, and 343 microM, respectively. The binding of Co2+ to the enzyme results in the appearance of an intense visible absorption spectrum of the metal ion with maxima at 633, 570, and 524 nm and extinction coefficients of 190, 194, and 150 M-1 cm-1, respectively, suggesting the formation of a pentacoordinate Co2+ complex. Optical titration with Co2+ yields a sigmoidal titration curve which is best fit by assuming the cooperative binding of three Co2+ ions with a K0.5 of 39.9 microM, comparable to the value of 16.6 microM obtained kinetically. Displacement of Co2+ by 1 equiv of Zn2+, which binds tightly to the A site of the 3',5'-exonuclease, shifts the optical spectrum to 524 nm and lowers the extinction coefficient to 30 -1 cm-1, indicative of octahedral coordination.2+ the formation of the binuclear complex.     

Construction and optimisation of a computer model for a bacterial membrane

The main steps in the construction of a computer model for a bacterial membrane are described. The membrane has been built of 72 lipid molecules, 54 of which being 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine (POPE) and 18--1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidyl-rac-glycerol (POPG) molecules (thus in the proportion of 3:1). The membrane was hydrated with 1955 water molecules (approximately 27 water molecules per lipid). To neutralise the electronic charge (-e) on each POPG molecule, 18 sodium ions (Na+) were added to the membrane close to the POPG phosphate groups. The atomic charges on the POPE and POPG headgroups were obtained from ab initio quantum mechanical restrained electrostatic potential fitting (RESP) (Bayly et al., 1993, J. Phys. Chem. 97, 10269) using the GAMESS program at the 6-31G* level (Schmidt et al., 1993, J. Comput. Chem. 14, 1347). The model constructed in this way provided an initial structure for subsequent molecular dynamics simulation studies intended to elucidate the atomic level interactions responsible for the structure and dynamics of the bacterial membrane.     

Modeling Study on the Cleavage Step of the Self-Splicing Reaction in Group I Introns

Abstract

A three-dimensional model of the Tetrahymena thermophila group I intron is used to further explore the catalytic mechanism of the transphosphorylation reaction of the cleavage step. Based on the coordinates of the catalytic core model proposed by Michel and Westhof (Michel, F., Westhof, E.J. Mol. Biol. 216, 585–610 (1990)), we first converted their ligation step model into a model of the cleavage step by the substitution of several bases and the removal of helix P9. Next an attempt to place a trigonal bipyramidal transition state model in the active site revealed that this modified model for the cleavage step could not accommodate the transition state due to insufficient space. A lowering of PI helix relative to surrounding helices provided the additional space required. Simultaneously, it provided a better starting geometry to model the molecular contacts proposed by Pyle et al. (Pyle, A M., Murphy, F. L., Cech, T. R. Nature 358, 123–128. (1992)), based on mutational studies involving the J8/7 segment Two hydrated Mg²⁺ complexes were placed in the active site of the ribozyme model, using the crystal structure of the functionally similar Klenow fragment (Beese, L.S., Steitz, T.A. EMBO J. 10, 25–33 (1991)) as a guide. Thepresence of two metal ions in the active site of the intron differs from previous models, which incorporate one metal ion in the catalytic site to fulfill the postulated roles of Mg²⁺ in catalysis. The reaction profile is simulated based on a trigonal bipyramidal transition state, and the role of the hydrated Mg²⁺ complexes in catalysis is further explored using molecular orbital calculations.     

Magnetic resonance studies of concanavalin A: assignment of histidine resonances in 220 MHz proton spectrum of complexes with Co2+ and Zn2+.

The low field regions of the 220 MHz proton magnetic resonance spectra of concanavalin A (Con A) complexes with metal ions show well resolved resonances from the C2 protons of histidine side chains. Shifts of these resonances are observed when Zn2+ ions at the transition metal ion binding site, S1, are replaced by CO2 ions. The magnitude of these shifts can be used to determine the orientation of axis of anisotropy of the Co2+ ligand field since the distances from the C2 protons of the histidines to S1 can be computed from the crystal structure coordinates. Assignment of the separate peaks in the spectrum of the Con A-Co2+-Ca2+ complex and of the Con A-Zn2+-Ca2+ complex, to particular histidines in the amino acid sequence of Con A then follows. The refined crystal coordinates of both Reeke et al. (Reeke, G. N., Jr., Becker, J. W., and Edelman, G. M. (1975) J. Biol. Chem, 250, 1525-1547) and of Hardman and Ainsworth (private communication) have been used. These two sets of coordinates both yield orientations for the axis of anisotropy which are approximately in the direction of the His 24 nitrogen ligand.     

Tubulin folding cofactor D is a microtubule destabilizing protein

A rapid switch between growth and shrinkage at microtubule ends is fundamental for many cellular processes. The main structural components of microtubules, the alphabeta-tubulin heterodimers, are generated through a complex folding process where GTP hydrolysis [Fontalba et al. (1993) J. Cell Sci. 106, 627-632] and a series of molecular chaperones are required [Sternlicht et al. (1993) Proc. Natl. Acad. Sci. USA 90, 9422-9426; Campo et al. (1994) FEBS Lett. 353, 162-166; Lewis et al. (1996) J. Cell Biol. 132, 1-4; Lewis et al. (1997) Trends Cell Biol. 7, 479-484; Tian et al. (1997) J. Cell Biol. 138, 821-823]. Although the participation of the cofactor proteins along the tubulin folding route has been well established in vitro, there is also evidence that these protein cofactors might contribute to diverse microtubule processes in vivo [Schwahn et al. (1998) Nature Genet. 19, 327-332; Hirata et al. (1998) EMBO J. 17, 658-666; Fanarraga et al. (1999) Cell Motil. Cytoskel. 43, 243-254]. Microtubule dynamics, crucial during mitosis, cellular motility and intracellular transport processes, are known to be regulated by at least four known microtubule-destabilizing proteins. OP18/Stathmin and XKCM1 are microtubule catastrophe-inducing factors operating through different mechanisms [Waters and Salmon (1996) Curr. Biol. 6, 361-363; McNally (1999) Curr. Biol. 9, R274-R276]. Here we show that the tubulin folding cofactor D, although it does not co-polymerize with microtubules either in vivo or in vitro, modulates microtubule dynamics by sequestering beta-tubulin from GTP-bound alphabeta-heterodimers.     

An interfacial equilibria model for the electrokinetic properties of a fat emulsion. Part III: A predictive computer model

Abstract

The semi-empirical thermodynamic model for the binding of metal ions to an emulsion (intralipid 20%) reported previously (Hall et al., 1991 ; Gaskin et al., 1993) is incorporated into a thermodynamic computer model. This permits the zeta potential and emulsion stability together with precipitate formation to be estimated for any intravenous nutrition regimen. A regimen frequently used in the intravenous nutrition of patients is considered in this modeling study. The effect of solution pH and calcium on the zeta potential of the emulsion is predicted. Calcium and magnesium are the only metal cations which are predicted to be of importance when considering stability of this emulsion.     

Evaluation by site-directed mutagenesis of aspartic acid residues in the metal site of pig heart NADP-dependent isocitrate dehydrogenase

Pig heart NADP-dependent isocitrate dehydrogenase requires a divalent metal cation for catalysis. On the basis of affinity cleavage studies [Soundar and Colman (1993) J. Biol. Chem. 268, 5267] and analysis of the crystal structure of E. coli NADP-isocitrate dehydrogenase [Hurley et al. (1991) Biochemistry 30, 8671], the residues Asp(253), Asp(273), Asp(275), and Asp(279) were selected as potential ligands of the divalent metal cation in the pig heart enzyme. Using a megaprimer PCR method, the Asp at each of these positions was mutated to Asn. The wild-type and mutant enzymes were expressed in Escherichia coli and purified. D253N has a specific activity, K(m) values for Mn(2+), isocitrate, and NADP, and also a pH-V(max) profile similar to those of the wild-type enzyme. Thus, Asp(253) is not involved in enzyme function. D273N has an increased K(m) for Mn(2+) and isocitrate with a specific activity 5% that of wild type. The D273N mutation also prevents the oxidative metal cleavage seen with Fe(2+) alone in the wild-type enzyme. As compared to wild type, D275N has greatly increased K(m) values for Mn(2+) and isocitrate, with a specific activity <0.1% that of wild type, and a large increase in pK(a) for the enzyme-substrate complex. D279N has only small increases in K(m) for Mn(2+) and isocitrate, but a specific activity <0.1% that of wild type and a major change in the shape of its pH-V(max) profile. These results suggest that Asp(273) and Asp(275) contribute to metal binding, whereas Asp(279), as well as Asp(275), is critical for catalysis. Asp(279) may function as the catalytic base. Using the Modeler program of Insight II, a structure for porcine NADP-isocitrate dehydrogenase was built based on the X-ray coordinates of the E. coli enzyme, allowing visualization of the metal-isocitrate site.     

Binding of N-acetyl-chitotriose to human lysozyme.

The interaction of N-acetyl-chitotriose ((GlcNAc)3) with human lysozyme [EC 3.2.1.17] was studied at various pH values by measuring changes in the circular dichroic (CD) band at 294 or 255 nm and the data were compared with the results for hen and turkey lysozymes reported previously (Kuramitsu et al. (1974) J. Biochem.76, 671-683; Kuramitsu et al. (1975) J. Biochem. 77, 291-301). The pH dependence of the binding constant of (GlcNAc)3 to human lysozyme was different from those for hen and turkey lysozymes. The catalytic carboxyls of human lysozyme, Asp 52 and Glu 35, were not perturbed on binding of (GlcNAc)3. This is consistent with the previous findings that the macroscopic pK values of Asp 52 and Glu 35 of human lysozyme are 3.4 and 6.8 at 0.1 ionic strength and 25 degrees and were unchanged on complexing with (GlcNAc)3. An ionizable group with pK 4.5, which participates in the binding of (GlcNAc)3 to hen lysozyme and was assigned as Asp 101, did not participate in the binding of the saccharide to human lysozyme. Between pH 9 and 11, the binding constants of (GlcNAc)3 to hen lysozyme remained unchanged, whereas perturbation of an ionizable group with pK 10.5 to 10.0 was observed for human lysozyme. This group may be Tyr 62 in the active-site cleft. The binding constants of (GlcNAc)3 to human lysozyme molecules having different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated using the binding constants obtained in the present experiments and the microscopic ionization constants of the catalytic carboxyls obtained previously. All four species of human lysozyme had similar binding constants to (GlcNAc)3. This result is different from those for hen and turkey lysozymes.     

Participation of active-site carboxylates of Escherichia coli DNA polymerase I (Klenow fragment) in the formation of a prepolymerase ternary complex

We have investigated the roles of four active-site carboxylates in the formation of a prepolymerase ternary complex of Escherichia coli DNA polymerase I (Klenow fragment), containing the template-primer and dNTP. The analysis of nine mutant enzymes with conserved and nonconserved substitutions of Asp(705), Glu(710), Asp(882), and Glu(883) clearly shows that both catalytically essential aspartates, Asp(705) and Asp(882), are required for the formation of a stable ternary complex. Of the two glutamates, only Glu(710) is required for ternary complex formation, while Glu(883) does not participate in this process. This investigation also reveals two interesting properties of the Klenow fragment with regard to enzyme-template-primer binary and enzyme-template-primer-dNTP ternary complex formation. These are (a) the significant resistance of enzyme-template-primer-dNTP ternary complexes to the addition of high salt or template-primer challenge and (b) the ability of the Klenow fragment to form ternary complexes in the presence of noncatalytic divalent cations such as Ca(2+), Co(2+), Ni(2+), and Zn(2+).     

Kinetics of association of anti-lysozyme monoclonal antibody D44.1 and hen-egg lysozyme

Association rate constants for antigen/antibody associations have been computed by Brownian Dynamics simulations of D. L. Ermak and J. A. McCammon, J. Chem. Phys. 69:1352-1360, 1978. The model of monoclonal antibody (mAb) D44.1 is based on crystallographic data (B. C. Braden et al., J. Mol. Biol. 243:767-781, 1994). Electrostatic forces that steer the antigen to the antibody-combining site are computed by solving the linearized Poisson-Boltzmann equation. D44. 1-HEL complex displays very similar association motifs to a related anti-lysozyme antibody, HyHEL-5-HEL system. The computed association rate constants are comparable in the two systems, although the experimental affinity constants differ by three orders of magnitude (D. Tello et al., Biochem. Soc. Trans. 21:943-946, 1993; K. A. Hibbits et al., Biochemistry. 33:3584-3590, 1994). Simulations suggest that the origin of the differences in the affinity come from dissociation rate constants. We have also carried out simulation experiments on a number of mutant antibody fragment-HEL associations to address the role of electrostatics and, to a limited extent, the orientational aspects of association.     

Catalyzed decomposition of urea. Molecular dynamics simulations of the binding of urea to urease

We present the results of molecular dynamics simulations on the urea/urease system. The starting structure was prepared from the 2.0 A crystal structure of Benini et al. [(1999) Struct. Folding Des. 7, 205-216] of DAP-inhibited urease (PDB code ), and the trimeric structure (2479 residues) resulted in 180K atoms after solvation by water. The force field parameters were derived using the bonded model approach described by Hoops et al. [(1991) J. Am. Chem. Soc. 113, 8262-8270]. Three different systems were analyzed, each one modeling a different protonation pattern for the His320 and His219 residues. In each case, the three monomers of urease have been analyzed separately. The time-averaged structures observed in the three monomers suggest that urease could follow two different competitive mechanisms. A "protein-assisted proton transfer" mechanism points to Asp221 as crucial for catalysis. An "Asp-mediated proton transfer" involves the transfer of a proton from the bridging OH to an NH2 moiety of urea, assisted by Asp360 in the active site. The impact of the simulation results on our understanding of urease catalysis is discussed in detail.