Molecular cloning and expression in Escherichia coli of a gene coding for bovine S100A1 protein and its Glu32-->Gln and Glu73-->Gln mutants.

Calcium binding S100A1 protein consists of two S100 alpha subunits. On the basis of sequence homology to other S100 proteins it is believed that the binding loops are formed by amino-acid residues 19-32 and 62-73 of S100 alpha polypeptide chain. In the oxidized form of the protein the subunits are linked covalently with each other by a disulphide bond between their Cys85 residues. A synthetic gene coding for bovine S100 alpha subunit was constructed and cloned into a derivative of pAED4 plasmid. The gene was expressed in Escherichia coli utilizing the T7 expression system. The expression products were purified and identified using mass spectrometry and by sequencing of their N- and C-termini. Three different forms (a, b, and c) of S100 alpha were produced: with the native sequence, with the initiator methionine at the N-terminus, and with an additional alanine at the C-terminus as well as with the initiator methionine. The material was partly oxidized. Interestingly, only the homodimers of a, b, and c species were formed. The total yield of the protein was about 50 mg/l of culture. Genes coding for Glu32-->Gln and Glu73-->Gln mutants of S100 alpha were obtained by site-directed mutagenesis and expressed in the same system. In both cases similar mixtures of oxidized and reduced a, b, and c species have been obtained. The total yield of E73Q mutant is similar to that of the native protein and that of E32Q lower by about a half. As expected, the mutants of S100 alpha subunits bind only one calcium ion.

Synthesis, cloning and expression in Escherichia coli of a gene coding for the Met8-->Leu CMTI I--a representative of the squash inhibitors of serine proteinases.

A chemically synthesized gene coding for a Cucurbita maxima trypsin inhibitor modified at position P'3 (Met8-->Leu CMTI I), i.e. at the third position downstream of the reactive site bond (Arg5-Ile), was cloned into a derivative of the plasmid pAED4 that utilizes a T7 expression system. The gene was expressed in Escherichia coli as a fusion protein that accumulates in inclusion bodies. After reduction and CNBr cleavage of the fusion protein followed by oxidative refolding and reverse-phase HPLC, about 5 mg of pure protein was obtained per 1 of cell culture. Association constants of recombinant Leu-8-CMTI I with bovine beta-trypsin and human cathepsin G are the same, within experimental error, as for CMTI I isolated from a natural source.

Indoleglycerol phosphate synthase-phosphoribosyl anthranilate isomerase: comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains.

Putative domain--domain interactions of the monomeric bifunctional enzyme indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase from Escherichia coli were probed by separating the domains on the gene level and expressing them as monofunctional proteins. The engineered monofunctional enzymes were found to be stable, monomeric proteins with virtually full catalytic activity. In addition, binding of indolyglycerol phosphate to the active site of indoleglycerol phosphate synthase and binding of reduced 1-[(2-carboxyphenyl)amino]-1-deoxyribulose 5-phosphate, a competitive inhibitor of both indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, were almost identical in both the mono- and bifunctional enzymes. Furthermore, no association between the monofunctional enzymes was found, neither in vitro, by sedimentation and gel filtration experiments, nor in vivo, by coexpression of the domains in the same cell. Thus, no selective advantages of the bifunctional enzyme from Escherichia coli over the respective monofunctional enzymes were found on a functional level. However, the phosphoribosyl anthranilate isomerase domain appears to stabilize the indoleglycerol phosphate synthase domain of the bifunctional enzyme from Escherichia coli by interactions that seem to subtly influence the kinetics of ligand binding.

Deoxydodecanucleotide heteroduplex d(TTTTATAATAAA). d(TTTATTATAAAA) containing the promoter Pribnow sequence TATAAT. I. Double-helix stability by UV spectrophotometry and calorimetry.

Thermally induced structural transition in the d(TTTTATAATAAA) d(TTTATTATAAAA) heteroduplex is characterized by UV-spectroscopy and differential scanning calorimetry. At low salt (less than 0.1 M) the occurrence of a cooperative transition in the lower temperature range, followed by a broad transition connected with small increase in absorbance is observed. At high salt (greater than or equal to 0.2 M) a single, monophasic transition appears. Linear dependence of the latter on log of salt concentration (dTm:dlogM = 14.2 degrees C) and of 1/Tm on log of oligomer concentration [derived therefrom delta H (v.H.) = 77.1 kcal/mole (duplex)] allows relating it to the melting of the heteroduplex helix. The non-cooperative transition, independent of oligomer concentration and similar to that of the single chain, was attributed to melting of short hairpin helices upon heteroduplex dissociation. Calorimetric enthalpy: 75.6 kcal/mole (duplex) proved significantly lower than predicted from known calorimetric data for poly[d(AT)] and poly d(A) X poly d(T).

Fluorescence quenching and spin label electron spin resonance studies of stacking self-association in aqueous solutions of 2-aminopurine riboside and its 5'-mono- and -diphosphate.

The autoassociation of 2-aminopurine riboside (rn2Pur) and its 5'-mono- (P-rn2Pur) and 5'-diphosphate (PP-rn2Pur) in neutral aqueous solutions was investigated using fluorescence quenching and ESR spin-label methods within the range 276-358 K. Respective equilibrium constants and thermodynamic functions were derived therefrom assuming two models of infinite autoassociation: (i) an isodesmic one (K2 = K3 = ... Kp), and (ii) one in which K2 no equal to K2 = K4 ... Kp. Comparative analysis of these data and that of the parent 2-aminopurine, obtained previously, allowed us to formulate the following conclusions: (1) the mechanism of autoassociation of rn2Pur varies with temperature in such a way that a T = 318 K the isodesmic model is fulfilled (K2 = Kp); at high temperatures Kp/K2 greater than 1, i.e. the process is cooperative, while at lower temperatures it becomes anticooperative (Kp/K2 greater less than 1); (2) at 298 K the tendency to autoassociation decreases in the order; rn2Pur greater than P-rn2Pur greater than PP-rn2Pur; (3) rn2Pur forms highly packed complexes with the bases stacked and the ribofuranose residues interacting via hydrogen bonds or water bridges; (4) autoassociation of P-rn2Pur and PP-rn2Pur is mainly governed by stacking of the bases, while the ribose phosphate residues attain a trans configuration corresponding to the lowest electrostatic repulsion between charged phosphate groups; even at high ionic strength (I = 0.8), a positive electrostatic contribution to the free enthalpy of autoassociation is observed; (5) the two methods employed gave similar results for P-rn2Pur, but somewhat different ones for rn2Pur because the presence of the spin label (nitroxide stable radical) at the 2'(3')-OH group of the ribose residues prevents its interaction via hydrogen bonding with an unlabeled one of an adjacent nucleoside.

Stacking self-association of pyrimidine nucleosides and of cytosines: effects of methylation and thiolation.

Stacking self-association equilibria in aqueous solutions of m3uridine, m34,2',3',5'uridine, 2'-deoxyuridine, m13,4,4cytosine, m14,4,4,5cytosine, s2cytidine and s4thymidine were studied at various temperatures by vapour-pressure osmometry. Equilibrium constants Kst's were computed on the assumption of the isodesmic model of self-association. Enthalpies of association were also obtained from the temperature dependence of Kst according to the van't Hoff equation. Analysis of the equilibrium and thermodynamic parameters demonstrated involvement of hydrophobic interactions in the stabilization of complexes of tetramethyluridine. Dipole-induced dipole interactions seem to predominate in the formation of s2C, s4T and of both dimethylaminocytosine complexes.

Osmometric studies on self-association of pyrimidines in aqueous solutions: evidence for involvement of hydrophobic interactions.

Vapour pressure osmometric studies were performed on stacking self-association of 25 uracil derivatives variously C- and N-substituted with polar and alkyl groups in aqueous solution at various temperatures. The respective equilibrium association constants Kst were computed on the assumption of the isodesmic model of self-association (K2=K3=...=Kn=Kst). Enthalpies of association for most of the compounds studied were obtained from the temperature-dependence of Kst, according to the van 't Hoff equation. Analysis of the equilibrium and thermodynamic parameters in terms of the association mechanism demonstrated the involvement of classical hydrophobic interactiors in the stabilization of complexes of di-and higher alkylated uracils. Data for the derivatives substituted with polar groups proved consistent with the predominant involvement of dipole-induced dipole forces in the association.

Barrier to rotation and conformation of the -NR2 group in cytosine and its derivatives. Part II. Experimental and theoretical dipole moments of methylated cytosines.

The dipole moments of several cytosine, methylaminocytosine and dime-thylaminocytosine derivatives with and without an ortho methyl group were determined experimentally in dioxane and benzene. Calculations of total energies and dipole moments were performed by the CNDO/2 and INDO methods for sp2 and sp3 hybridization of exocyclic nitrogen for different values of rotational angle phiC-N. Comparison of the experimental dipole moments with those calculated for the energy minima suggests that the conformation of the dimethylamino group is not planar and differs from that found in cytosine. 1,5,7-Trimethylcytosine, with the dipole moment of 7 Deby units, was considered to be the model compound which closely reproduces the dipole moment of cytosine.