Synthesis and acid ionization constants of cyclic cystine peptides H-Cys-(Gly)n-Cys-OH (n = 0-4)

Cyclic peptide disulfides of the general formula H-Cys-(Gly)n-Cys-OH (n = 0-4) were synthesized from the corresponding peptide derivatives [Boc-Cys(Trt)(Gly)-n-Cys(Trt)-OBut] by oxidation with iodine in methanol and by subsequent removal of the terminal groups with trifluoroacetic acid. Acid ionization constants of the obtained peptides were determined by potentiometric titration in aqueous KCl (0.1 mol/L) medium. All compounds have two dissociable hydrogens, corresponding to carboxyl (pK1 = 2.35-2.84) and to terminal amino group (pK2 = 5.61-6.93); pK1 values show first an upward and then a downward trend with the increase in ring size; the opposite is true for pK2 values. These trends could be tentatively attributed to the intramolecular salt bridge (-COO- ----NH+3-) formation.     

Application of the two-dimensional nmr techniques on a des(Ala,Gly)-somatostatin analog

Two-dimensional nmr techniques have been carried out for the peak assignment of the spectrum of a somatostatin analog. Two-dimensional J-resolved spectroscopy simplified the rather broad and complicated spectrum to show the center of chemical shifts of each resonance and gave information on the coupling profiles. Another technique, two-dimensional spin-echo correlated spectroscopy, revealed the connectivities between protons which are correlated by weak spin–spin couplings. The combination of the results of these two complementary techniques made it possible for us to assign almost all peaks of the spectrum of the 11-residue somatostatin analog.     

Conformations of proline-containing cyclic peptides. 1H and 13C n.m.r. evidence for a solvent stabilized All-Cis X-Pro conformer of Cyclo-(Pro-Gly-Gly-Pro)2

As inferred from 13C, 1H n.m.r. data, CD measurements and ion-binding experiments, the title molecule can assume two major C2 symmetric conformations. One of these has an all-trans X-Pro peptide backbone and two 1 comes from 4 intramolecular H-bonds and represents the predominant (greater than or equal to 95%) form in D2O and nonpolar (CD3CN) solvents. Stabilized by specific solvent-solute interactions, the other conformer becomes competitive (45%) in DMSO solution. It is shown to possess a four-cis X-Pro skeleton and no intramolecular H-bonds. The Mg++ complex of the cyclic peptide in CD3CN is again C2 symmetric and its formation proceeds via a slow trans leads to cis isomerization of two X-Pro peptide bonds.     

Conformation dynamics of cyclic disulfides and selenosulfides in CXXC(U) (X = Gly,Ala) tetrapeptide redox motifs

Thioredoxin fold proteins often contain a Cys‐(Xxx)_n‐Cys(Sec) or CX_nC(U) motif, where the active cysteine (C) or selenocysteine (U) is bridged by X residues, which vary with protein function. The effect of the X residues on the conformation space of the oxidized disulfide and selenosulfide forms of the CXXC(U) motif has been investigated using molecular dynamics (MD) and density functional theory. Multi‐microsecond‐length MD simulations of the CGGC, CGAC, and CAGC cyclic peptides show that CGGC rings readily exchange between several conformations over the course of the simulation, but steric interactions with the methyl group of Ala limit the conformation space available to the cyclic peptide, especially for CGAC. The potential for the motif to be reduced, as measured by the energy of the lowest unoccupied molecular orbitals, is dependent upon the ring conformation. These results suggest that control of available conformations by the bridging residues and the protein tertiary structure may be important for defining the function of the CXXC motif. Theoretical ⁷⁷Se chemical shifts of the selenosulfide moiety are dependent upon the conformation and/or intramolecular Se···O interactions with the backbone carbonyl group of the C‐terminal U residue.     

Cisplatin mediates selective downstream hydrolytic cleavage of Met-(Gly)(n)-His segments (n=1,2) in methionine- and histidine-containing peptides: the role of ammine loss trans to the initial Pt-S(Met) anchor in facilitating amide hydrolysis

The pH- and time-dependent reactions of the antitumor drug cisplatin, cis-[PtCl(2)(NH(3))(2)], with the methionine- and histidine-containing pentapeptides Ac-Met-Gly-His-Gly-Gly-OH, Ac-Met-Gly-Gly-His-Gly-OH and Ac-Gly-Met-Gly-His-Gly-OH (Gly=glycyl, Met=L-methionyl, His=L-histidyl) at 313K have been investigated by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. Cisplatin mediates a rapid "downstream" hydrolytic cleavage of the Met-Gly amide bond in weakly acid solution (pH < or =5) for all three peptides, leading to release of H-Gly-His-Gly-Gly-OH, H-Gly-Gly-His-Gly-OH and H-Gly-His-Gly-OH, respectively, and formation of kappa(2)S,N(M) chelate complexes of the methionine-containing residuals Ac-Met-OH or Ac-Gly-Met-OH. An alternative reaction pathway affords tridentate kappa(3)S,N(M),N(imidazole) macrochelates of the original pentapeptide following ammine loss. The downstream cleavage pathway is competitive with the likewise cisplatin-mediated upstream cleavage of the Ac-Gly linkage in the pentapeptide Ac-Gly-Met-Gly-His-Gly-OH. This leads to formation of both the kappa(3)S,N(M),N(G1) complex of H-Gly-Met-Gly-His-Gly-OH due to upstream cleavage and the analogous tridentate complex for H-Gly-Met-OH due to initial downstream loss of H-Gly-His-Gly-OH followed by upstream loss of acetic acid. As downstream cleavage is not observed for Ac-(Gly)(2)-Met-(Gly)(2)-OH under similar conditions, it may be concluded that rapid histidine imidazole substitution of the ammine ligand in trans-position to an anchoring methionine S atom must assist hydrolytic cleavage of the Met-Gly amide bond.     

Resin effects in solid phase S(n)Ar and S(n)2 macrocyclizations

Seven different supports were compared in solid-phase S(N)Ar and S(N)2 macrocyclization reactions. Product purities were assayed for a relatively facile ring-closure process to give products 1 and 3. Some less-facile ring-closure reactions give the undesired dimeric macrocyclization by-products 2; some of these more-demanding ring closures were also examined. Finally, experiments were performed to gauge the rate of cyclizations on different resins, and some qualitative data were obtained for this.     

Novel mutations (Asn 484 Lys,Thr 500 Ala,Gly 438 Glu) in Morquio B disease

Primary deficiency of beta-galactosidase results in GM1 gangliosidosis and Morquio B disease. Of the more than 40 disease-causing mutations described in the Gal gene to date, about 75% are of the missense type and are scattered along the length of the gene. No single, major common mutation has been associated with GM1 gangliosidosis. However, a Trp 273 Leu mutation has been commonly found in the majority of patients with Morquio B disease defined genotypically to date.We now report three new mutations in three Morquio B patients where the Trp 273 Leu mutation is absent. Two of the mutations, C1502G (Asn 484 Lys) and A1548G (Thr 500 Ala), were found in twins (one male, one female) who display a mild form of Morquio B disease and keratan sulfate in the urine. In their fibroblasts, residual activity was 1.9% and 2.1% of controls. On Western blots, the 84-kDa precursor and the 64-kDa mature protein were barely detectable. The occurrence of a 45-kDa degradation product indicates that the mutated protein reached the lysosome but was abnormally processed. In the third case, we identified only a G1363A (Gly 438 Glu) mutation (a major deletion on the second allele has not been ruled out). This female patient too displays a very mild form of the disease with a residual activity of 5.7% of control values. In fibroblasts from this case, the 84-kDa precursor and the 45-kDa degradation product were present, while the mature 64-kDa form was barely detectable. The occurrence of these three mutations in the same area of the protein may define a domain involved in keratan sulfate degradation.     

Conformations of model peptides in membrane-mimetic environments.

The influence of a membrane environment on the conformational energetics of a polypeptide chain has been investigated through studies of model peptides in a variety of membrane-mimetic media. Nuclear magnetic resonance (NMR) and circular dichroism (CD) data have been obtained for the peptides in bulk hydrophobic solvents, normal micelles, and reversed micelles. Several hydrophobic peptides which are sparingly soluble in water have been solubilized in aqueous sodium dodecyl sulfate (SDS) solution. NMR and CD data indicate that the micelle-solubilized peptides experience an environment with the conformational impact of bulk methanol, and have decreased conformational freedom. The site of residence of the peptides interacting with the micelles appears to be near the surfactant head groups, in a region permeated by water, and not in the micelle core. Strongly hydrophilic peptides have been solubilized in nonpolar solvents by reversed micelles. These peptides are located in small water pools in close association with the head groups of the surfactant. NMR and CD data show that there is a conformational impact of this interfacial water region on peptide solubilizates distinct from that of bulk water.     

Characterization of divalent cation localization in the minor groove of the A(n)T(n) and T(n)A(n) DNA sequence elements by (1)H NMR spectroscopy and manganese(II)

The localization of Mn(2+) in A-tract DNA has been studied by (1)H NMR spectroscopy using a series of self-complementary dodecamer oligonucleotides that contain the sequence motifs A(n)(n) and T(n)A(n), where n = 2, 3, or 4. Mn(2+) localization in the minor groove is observed for all the sequences that have been studied, with the position and degree of localization being highly sequence-dependent. The site most favored for Mn(2+) localization in the minor groove is near the 5'-most ApA step for both the T(n)A(n) and the A(n)T(n) series. For the T(n)A(n) series, this results in two closely spaced symmetry-related Mn(2+) localization sites near the center of each duplex, while for the A(n)T(n) series, the two symmetry-related sites are separated by as much as one half-helical turn. The degree of Mn(2+) localization in the minor groove of the T(n)A(n) series decreases substantially as the AT sequence element is shortened from T(4)A(4) to T(2)A(2). The A(n)T(n) series also exhibits length-dependent Mn(2+) localization; however, the degree of minor groove occupancy by Mn(2+) is significantly less than that observed for the T(n)A(n) series. For both A(n)T(n) and T(n)A(n) sequences, the 3'-most AH2 resonance is the least broadened of the AH2 resonances. This is consistent with the observation that the minor groove of A-tract DNA narrows in the 5' to 3' direction, apparently becoming too narrow after two base pairs for the entry of a fully hydrated divalent cation. The results that are reported illustrate the delicate interplay that exists between DNA nucleotide sequence, minor groove width, and divalent cation localization. The proposed role of cation localization in helical axis bending by A-tracts is also discussed.     

Mutations of Arg440 and Gly455/Gly456 oppositely change pH sensing of Na+/H+ exchanger 1

To identify important amino acid residues involved in intracellular pH (pH(i)) sensing of Na(+)/H(+) exchanger 1, we produced single-residue substitution mutants in the region of the exchanger encompassing the putative 11th transmembrane segment (TM11) and its adjacent intracellular (intracellular loop (IL) 5) and extracellular loops (extracellular loop 6). Substitution of Arg(440) in IL5 with other residues except positively charged Lys caused a large shift in pH(i) dependence of (22)Na(+) uptake to an acidic side, whereas substitution of Gly(455) or Gly(456) within the highly conserved glycine-rich sequence of TM11 shifted pH(i) dependence to an alkaline side. The observed alkaline shift was larger with substitution of Gly(455) with residues with increasing sizes, suggesting the involvement of the steric effect. Interestingly, mutation of Arg(440) (R440D) abolished the ATP depletion-induced acidic shift in pH(i) dependence of (22)Na(+) uptake as well as the cytoplasmic alkalinization induced by various extracellular stimuli, whereas with that of Gly(455) (G455Q) these functions were preserved. These mutant exchangers did not alter apparent affinities for extracellular transport substrates Na(+) and H(+) and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride. These results suggest that positive charge at Arg(440) is required for normal pH(i) sensing, whereas mutation-induced perturbation of the TM11 structure may be involved in the effects of Gly mutations. Thus, both Arg(440) in IL5 and Gly residues in the conserved segment of TM11 appear to constitute important elements for proper functioning of the putative "pH(i) sensor" of Na(+)/H(+) exchanger 1.     

Mutations in Ser174 and the glycine-rich sequence (Gly149, Gly150, and Thr156) in the beta subunit of Escherichia coli H(+)-ATPase.

A sequence motif in the beta subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta Gly149 by Ser suppressed the effect of the beta Ser174----Phe mutation (defective H(+)-ATPase), but beta Gly150----Ser substitution did not have this effect. A single mutation (beta Gly149----Ser or beta Gly150----Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta Gly149----Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca(2+)-dependent activity, but had the wild-type level of Mg(2+)-dependent activity with active oxidative phosphorylation. Introduction of a beta Gly149----Ser or beta Gly150----Ser mutation with the beta Ser174----Phe mutation also lowered the Ca(2+)-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta Thr156----Ala or Cys mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity. The possible location of the catalytic site including divalent cation binding site(s) is discussed.     

The use of phase diagrams in the crystallization of oligonucleotide duplexes. I. A model of crystallization of the (pGpT)n.(pApC)n + spermine system

A set of experimental phase diagrams revealing the region of existence of microcrystals in mixture "(pGpT)n.(pApC)n+spermine", n = 2,3,4, was obtained. All diagrams are wedge-like with the slope of the upper branch and the level of the lower one depending on the oligonucleotidd length. The presence of MPD, MgCl2 and NaCl changes the form of the diagrams in a different manner. A model explaining the peculiar features of the diagrams for mixture "oligonucleotide duplex+spermine" is proposed. The analysis of the diagrams was carried out on the basis of this model and the values of the binding constants for binding of spermine and Mg2+ to duplexes were estimated. Some conclusions about the types of complexes, which may form microcrystals in different regions of diagrams were made.     

Conformations of copoly(gamma-benzyl L-glutamate-O-benzyl-L-serine)

Four samples of copolymers of γ-benzyl is L -glutamic (BLG) and O-benxyl-L -serine (BLS) were synthesized by changing the mixing ratio of two monomer anhydrides. The conformations of these copolymers in CHCl₃, containing very small amount of dichloroacetic acid (DCA) necessary to dissolve the sample, were determined by their compositions; the copolymer which is rich in BLG is mostly helical and one which is rich in BLS is mostly in the β conformation. However, in DCA solution the conformations of these copolymers are independent of their composition. The interesting observation was that the copolymer which has least con tent of BLS is richest in β-conformation.     

Conformations and pharmacophores of cyclic RGD containing peptides which selectively bind integrin alpha(v)beta3.

This paper reports a detailed conformational characterization in solution by 1H-NMR in H2O and DMSO-d6 and molecular modeling simulations of cyclic peptides containing the RGDDV pharmacophore and the RGDY(Me)R pharmacophore. These two pentapeptide sequences when properly constrained in cyclic peptides are low to sub-nanomolar inhibitors of integrin alpha(v)beta3. The peptides containing the RGDDY(Me)R sequence bind potently to integrin alphaIIb3 as well. The conformations found in H2O and in DMSO-d6 solutions are valuable for the design of peptidomimetics of these two pharmacophores. The structure-activity relationships of the RGDDV and RGDY(Me)R pharmacophores within cyclic peptides are discussed. Specifically, the orientation of surface-accessible chemical features on the ligand, such as hydrophobic, positive and negative ionizable groups, which are considered to be responsible for the desired biological activity, is focused on.     

Isolation of novel tRNA(Ala) mutants by library selection in a tRNA(Ala) knockout strain

The relationship between tRNA structure and function has been widely investigated by site-directed mutagenesis. This method has been a very useful tool to reveal the critical bases in tRNAs that are important for recognition and aminoacylation, but has been limited by the large number of possible base combinations in tRNA molecules. We have devised a new method that uses tRNA knockout cells for selection of functional tRNAs from a mutant tRNA gene library to overcome this limitation. To explore the mechanism of tRNA(Ala) recognition, the bases of the acceptor-stem region were randomized and active mutants were selected in a tRNA(Ala) knockout strain. Mutants of tRNA(Ala) having diverse sequence combinations in the acceptor-stem region and a broad range of functional activity to support knockout cell growth were isolated. The mutant tRNAs selected by the method included molecules containing novel base substitutions as well as extensively altered base combinations that would not be readily generated by rationally designed site-directed mutagenesis. Our results emphasize the importance of the acceptor stem as a structural unit in which some nucleotides may carry more weight than others, but in summation every nucleotide contributes to the interaction with the enzyme.     

Coordination and thermodynamics of stable Zn(II) complexes in the gas phase

Ab initio molecular orbital methods in combination with DFT calculations were used to study the structural and thermodynamic properties of 17 complexes containing zinc cation and four first-shell ligands as models of active site of metalloenzymes (e.g. angiotensin converting enzyme, thermolysin). The geometry of the complexes was relaxed by complete optimization by ab initio molecular orbital methods at Hertree-Fock level with 3-21G* basis set. Following single point calculation with tight SCF criteria at the B3LYP level with 6-311+G(2d,p) basis set was used to calculate accurate interaction enthalpies. The structure and thermodynamics of optimized complexes are discussed from the point of view of their biological importance.