Combinations of turns in proteins.

We observed that beta- and gamma-turns in protein structure may be associated as peptides representing combinations of turns that span between nine and 26 amino acid residues along the polypeptide backbone chain and often correspond to loops in the protein structure. Around 475 peptides resulted from the analysis of a non-redundant data set corresponding to 248 protein crystal structures selected from the Protein Data Bank. Nearly 40% protein chains are associated with two or more peptides and the peptides with nine and 10 amino acid residues are more frequent. A maximum of four distinct peptides varying in number of amino acid residues were observed in at least 10 proteins along the same protein chain. Nearly 80% peptides comprise type IV beta-turns that are associated with irregular dihedral angle values suggesting this may be important for the conformational diversity associated with the loops in proteins. In general, predominant interactions that possibly stabilize these peptides involve main-chain and side-chain interactions with solvent, in addition to hydrogen bond, salt-bridge and non-bonded interactions. Majority of the peptides were observed in hydrolase, oxidoreductase, transferase, serine proteinase/inhibitor complex, electron transport/electron transfer and lyase proteins.

Interspecies hybrid HbS: complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains.

Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitro from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O(2) affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O(2) affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The (1)H-NMR spectroscopy detected distinct differences between the heme environments and alpha(1)beta(1) interfaces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; the pig beta-chain dictated the conformation of the heme environment of the human alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the conformation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)beta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in the cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dimer. The semisynthetically generated chimeric pig-human and human-pig alpha-chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic potential energy surfaces on the alpha-chain of alpha(2)(P)beta(2)(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig alpha-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric alpha-chains can serve as blueprints for the design of a new generation of variants of alpha-chain(s) suitable for the gene therapy of sickle cell disease.

Chimeric hemoglobins--hybrids of human and swine hemoglobin: assembly and stability of interspecies hybrids.

Transgenic swine expressing human HbA contained only one of two types of the anticipated interspecies hybrids, namely H alpha 2 P beta 2 (H = human, P = swine). In an attempt to establish whether the absence of the swine alpha and human beta (P alpha 2 H beta 2) hybrid in vivo is a reflection of the lack of complementarity between the interspecies chains to generate appropriate interfaces, we have undertaken the in vitro assembly of swine alpha and human beta chimeric tetramer. In contrast to the in vivo transgenic swine system, in vitro the hybrid of swine alpha human beta chain is assembled readily and the hybrid exhibits normal cooperative oxygen binding. Both the swine alpha human beta and the human alpha swine beta interspecies hybrids are stable around neutral pH and do not segregate into parent tetramers even when mixed together. On the other hand, nearly complete exchange of P alpha chain of P alpha 2 H beta 2 hybrid occurs in the presence of H alpha chain at pH 6.0 and room temperature, resulting in the formation of HbA. However, very little of such an exchange reaction takes place at pH 7.0. These results suggest that the thermodynamic stability of P alpha 2 H beta 2 hybrid is lower compared to that of HbA. In contrast, P beta chain of H alpha 2 P beta 2 hybrid is refractory to exchange with H beta chain at pH 7.0 as well as at pH 6.0, suggesting that the stability of H alpha 2 P beta 2 is higher compared to that of HbA (H alpha 2 H beta 2). The swine alpha human beta chimeric Hb undergoes subunit exchange reaction with human alpha-chain in the presence of 0.9 M MgCl2, at pH 7.0. This demonstrates the lower thermodynamic stability of the intradimeric interactions of the heterodimer even at neutral pH. A synergistic coupling of the intra- and interdimeric interactions of the swine alpha and human beta chain heterodimer is essential for the thermodynamic stability of the chimeric Hb under the physiological conditions. Accordingly, we speculate that the lower thermodynamic stability of P alpha H beta heterodimer (compared to the homodimers H alpha H beta and P alpha P beta) facilitates its segregation into the homodimers by subunit exchange reaction involving either H alpha or P beta. This molecular aspect by itself or possibly along with other cellular aspects of the swine system results in the absence of P alpha 2 H beta 2 hybrid in transgenic swine expressing HbA.

Polymerization of hemoglobin S. Quinary interactions of Glu-43(beta).

Hemoglobin S (HbS) Hoshida and three substituted forms of HbS Hoshida (the substituents being on the amide nitrogen of Gln-43(beta) have been prepared by the amidation of Glu-43(beta) of HbS with ammonia, methylamine, glycine ethyl ester, and galactosamine. The O2 affinity of HbS is increased slightly on amidation of Glu-43(beta). All the four amidated derivatives exhibited nearly the same oxygen affinity. On the other hand, the influence of amidation on the solubility exhibits some sensitivity to the chemical nature of the substituent on the Gln-43(beta). The solubility of HbS Hoshida (a case with no substitution on Gln-43(beta), and the methyl-substituted derivatives are about 33 and 36% higher than that of HbS. The solubility of the HbS modified with the glycine ethyl ester or galactosamine is increased to 41 and 47%, respectively. The first derivative UV spectra of HbS Hoshida and its methyl derivative reflect very little perturbations in their alpha 1 beta 2 interface as compared with that of HbS, whereas the amidated derivatives with larger substituents on Gln-43(beta) reflected noticeable difference. Thus, the increase in the solubility and the oxygen affinity of HbS on the amidation of Glu-43(beta) is primarily a consequence of the loss of the negative charge at 43(beta), a residue proximal to the alpha 1 beta 2 interface. The copolymerization studies of amidated HbS with HbA, and HbS with amidated HbA demonstrate that cis Glu-43(beta) is the "active" residue. This assignment is discrepant with the earlier implication of a trans configuration for this residue in the polymer (Edelstein, S. J. (1981) J. Mol. Biol. 150, 557-575). However, it is consistent with the solution studies of Nagel et al. (Nagel, R. L., Bookchin, R. M., Johnson, J., Labie, D., Wajcman, H.., Isaac-Sodeye, W. A., Honig, G. R., Schiliro, G., Crookstan, J. H., and Matsutomo, K. (1979) Proc. Nat. Acad. Sci. U.S.A. 76, 670-672) and McCurdy et al. (McCurdy, P. R., Lorkin, P. A., Casey, R., Lehmann, H., Uddin, D. E., and Dickson, L. G. (1974) Am. J. Med. 57, 665-760).

Recombinant hemoglobin A produced in transgenic swine: structural equivalence with human hemoglobin A.

Recombinant human hemoglobin A produced by coexpressing human alpha and beta globin genes in swine, and purified from the lysate of transgenic swine has been subjected to detailed protein chemical analysis. These structural studies involving laser desorption mass spectrometry, separation of globin chains by RPHPLC, amino terminal sequence analysis of the isolated globin chains, the tryptic peptide mapping of the purified globin chains and the amino acid composition analysis of the purified tryptic peptides of globin chains have established the primary structural equivalence of the globin chains of the transgenic swine derived hemoglobin A with that of human hemoglobin A. These results demonstrate that the transgenic swine system correctly translates the human alpha and beta globin m-RNA; carries out the correct cotranslational processing of globin chains, and does not introduce any unwanted post translational modifications into the mature chains. Thus, the transgenic swine expression system is an excellent approach for the production of HbA for developing an effective hemoglobin based oxygen carrier.

Contribution of the gamma-carboxyl group of Glu-43(beta) to the alkaline Bohr effect of hemoglobin A.

Glu-43(beta) of hemoglobin A exhibits a high degree of chemical reactivity around neutral pH for amidation with nucleophiles in the presence of carbodiimide. Such a reactivity is unusual for the side-chain carboxyl groups of proteins. In addition, the reactivity of Glu-43(beta) is also sensitive to the ligation state of the protein [Rao, M. J., & Acharya, A. S. (1991) J. Protein Chem. 10, 129-138]. The influence of deoxygenation of hemoglobin A on the chemical reactivity of the gamma-carboxyl group of Glu-43(beta) has now been investigated as a function of pH (from 5.5 to 7.5). The chemical reactivity of Glu-43(beta) for amidation increases upon deoxygenation only when the modification reaction is carried out above pH 6.0. The pH-chemical reactivity profile of the amidation of hemoglobin A in the deoxy conformation reflects an apparent pKa of 7.0 for the gamma-carboxyl group of Glu-43(beta). This pKa is considerably higher than the pKa of 6.35 for the oxy conformation. The deoxy conformational transition mediated increase in the pKa of the gamma-carboxyl group of Glu-43(beta) implicates this carboxyl group as an alkaline Bohr group. The amidated derivative of hemoglobin A with 2 mol of glycine ethyl ester covalently bound to the protein was isolated by CM-cellulose chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Antifungal potential of binary and mixed-ligand complexes of N,2'-diphenyl acetohydroxamic acid.

Chelating potential of N,2'-DPAHA with 3d metal ions such as Cu(II), Ni(II), Zn(II), and Cd(II) in the presence of Gly and Phen has been investigated. These experiments were designed to study the role of the stability of mixed-ligand complexes in the modulation of its fungicidal potential. The mixed-ligand complexes were found to be more stable than binary complexes. Enhanced stability of mixed-ligand complexes of Ni(II), Co(II), Zn(II), and Cd(II) is presumably due to pi-bonding effects. In the stabilization of the Cu(II) mixed-ligand complex system, the Jahn-Tellar effect may play a vital role, in addition to pi-bonding effects. Fungicidal activity of N,2'-DPAHA and its binary complexes with Cu(II), Ni(II), and Co(II) was examined against Fusarium oxysporum using the inhibition zone technique. Binary complexes of Zn(II) and Cd(II) with N,2'-DPAHA and mixed-ligand complexes M(II)-Gly or Phen-N,2'-DPAHA, where M(II) = Cu(II), Ni(II), Zn(II), Co(II), and Cd(II) were screened against Alternaria alternata by slide germination technique. All mixed-ligand complexes exhibited fungicidal activity but did not improve significantly compared to binary complexes. Synergistic action of primary and secondary ligands has increased the stability of the mixed-ligand complex compared to the binary complex (1:1) of the secondary ligand (N,2'-DPAHA), and the fungicidal potential of the mixed-ligand complex involving N,2'-DPAHA as secondary ligand was not increased.

Basic carboxyl groups of hemoglobin S: influence of oxy-deoxy conformation on the chemical reactivity of Glu-43(beta).

The gamma-carboxyl groups of Glu-43(beta) and Glu-22(beta) of hemoglobin-S (HbS), two intermolecular contact residues of deoxy protein, are activated by carbodiimide at pH 6.0. The selectivity of the modification by the two nucleophiles, glycine ethyl ester (GEE) and glucosamine, is distinct. Influence of N-hydroxysulfosuccinimide, a reagent that rescues carbodiimide-activated carboxyl (O-acyl isourea) as sulfo-NHS ester, on the overall selectivity and efficiency of the coupling of Glu-22(beta) and Glu-43(beta) with nucleophiles has been investigated. Sulfo-NHS increases the extent of coupling of nucleophiles to HbS. The rescuing efficiency of sulfo-NHS(increase in modification) with GEE and galactosamine as nucleophiles is 2.0 and 2.8, respectively. In the presence of sulfo-NHS, the extent of modification of a carboxyl group is a direct reflection of the extent to which it is activated (i.e., the protonation state of the carboxyl group). The modification reaction exhibits very high selectivity for Glu-43(beta) with GEE and galactosamine (GA) in the presence of sulfo-NHS. From the studies of the kinetics of amidation of oxy-HbS at its Glu-43(beta) (i.e., chemical reactivity) as a function of the pH in the region of 5.5-7.5, the apparent pKa of its gamma-carboxyl group has been calculated to be 6.35. Deoxygenation of HbS, nearly doubles the chemical reactivity of Glu-43(beta) of HbS at pH 7.0. It is suggested that the increased hydrophobicity of the microenvironment of Glu-43(beta), which occurs on deoxygenation of the protein, is reflected as the increased chemical reactivity of the gamma-carboxyl group and could be one of the crucial preludes to the polymerization process.

Geometry of proline and hydroxyproline I: An analysis of X-ray crystal structure data.

We have carried out an analysis of crystal structure data on prolyl and hydroxyprolyl moieties in small molecules. The flexibility of the pyrrolidine ring due to the pyramidal character of nitrogen has been defined in terms of two projection angles delta 1 and delta 2. The distribution of these parameters in the crystal structures is found to be consistent with results of the energy calculations carried out on prolyl moieties in our laboratory.

Synthesis and spectroscopic and fungicidal characterization of hydroxamic acids and their metal chelates.

Hydroxamic acid chelates of the type ML2, ML2', and ML2" where M = Cu(II), Ni(II) or Co(II) and L = N,2'-diphenylacetohydroxamic acid (N,2'-DPAHA), L' = 2,2'-diphenylacetohydroxamic acid (2,2'-DPAHA), and L" = 2-phenylacetohydroxamic acid (2-PAHA) have been isolated and characterized on the basis of elemental analysis and infrared and magnetic data. These metal chelates were screened for their fungicidal activity. The testing against fungi has been carried out by slide germination technique against Alternaria alternata and by inhibition zone technique against Fusarium oxysporum and Aspergillus flavus. The fungicidal activity of chelates and their parent ligand has been compared with the commercial fungicide, Dithane M-45, screened under similar conditions.