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Sperka T Boross P Eizert H Tözsér J Bagossi P 《Protein engineering, design & selection : PEDS》2006,19(8):369-375
To explore the role of residues being close to the catalytic aspartates in the higher pH optimum and in the lower dimer stability of human foamy virus (HFV) protease (PR) in comparison with human immunodeficiency virus type 1 (HIV-1) protease, single (Q8R, H22L, S25T, T28D) and double (Q8R-T28D, H22L-T28D) mutants were created based on sequence alignments and on the molecular model of HFV PR. The wild-type and mutant enzymes were expressed in fusion with maltose binding protein in Escherichia coli and the fusion proteins were purified by affinity chromatography. Specificity constant of most mutants was lower, but the value of Q8R-T28D double mutant enzyme was higher than that of the wild-type HFV PR. Furthermore, urea denaturation at two pH values and pH optimum values showed an increased stability and pH optimum for most mutants. These results suggest that the mutated residues may not be responsible for the higher pH optimum of HFV PR, but they may contribute to the lower dimer stability as compared with that of HIV-1 PR. 相似文献
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Miklóssy G Tözsér J Kádas J Ishima R Louis JM Bagossi P 《Protein engineering, design & selection : PEDS》2008,21(7):453-461
An intracellularly expressed defective human immunodeficiency virus type-1 (HIV-1) protease (PR) monomer could function as a dominant-negative inhibitor of the enzyme that requires dimerization for activity. Based on in silico studies, two mutant PRs harboring hydrophilic mutations, capable of forming favorable inter- and intra-subunit interactions, were selected: PR(RE) containing Asp25Arg and Gly49Glu mutations, and PR(RER) containing an additional Ile50Arg mutation. The mutants were expressed and tested by PR assays, nuclear magnetic resonance (NMR) and cell culture experiments. The mutant PRs showed dose-dependent inhibition of the wild-type PR in a fluorescent microtiter plate PR assay. Furthermore, both mutants were retained by hexahistidine-tagged wild-type HIV-1 PR immobilized on nickel-chelate affinity resin. For the first time, heterodimerization between wild-type and dominant-negative mutant PRs were also demonstrated by NMR spectroscopy. (1)H-(15)N Heteronuclear Single Quantum Coherence NMR spectra showed that although PR(RE) has a high tendency to aggregate, PR(RER) exists mainly as a folded monomer at 25-35 microM concentration, but in the presence of wild-type PR in a ratio of 1:1, heterodimerization occurs with both mutants. While the recombinant virus containing the PR(RE) sequence showed only very low level of expression, expression of the viral proteins of the virus with the PR(RER) sequence was comparable with that of the wild-type. In cell culture experiments, infectivity of viral particles containing PR(RER) protein was reduced by 82%, at mutant to wild-type infective DNA ratio of 2:1. 相似文献
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Bagossi P; Cheng YS; Oroszlan S; Tozser J 《Protein engineering, design & selection : PEDS》1998,11(6):439-445
The specificity of linked homo- and heterodimeric HIV-1 and HIV-2
proteinases was characterized by using oligopeptide substrates. For two
substrates the k(cat)/Km values for the heterodimers were the mean values
for those of the homodimers, suggesting that these substrates could
productively bind into the heterodimers in both directions. However, for
two other substrates the k(cat)/Km values for the heterodimers were higher
than those of the homodimers, suggesting that these substrates could
productively bind into the enzymes in a preferable direction. However, the
mode of binding does not seem to depend on the sequential position of the
subunits. The studied linked homo- and heterodimers may represent
intermediate stages in the evolution of bilobal aspartic proteinases. As
divergence in sequence of the two halves of such a proteinase increases,
the possibility of bidirectional binding is likely lost at the expense of
the optimized side-chain subsite interactions. The differences in observed
and calculated k(cat)/Km values revealed dependence of the substrate
specificity at one subsite of the enzyme from the next residue in sequence
of substrate. These findings were also supported by molecular modeling
studies.
相似文献
4.
Bagossi Peter; Cheng Yin-Shyun E.; Oroszlan Stephen; Tozser Jozsef 《Protein engineering, design & selection : PEDS》1996,9(11):997-1003
Mutations were introduced into the active site triplet (AspThrGly)of one or both subunits of a linked dimer of human immunodeficiencyvirus type 1 proteinase. Mutation of Thr to Ser in one or bothsubunits did not alter the activity of the enzyme substantially,whereas its mutation to Asn in one subunit caused a dramaticdecrease in catalytic efficiency. Mutation of Gly to Val inone subunit also yielded an enzyme with very low activity. Theenzymes containing Thr Asn and Gly Val mutations in both subunitsresulted in inactive enzymes, based on their inability to self-processand on assay with an oligopeptide substrate. The dramatic decreasein enzyme efficiency of the mutants was interpreted using molecularmodels of the enzymes. 相似文献
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