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Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis 总被引:16,自引:2,他引:14
Mariagrazia Pizza Mario Domenighini Wim Hol Valentine Giannelli Maria Rita Fontana Marzia M. Giuliani Claudia Magagnoli Samuele Peppoloni Roberto Manetti Rino Rappuoli 《Molecular microbiology》1994,14(1):51-60
Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases. 相似文献
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The ClpXP ATP-Dependent Protease Regulates Flagellum Synthesis in Salmonella enterica Serovar Typhimurium 总被引:6,自引:0,他引:6
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Toshifumi Tomoyasu Tomiko Ohkishi Yoshifumi Ukyo Akane Tokumitsu Akiko Takaya Masato Suzuki Kachiko Sekiya Hidenori Matsui Kazuhiro Kutsukake Tomoko Yamamoto 《Journal of bacteriology》2002,184(3):645-653
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Identification of amino acids in the gamma-carboxylation recognition site on the propeptide of prothrombin 总被引:2,自引:0,他引:2
P Huber T Schmitz J Griffin M Jacobs C Walsh B Furie B C Furie 《The Journal of biological chemistry》1990,265(21):12467-12473
A gamma-carboxylation recognition site on the propeptide of the vitamin K-dependent blood coagulation proteins directs the carboxylation of glutamic acid residues by binding to the vitamin K-dependent carboxylase. To determine residues that define this site, we evaluated the effect of mutation of certain residues in the prothrombin propeptide on the extent of carboxylation. The prothrombin cDNA modified by site-specific mutagenesis was expressed in Chinese hamster ovary cells using a system that yields functional fully carboxylated prothrombin. The cell supernatants containing recombinant prothrombin were evaluated for the extent of gamma-carboxylation by immunoassay. Conformation-specific anti-prothrombin:Ca(II)-specific antibodies measure native completely carboxylated prothrombin; anti-prothrombin:total antibodies measure all forms of prothrombin, regardless of gamma-carboxyglutamic acid content. Mutation of His-18 to Gly, Val-17 to Ser, Leu-15 to Gly or Asp, or Ala-10 to Asp was associated with a partial (30-65%) inhibition of gamma-carboxylation. Mutation of Ala-14 to Ser or Ser-8 to Val did not inhibit gamma-carboxylation. From this and earlier work, residues whose mutation leads to a significant impairment of carboxylation include His-18, Val-17, Phe-16, Leu-15, and Ala-10. Residues whose mutation does not alter the carboxylation recognition site include Ala-14, Ser-8, Arg-4, and Arg-1. To determine the size of the recognition site, the in vitro carboxylation of propeptide-containing synthetic peptides was compared. A 28-residue peptide, based upon residues -18 to +10 of prothrombin, and a 54-residue peptide, based upon residues -18 to +36 of prothrombin, were carboxylated by partially purified bovine carboxylase with similar Km values of 2-5 microM. These results indicate that the gamma-carboxyglutamic acid-rich region of prothrombin makes a minimal contribution to carboxylase binding. A molecular surface of about five amino acids located within the propeptide appears to define the carboxylation recognition site on the precursor forms of the vitamin K-dependent proteins. 相似文献
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Putative transmembrane helix 3 of the tetracycline/H+ antiporter encoded by a transposon, Tn10, contains four serine residues, Ser-77, Ser-82, Ser-91 and Ser-92. Each of these serine residues was replaced by site-directed mutagenesis. Of these four serine residues, Ser-77 was important for the transport function, and a bulky side chain at position 91 hindered substrate translocation, whereas Ser-82 and Ser-92 did not play any role. Ser-77 and Ser-91 are on the same vertical stripe, that includes the essential Asp-84, on the hydrophilic side of putative helix 3. These observations suggest that helix 3 is part of the tetracycline translocation channel across the membrane. 相似文献
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Megan K Townsend Nathan J Carr Jyoti G Iyer Shelley M Horne Penelope S Gibbs Birgit M Prüß 《BMC microbiology》2008,8(1):12