Delineation via site-directed mutagenesis of the carboxyl-terminal region of human choriogonadotropin beta required for subunit assembly and biological activity.

The v-sis oncogene and its cellular homolog c-sis encode chain B of platelet-derived growth factor. Cells transformed by v-sis produce a platelet-derived growth factor-related molecule which is able to stimulate the platelet-derived growth factor receptor in an autocrine fashion. Site-directed mutagenesis was used to construct several mutations which substitute charged residues for hydrophobic residues in the proposed signal sequence of the v-sis gene product. Two of these mutations resulted in the synthesis of altered v-sis gene products with an unexpected nuclear location and a loss of biological activity. We also report here the intracellular localization of the v-sis gene product to the endoplasmic reticulum-Golgi compartment, where signal sequence cleavage and N-linked glycosylation occur. The v-sis gene product contains no transmembrane regions, as it is completely protected within isolated microsomes from trypsin proteolysis. Site-directed mutagenesis was also used to alter a proposed proteolytic processing site in the v-sis gene product. This mutant v-sis gene, which encodes Asn-Ser in place of Lys-Arg at residues 110 to 111, was found to retain full biological activity.

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Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2

Biochemical Journal ◽

10.1042/bj20020092 ◽

2002 ◽

Vol 366 (3) ◽

pp. 971-976 ◽

Cited By ~ 53

Author(s):

Lucimara CHIOATO ◽

Arthur H.C. de OLIVEIRA ◽

Roberto RULLER ◽

Juliana M. SÁ ◽

Richard J. WARD

Keyword(s):

Phospholipase A2 ◽

Lipid Membrane ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Phospholipid Bilayer ◽

Directed Mutagenesis ◽

Aromatic Residues ◽

Loop Region ◽

Terminal Loop ◽

Arginine Residues

Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca2+-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117—122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys122, these same substitutions did not significantly alter the Ca2+-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca2+-independent membrane-damaging activity but, with the exception of Lys122, had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115—119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A2, and support a model of a Ca2+-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.

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Carboxyl-Terminal Truncation and Site-Directed Mutagenesis of the EF Hand Structure-Domain of the Small Subunit of Rabbit Calcium-Dependent Protease1

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a122585 ◽

1988 ◽

Vol 104 (6) ◽

pp. 927-933 ◽

Cited By ~ 30

Author(s):

Yasufumi Minami ◽

Yasufumi Emori ◽

Shinobu Imajoh-Ohmi ◽

Hiroshi Kawasaki ◽

Koichi Suzuki

Keyword(s):

Small Subunit ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Calcium Dependent ◽

Ef Hand ◽

Carboxyl Terminal ◽

Structure Domain

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Domain mapping of a claudin-4 modulator, the C-terminal region of C-terminal fragment of Clostridium perfringens enterotoxin, by site-directed mutagenesis

Biochemical Pharmacology ◽

10.1016/j.bcp.2007.12.016 ◽

2008 ◽

Vol 75 (8) ◽

pp. 1639-1648 ◽

Cited By ~ 51

Author(s):

Azusa Takahashi ◽

Eriko Komiya ◽

Hideki Kakutani ◽

Takeshi Yoshida ◽

Makiko Fujii ◽

...

Keyword(s):

Clostridium Perfringens ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Clostridium Perfringens Enterotoxin ◽

Terminal Fragment ◽

Domain Mapping

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Involvement of various amino- and carboxyl-terminal residues in the active site of the histidine-containing protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus: site-directed mutagenesis with the ptsH gene, biochemical characterization and NMR studies of the mutant proteins

Protein Engineering Design and Selection ◽

10.1093/protein/6.4.417 ◽

1993 ◽

Vol 6 (4) ◽

pp. 417-423 ◽

Cited By ~ 12

Author(s):

R. Kruse ◽

W. Hengstenberg ◽

W. Beneicke ◽

H.R. Kalbitzer

Keyword(s):

Active Site ◽

Biochemical Characterization ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Phosphotransferase System ◽

Nmr Studies ◽

Mutant Proteins ◽

Staphylococcus Carnosus ◽

Carboxyl Terminal

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Evidence for crucial electrostatic interactions between Bcl-2 homology domains BH3 and BH4 in the anti-apoptotic Nr-13 protein

Biochemical Journal ◽

10.1042/bj20020836 ◽

2002 ◽

Vol 368 (1) ◽

pp. 213-221 ◽

Cited By ~ 8

Author(s):

Philippe LALLE ◽

Abdel AOUACHERIA ◽

Agnès DUMONT-MISCOPEIN ◽

Martin JAMBON ◽

Séverine VENET ◽

...

Keyword(s):

Energy Minimization ◽

Electrostatic Interactions ◽

Family Members ◽

Biological Significance ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Conserved Domains ◽

Apoptotic Activity ◽

Interacting Domain

Nr-13 is an anti-apoptotic member of the Bcl-2 family previously shown to interact with Bax. The biological significance of this interaction was explored both in yeast and vertebrate cells and revealed that Nr-13 is able to counteract the pro-apoptotic activity of Bax. The Bax-interacting domain has been identified and corresponds to α-helices 5 and 6 in Nr-13. Site-directed mutagenesis has revealed that the N-terminal region of Nr-13 is essential for activity and corresponds to a genuine Bcl-2 homology domain (BH4). The modelling of Nr-13, based on its similarity with other Bcl-2 family proteins and energy minimization, suggests the possibility of electrostatic interactions between the two N-terminal-conserved domains BH4 and BH3. Disruption of these interactions severely affects Nr-13 anti-apoptotic activity. Together our results suggest that electrostatic interactions between BH4 and BH3 domains play a role in the control of activity of Nr-13 and a subset of Bcl-2 family members.

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Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin

Applied and Environmental Microbiology ◽

10.1128/aem.69.7.3777-3783.2003 ◽

2003 ◽

Vol 69 (7) ◽

pp. 3777-3783 ◽

Cited By ~ 61

Author(s):

Christiane Szekat ◽

Ralph W. Jack ◽

Dirk Skutlarek ◽

Harald Färber ◽

Gabriele Bierbaum

Keyword(s):

Biological Activity ◽

Expression System ◽

Biosynthetic Gene Cluster ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Lipid Ii ◽

Type Gene ◽

Self Protection ◽

Acid Function ◽

Reduced Activity

ABSTRACT The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.

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