Characterization of the functional role of a flexible loop in the .alpha.-subunit of tryptophan synthase from Salmonella typhimurium by rapid-scanning, stopped-flow spectroscopy and site-directed mutagenesis

Biochemistry ◽  
1993 ◽  
Vol 32 (39) ◽  
pp. 10404-10413 ◽  
Author(s):  
Peter S. Brzovic ◽  
C. Craig Hyde ◽  
Edith W. Miles ◽  
Michael F. Dunn
Keyword(s):  
Functional Role ◽  
Site Directed Mutagenesis ◽  
Alpha Subunit ◽  
Directed Mutagenesis ◽  
Stopped Flow ◽  
Tryptophan Synthase ◽  
Flexible Loop ◽  
Rapid Scanning

Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis

Biochemistry ◽  
1991 ◽  
Vol 30 (46) ◽  
pp. 11092-11103 ◽  
Author(s):  
Mark S. Warren ◽  
Katherine A. Brown ◽  
Martin F. Farnum ◽  
Elizabeth E. Howell ◽  
Joseph Kraut
Keyword(s):  
Escherichia Coli ◽  
Dihydrofolate Reductase ◽  
Functional Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Probing the functional role of phenylalanine-31 of Escherichia coli dihydrofolate reductase by site-directed mutagenesis

Biochemistry ◽  
1987 ◽  
Vol 26 (13) ◽  
pp. 4093-4100 ◽  
Author(s):  
Jin Tann Chen ◽  
Kazunari Taira ◽  
Chen Pei D. Tu ◽  
Stephen J. Benkovic
Keyword(s):  
Escherichia Coli ◽  
Dihydrofolate Reductase ◽  
Functional Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Characterization of Recombinant von Willebrand Factors Mutated on Cysteine 509 or 695

1996 ◽  
Vol 76 (03) ◽  
pp. 453-459 ◽  
Author(s):  
Virginie Siguret ◽  
Anne-Sophie Ribba ◽  
Olivier Christophe ◽  
Ghislaine Chérel ◽  
Bernadette Obert ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Mutant Proteins ◽  
Von Willebrand ◽  
High Molecular Weight Multimers

SummaryThe interacting domain of vWF with platelet GPIb has been shown to overlap the large A1 loop formed by the intra-chain disulfide bond linking Cys 509 to Cys 695. In order to further investigate the role of the conformation of this region, we have expressed in COS-7 cells three mutated full-length recombinant vWFs (rvWFs) in which the substitutions Cys509Gly, Cys509Arg or Cys695Gly have been introduced by site-directed mutagenesis. SDS-agarose gel electrophoresis demonstrated an impaired multimerization of the mutants with undetectable high molecular weight multimers and a decrease of the relative amounts of the intermediate sized multimers. Binding analysis showed that rvWFC509G and rvWFC509R did not interact with botrocetin but spontaneously interacted with GPIb; the latter binding remained unchanged in the presence of ristocetin. This indicates that the substitution of Cys509 by Gly or Arg creates a conformation of vWF that increases its binding to GPIb. In contrast, rvWFC695G which did not react with botrocetin was also unable to interact with GPIb even in the presence of ristocetin, indicating that sequences interacting with GPIb are masked and/or disrupted. In conclusion, the substitution of each of the Cys509 and 695 results in mutant proteins which may be “locked” into active or inactive conformations in regard to the binding to platelet GPIb receptor.

The Functional Role of Positively Charged Amino Acid Side Chains in α-Bungarotoxin Revealed by Site-Directed Mutagenesis of a His-Tagged Recombinant α-Bungarotoxin†

Biochemistry ◽  
1999 ◽  
Vol 38 (24) ◽  
pp. 7847-7855 ◽  
Author(s):  
Julie A. Rosenthal ◽  
Mark M. Levandoski ◽  
Belle Chang ◽  
Jerald F. Potts ◽  
Qing-Luo Shi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Functional Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Side Chains ◽  
Amino Acid Side Chains ◽  
Positively Charged
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