Contribution of a Salt Bridge to the Thermostability of Adrenodoxin Determined by Site-Directed Mutagenesis

2001 ◽  
Vol 396 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Asya V. Grinberg ◽  
Rita Bernhardt
Keyword(s):  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Molecular Mechanism of Action of Pharmacoperone Rescue of Misrouted GPCR Mutants: The GnRH Receptor

2009 ◽  
Vol 23 (2) ◽  
pp. 157-168 ◽  
Author(s):  
Jo Ann Janovick ◽  
Akshay Patny ◽  
Ralph Mosley ◽  
Mark T. Goulet ◽  
Michael D. Altman ◽  
...  
Keyword(s):  
Quality Control ◽  
Salt Bridge ◽  
Gnrh Receptor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Pharmacological Chaperones ◽  
Naturally Occurring ◽  
Protein Mutants ◽  
Molecular Mechanism Of Action ◽  
G Protein Coupled

Abstract The human GnRH receptor (hGnRHR), a G protein-coupled receptor, is a useful model for studying pharmacological chaperones (pharmacoperones), drugs that rescue misfolded and misrouted protein mutants and restore them to function. This technique forms the basis of a therapeutic approach of rescuing mutants associated with human disease and restoring them to function. The present study relies on computational modeling, followed by site-directed mutagenesis, assessment of ligand binding, effector activation, and confocal microscopy. Our results show that two different chemical classes of pharmacoperones act to stabilize hGnRHR mutants by bridging residues D98 and K121. This ligand-mediated bridge serves as a surrogate for a naturally occurring and highly conserved salt bridge (E90–K121) that stabilizes the relation between transmembranes 2 and 3, which is required for passage of the receptor through the cellular quality control system and to the plasma membrane. Our model was used to reveal important pharmacophoric features, and then identify a novel chemical ligand, which was able to rescue a D98 mutant of the hGnRHR that could not be rescued as effectively by previously known pharmacoperones.

scholarly journals Structure-function analysis of the Saccharomyces cerevisiae G1 cyclin Cln2.

1997 ◽  
Vol 17 (8) ◽  
pp. 4654-4666 ◽  
Author(s):  
K N Huang ◽  
S A Odinsky ◽  
F R Cross
Keyword(s):  
Function Analysis ◽  
Salt Bridge ◽  
Cyclin A ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Alanine Scanning Mutagenesis ◽  
The Third ◽  
Functional Assays ◽  
New Alleles ◽  
Made In

We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function.

The site-directed mutagenesis and prokaryotic expression of midkine gene in Cynoglossus semilaevis

2013 ◽  
Vol 37 (3) ◽  
pp. 330
Author(s):  
Yanan WANG ◽  
Xudong LIU ◽  
Linlin MU ◽  
Zhipeng LIU ◽  
Chunmei LI ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Cynoglossus Semilaevis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Site-directed mutagenesis on alkaline phosphatasefrompseudomonas aeruginosapao1 for enhanced activity

2016 ◽  
Vol 7 (4) ◽  
Author(s):  
UMA SELVARAJ ◽  
THIRUMALAI MUTHUKUMARESAN ◽  
GAYATHRI VIJAYENDRAN ◽  
SENTHIL KUMAR DEVAN ◽  
VENU BABU P ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Enhanced Activity
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