scholarly journals Comprehensive Alanine-scanning Mutagenesis of Escherichia coli CsrA Defines Two Subdomains of Critical Functional Importance

2006 ◽  
Vol 281 (42) ◽  
pp. 31832-31842
Author(s):  
Jeffrey Mercante ◽  
Kazushi Suzuki ◽  
Xiaodong Cheng ◽  
Paul Babitzke ◽  
Tony Romeo
Keyword(s):  
Escherichia Coli ◽  
Functional Importance ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning

scholarly journals Systematic alanine scanning of PAX8 paired domain reveals functional importance of the N-subdomain

2019 ◽  
Vol 62 (3) ◽  
pp. 129-135 ◽  
Author(s):  
Megumi Iwahashi ◽  
Satoshi Narumi
Keyword(s):  
Structure Function ◽  
Congenital Hypothyroidism ◽  
Loss Of Function ◽  
Paired Domain ◽  
Functional Importance ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Pathogenicity Prediction ◽  
Pax Family

Thyroid-specific transcription factor PAX8 has an indispensable role in the thyroid gland development, which is evidenced by the facts thatPAX8/Pax8mutations cause congenital hypothyroidism in humans and mice. More than 90% of knownPAX8mutations were located in the paired domain, suggesting the central role of the domain in exerting the molecular function. Structure-function relationships of PAX8, as well as other PAX family transcription factors, have never been investigated in a systematic manner. Here, we conducted the first alanine scanning mutagenesis study, in which 132 alanine variants located in the paired domain of PAX8 were created and systematically evaluatedin vitro. We found that 76 alanine variants (55%) were loss of function (LOF) variants (defined by <30% activity as compared with wild type PAX8). Importantly, the distribution of LOF variants were skewed, with more frequently observed in the N-subdomain (65% of the alanine variants in the N-subdomain) than in the C-subdomain (45%). Twelve out of 13 alanine variants in residues that have been affected in patients with congenital hypothyroidism were actually LOF, suggesting that the alanine scanning data can be used to evaluate the functional importance of mutated residues. Using ourin vitrodata, we tested the accuracy of seven computational algorithms for pathogenicity prediction, showing that they are sensitive but not specific to evaluate on the paired domain alanine variants. Collectively, our experiment-based data would help better understand the structure-function relationships of the paired domain, and would provide a unique resource for pathogenicity prediction of futurePAX8variants.

scholarly journals Comprehensive Alanine-scanning Mutagenesis ofEscherichia coliCsrA Defines Two Subdomains of Critical Functional Importance

2006 ◽  
Vol 281 (42) ◽  
pp. 31832-31842 ◽  
Author(s):  
Jeffrey Mercante ◽  
Kazushi Suzuki ◽  
Xiaodong Cheng ◽  
Paul Babitzke ◽  
Tony Romeo
Keyword(s):  
Functional Importance ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning
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