Document Detail


Comprehensive analysis of surface charged residues involved in thermal stability in Alicyclobacillus acidocaldarius esterase 2.
MedLine Citation:
PMID:  23035254     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Here we report a comprehensive analysis through alanine-scanning mutagenesis of the contribution of surface ion pairs to the thermal stability of Alicyclobacillus acidocaldarius esterase 2 (EST2). We produced 16 single mutants, 4 double mutants corresponding to selected ion pairs R31/E118, E43/K102, R58/D130, D145/R148, 2 double mutants (R63A/R98A and E50A/D94A) involving residues of a large ion network on the protein surface and the double-mutant R98A/R148A meant to disrupt the R98 interactions within the said network and, contextually, the interaction between R148 and D145. The double-mutant E43A/E273K was obtained by chance. All selected residues were replaced with alanine except E91, which was mutated to a glycine and K102, which was changed to a glutamine. All 24 proteins were over-expressed in Escherichia coli, purified and characterized with respect to the main features. Structural stability data were compared with an in silico prediction of ΔΔG values. Our study of the individual factors involved in thermostability and their structural interpretation reveals that the great stability of this thermophilic protein can be explained by the contribution of a few residues at the protein surface.
Authors:
Margherita Pezzullo; Pompea Del Vecchio; Luigi Mandrich; Roberto Nucci; Mosè Rossi; Giuseppe Manco
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-10-3
Journal Detail:
Title:  Protein engineering, design & selection : PEDS     Volume:  -     ISSN:  1741-0134     ISO Abbreviation:  Protein Eng. Des. Sel.     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-4     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101186484     Medline TA:  Protein Eng Des Sel     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Istituto di Biochimica delle Proteine-Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy.
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