Thermally denatured state determines refolding in lipase: mutational analysis.


Abstract

Irreversibility of thermally denatured proteins due to aggregation limits thermodynamic characterization of proteins and also confounds the identification of thermostable mutants in protein populations. Identification of mutations that prevent the aggregation of unfolded proteins provides insights into folding pathways. In a lipase from Bacillus subtilis, evolved by directed evolution procedures, the irreversibility due to temperature-mediated aggregation was completely prevented by a single mutation, M137P. Though the parent and the mutants unfold completely on heating, mutants having substitutions M137P, along with M134E and S163P, completely or partially prevent the formation of aggregation-prone intermediate(s) at 75 degrees C. The three mutants show only a marginal increase in free energy of unfolding (DeltaG(H(2)O)), however, the profiles of the residual activity with temperature shows remarkable shift to higher temperature compared to parent. The intermediate(s) were characterized by enhanced binding of bis-ANS, a probe to titrate surface hydrophobicity, aggregation profiles and by estimation of soluble protein. Inclusion of salt in the refolding conditions prevents the reversibility of mutant having charge substitution, while the reversibility of mutant with the introduction of proline was unaffected, indicating the role of charge mediated interaction in M134E in preventing aggregation. Partial prevention of thermal aggregation in wild-type lipase with single substitution, M137P, incorporated by site-directed mutagenesis, suggests that the affect of M137P is independent of the intrinsic thermostability of lipase. Various effects of the mutations suggest their role is in prevention of the formation of aggregation prone intermediate(s). These mutations, describe yet another strategy to enhance the thermotolerance of proteins, where their influence is observed only on the denatured ensemble. Study holds ProTherm entries: 24955, 24956, 24957, 24958, 24959, 24960, 24961, 24962, 24963, 24964 Extra Details: lipase; site-saturation mutagenesis; thermal denaturation; aggregation; intermediate; bis-ANS binding; denatured ensemble

Submission Details

ID: SKUCVt3a

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:55 p.m.

Version: 1

Publication Details
Ahmad S;Rao NM,Protein Sci. (2009) Thermally denatured state determines refolding in lipase: mutational analysis. PMID:19472328
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1I6W 2001-03-05T00:00:00+0000 1.5 THE CRYSTAL STRUCTURE OF BACILLUS SUBTILIS LIPASE: A MINIMAL ALPHA/BETA HYDROLASE ENZYME
1ISP 2001-12-19T00:00:00+0000 1.3 Crystal structure of Bacillus subtilis lipase at 1.3A resolution
1R4Z 2003-10-09T00:00:00+0000 1.8 Bacillus subtilis lipase A with covalently bound Rc-IPG-phosphonate-inhibitor
1R50 2003-10-09T00:00:00+0000 1.45 Bacillus subtilis lipase A with covalently bound Sc-IPG-phosphonate-inhibitor
1T2N 2004-04-22T00:00:00+0000 1.8 Structure of a thermostable triple mutant of Bacillus subtilis lipase obtained through directed evolution
1T4M 2004-04-30T00:00:00+0000 2.0 STRUCTURE OF A THERMOSTABLE DOUBLE MUTANT OF BACILLUS SUBTILIS LIPASE OBTAINED THROUGH DIRECTED EVOLUTION
2QXT 2007-08-13T00:00:00+0000 2.0 Crystal Structure Analysis of the Bacillus subtilis lipase crystallized at pH 4.5
2QXU 2007-08-13T00:00:00+0000 1.9 Crystal Structure Analysis of the Bacillus subtilis lipase crystallized at pH 5.0
3D2A 2008-05-08T00:00:00+0000 1.73 Structure of 1-17A4, a thermostable mutant of Bacillus subtilis lipase obtained through directed evolution
3D2B 2008-05-08T00:00:00+0000 1.95 Structure of 2D9, a thermostable mutant of Bacillus subtilis lipase obtained through directed evolution

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Lipase EstA P37957 ESTA_BACSU