Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding.


Abstract

Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 2525, 2526, 2527, 2528, 2529, 2530, 2531, 14027, 14028, 14029, 14030, 14031, 14032 Extra Details: additive : EDTA(10 mM), subtilisin BPN'; amino acid substitutions; hydrophobic; structural changes;,thermodynamic stability ; electrostatic interactions

Submission Details

ID: dtvShhFm

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:19 p.m.

Version: 1

Publication Details
Pantoliano MW;Whitlow M;Wood JF;Dodd SW;Hardman KD;Rollence ML;Bryan PN,Biochemistry (1989) Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding. PMID:2684274
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
1A2Q 1998-01-08T00:00:00+0000 1.8 SUBTILISIN BPN' MUTANT 7186
1AK9 1997-05-30T00:00:00+0000 1.8 SUBTILISIN MUTANT 8321
1AQN 1997-07-31T00:00:00+0000 1.8 SUBTILISIN MUTANT 8324
1AU9 1997-09-12T00:00:00+0000 1.8 SUBTILISIN BPN' MUTANT 8324 IN CITRATE
1DUI 2000-01-17T00:00:00+0000 2.0 Subtilisin BPN' from Bacillus amyloliquefaciens, crystal growth mutant
1GNS 2001-10-06T00:00:00+0000 1.8 SUBTILISIN BPN'
1GNV 2001-10-10T00:00:00+0000 1.9 CALCIUM INDEPENDENT SUBTILISIN BPN' MUTANT
1LW6 2002-05-30T00:00:00+0000 1.5 Crystal Structure of the Complex of Subtilisin BPN' with Chymotrypsin Inhibitor 2 at 1.5 Angstrom Resolution
1S01 1989-08-21T00:00:00+0000 1.7 LARGE INCREASES IN GENERAL STABILITY FOR SUBTILISIN BPN(PRIME) THROUGH INCREMENTAL CHANGES IN THE FREE ENERGY OF UNFOLDING
1S02 1991-02-20T00:00:00+0000 1.9 EFFECTS OF ENGINEERED SALT BRIDGES ON THE STABILITY OF SUBTILISIN BPN'

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Subtilisin BPN' P00782 SUBT_BACAM