Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond.


Abstract

Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in a 1.3-A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1 degrees C higher than that of the wild-type protein and 5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiments performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers. Study holds ProTherm entries: 3031, 3032, 3033, 3034 Extra Details: additive : EDTA(10 mM), subtilisin BPN'; disulfide bond; protein engineering;,cysteine; statistical thermodynamics; stabilization

Submission Details

ID: X4NSdDWh

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:20 p.m.

Version: 1

Publication Details
Pantoliano MW;Ladner RC;Bryan PN;Rollence ML;Wood JF;Poulos TL,Biochemistry (1987) Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond. PMID:3476160
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
1TO2 2004-11-09 1.3 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59K, in pH 9 cryosoak
1TM5 2004-11-09 1.45 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59A mutant
1V5I 2005-03-08 1.5 Crystal structure of serine protease inhibitor POIA1 in complex with subtilisin BPN'
1LW6 2002-08-21 1.5 Crystal Structure of the Complex of Subtilisin BPN' with Chymotrypsin Inhibitor 2 at 1.5 Angstrom Resolution
1Y34 2005-05-17 1.55 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 E60A mutant
1Y1K 2005-05-17 1.56 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 T58A mutant
1TM3 2004-11-09 1.57 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59k mutant
1TM7 2004-11-09 1.59 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59Y mutant
1Y4A 2005-05-17 1.6 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59R/E60S mutant
1SUP 1995-11-14 1.6 SUBTILISIN BPN' AT 1.6 ANGSTROMS RESOLUTION: ANALYSIS OF DISCRETE DISORDER AND COMPARISON OF CRYSTAL FORMS
1TMG 2004-11-09 1.67 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59F mutant
1TO1 2004-11-09 1.68 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 Y61A mutant
1Y3C 2005-05-17 1.69 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 R62A mutant
1TM1 2004-11-09 1.7 CRYSTAL STRUCTURE OF THE COMPLEX OF SUBTILISIN BPN' WITH CHYMOTRYPSIN INHIBITOR 2
1S01 1990-10-15 1.7 LARGE INCREASES IN GENERAL STABILITY FOR SUBTILISIN BPN(PRIME) THROUGH INCREMENTAL CHANGES IN THE FREE ENERGY OF UNFOLDING
3F49 2008-12-16 1.7 Anion-triggered Engineered Subtilisin SUBT_BACAM
1TM4 2004-11-09 1.7 crystal structure of the complex of subtilsin BPN'with chymotrypsin inhibitor 2 M59G mutant
3CNQ 2008-05-06 1.71 Prosubtilisin Substrate Complex of Subtilisin SUBT_BACAM
1Y3F 2005-05-17 1.72 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 F69A mutant
1SUB 1994-01-31 1.75 CALCIUM-INDEPENDENT SUBTILISIN BY DESIGN
1SUC 1994-01-31 1.8 CALCIUM-INDEPENDENT SUBTILISIN BY DESIGN
1Y33 2005-05-17 1.8 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 T58P mutant
1AU9 1997-12-31 1.8 SUBTILISIN BPN' MUTANT 8324 IN CITRATE
1SUE 1998-10-14 1.8 SUBTILISIN BPN' FROM BACILLUS AMYLOLIQUEFACIENS, MUTANT
2ST1 1991-07-15 1.8 THE THREE-DIMENSIONAL STRUCTURE OF BACILLUS AMYLOLIQUEFACIENS SUBTILISIN AT 1.8 ANGSTROMS AND AN ANALYSIS OF THE STRUCTURAL CONSEQUENCES OF PEROXIDE INACTIVATION
1A2Q 1998-04-29 1.8 SUBTILISIN BPN' MUTANT 7186
3BGO 2008-06-03 1.8 Azide complex of Engineered Subtilisin SUBT_BACAM
3SIC 1994-01-31 1.8 MOLECULAR RECOGNITION AT THE ACTIVE SITE OF SUBTILISIN BPN': CRYSTALLOGRAPHIC STUDIES USING GENETICALLY ENGINEERED PROTEINACEOUS INHIBITOR SSI (STREPTOMYCES SUBTILISIN INHIBITOR)
1AQN 1998-01-14 1.8 SUBTILISIN MUTANT 8324
1Y3D 2005-05-17 1.8 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 R67A mutant
1YJB 1996-07-11 1.8 SUBTILISIN BPN' 8397+1 (E.C. 3.4.21.14) (MUTANT WITH MET 50 REPLACED BY PHE, ASN 76 REPLACED BY ASP, GLY 169 REPLACED BY ALA, GLN 206 REPLACED BY CYS, ASN 218 REPLACED BY SER AND LYS 256 REPLACED BY TYR) (M50F, N76D, G169A, Q206C, N218S, AND K256Y) IN 35% DIMETHYLFORMAMIDE
2SIC 1993-04-15 1.8 REFINED CRYSTAL STRUCTURE OF THE COMPLEX OF SUBTILISIN BPN' AND STREPTOMYCES SUBTILISIN INHIBITOR AT 1.8 ANGSTROMS RESOLUTION
1Y3B 2005-05-17 1.8 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 E60S mutant
1AK9 1997-11-12 1.8 SUBTILISIN MUTANT 8321
1GNS 2002-06-27 1.8 SUBTILISIN BPN'
1SBH 1995-12-07 1.8 SUBTILISIN BPN' 8397+1 (E.C. 3.4.21.14) MUTANT (M50F, N76D, G169A, Q206C, N218S, K256Y)
1YJC 1996-07-11 1.8 SUBTILISIN BPN' 8397+1 (E.C. 3.4.21.14) (MUTANT WITH MET 50 REPLACED BY PHE, ASN 76 REPLACED BY ASP, GLY 169 REPLACED BY ALA, GLN 206 REPLACED BY CYS, ASN 218 REPLACED BY SER AND LYS 256 REPLACED BY TYR) (M50F, N76D, G169A, Q206C, N218S, AND K256Y) IN 50% DIMETHYLFORMAMIDE
1YJA 1996-07-11 1.8 SUBTILISIN BPN' 8397+1 (E.C. 3.4.21.14) (MUTANT WITH MET 50 REPLACED BY PHE, ASN 76 REPLACED BY ASP, GLY 169 REPLACED BY ALA, GLN 206 REPLACED BY CYS, ASN 218 REPLACED BY SER AND LYS 256 REPLACED BY TYR) (M50F, N76D, G169A, Q206C, N218S, AND K256Y) IN 20% DIMETHYLFORMAMIDE
1Y48 2005-05-17 1.84 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 R65A mutant
1GNV 2002-06-24 1.9 CALCIUM INDEPENDENT SUBTILISIN BPN' MUTANT
1S02 1992-01-15 1.9 EFFECTS OF ENGINEERED SALT BRIDGES ON THE STABILITY OF SUBTILISIN BPN'
1SUD 1994-01-31 1.9 CALCIUM-INDEPENDENT SUBTILISIN BY DESIGN
3CO0 2008-10-14 1.93 Substrate Complex of Fluoride-sensitive Engineered Subtilisin SUBT_BACAM
1DUI 2000-01-28 2.0 Subtilisin BPN' from Bacillus amyloliquefaciens, crystal growth mutant
1SPB 1995-10-15 2.0 SUBTILISIN BPN' PROSEGMENT (77 RESIDUES) COMPLEXED WITH A MUTANT SUBTILISIN BPN' (266 RESIDUES). CRYSTAL PH 4.6. CRYSTALLIZATION TEMPERATURE 20 C DIFFRACTION TEMPERATURE-160 C
1Y4D 2005-05-17 2.0 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59R/E60S mutant
1ST2 1991-07-15 2.0 THE THREE-DIMENSIONAL STRUCTURE OF BACILLUS AMYLOLIQUEFACIENS SUBTILISIN AT 1.8 ANGSTROMS AND AN ANALYSIS OF THE STRUCTURAL CONSEQUENCES OF PEROXIDE INACTIVATION
1SBN 1994-01-31 2.1 REFINED CRYSTAL STRUCTURES OF SUBTILISIN NOVO IN COMPLEX WITH WILD-TYPE AND TWO MUTANT EGLINS. COMPARISON WITH OTHER SERINE PROTEINASE INHIBITOR COMPLEXES
2SNI 1988-09-07 2.1 STRUCTURAL COMPARISON OF TWO SERINE PROTEINASE-PROTEIN INHIBITOR COMPLEXES. EGLIN-C-SUBTILISIN CARLSBERG AND CI-2-SUBTILISIN NOVO
1SUA 1998-01-14 2.1 SUBTILISIN BPN'
1SBI 1995-12-07 2.2 SUBTILISIN BPN' 8397 (E.C. 3.4.21.14) MUTANT (M50F, N76D, G169A, Q206C, N218S)
5SIC 1994-01-31 2.2 MOLECULAR RECOGNITION AT THE ACTIVE SITE OF SUBTILISIN BPN': CRYSTALLOGRAPHIC STUDIES USING GENETICALLY ENGINEERED PROTEINACEOUS INHIBITOR SSI (STREPTOMYCES SUBTILISIN INHIBITOR)
5OX2 2018-01-10 2.24 Crystal structure of thymoligase, a substrate-tailored peptiligase variant
1UBN 1999-06-08 2.4 SELENOSUBTILISIN BPN
1SIB 1993-10-31 2.4 REFINED CRYSTAL STRUCTURES OF SUBTILISIN NOVO IN COMPLEX WITH WILD-TYPE AND TWO MUTANT EGLINS. COMPARISON WITH OTHER SERINE PROTEINASE INHIBITOR COMPLEXES
1SBT 1977-01-06 2.5 ATOMIC COORDINATES FOR SUBTILISIN BPN (OR NOVO)
2SBT 1976-10-06 2.8 A COMPARISON OF THE THREE-DIMENSIONAL STRUCTURES OF SUBTILISIN BPN AND SUBTILISIN NOVO

Relevant UniProtKB Entries

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