Energetics of folding subtilisin BPN'.


Abstract

Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. Here we document for the first time the in vitro folding of the mature form of subtilisin. Subtilisin was modified by site-directed mutagenesis to be proteolytically inactive, allowing the impediments to folding to be systematically examined. First, the thermodynamics and kinetics of calcium binding to the high-affinity calcium A-site have been measured by microcalorimetry and fluorescence spectroscopy. Binding is an enthalpically driven process with an association constant (Ka) equal to 7 x 10(6) M-1. Furthermore, the kinetic barrier to calcium removal from the A-site (23 kcal/mol) is substantially larger than the standard free energy of binding (9.3 kcal/mol). The kinetics of calcium dissociation from subtilisin (e.g., in excess EDTA) are accordingly very slow (t1/2 = 1.3 h at 25 degrees C). Second, to measure the kinetics of subtilisin folding independent of calcium binding, the high-affinity calcium binding site was deleted from the protein. At low ionic strength (I = 0.01) refolding of this mutant requires several days. The folding rate is accelerated almost 100-fold by a 10-fold increase in ionic strength, indicating that part of the free energy of activation may be electrostatic. At relatively high ionic strength (I = 0.5) refolding of the mutant subtilisin is complete in less than 1 h at 25 degrees C. We suggest that part of the electrostatic contribution to the activation free energy for folding subtilisin is related to the highly charged region of the protein comprising the weak ion binding site (site B).(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4316 Extra Details: kinetic barrier; calcium binding; ionic strength;,electrostatic; highly charged region

Submission Details

ID: HrGiQRfn

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Bryan P;Alexander P;Strausberg S;Schwarz F;Lan W;Gilliland G;Gallagher DT,Biochemistry (1992) Energetics of folding subtilisin BPN'. PMID:1599918
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
1LW6 2002-08-21 1.5 Crystal Structure of the Complex of Subtilisin BPN' with Chymotrypsin Inhibitor 2 at 1.5 Angstrom Resolution
1V5I 2005-03-08 1.5 Crystal structure of serine protease inhibitor POIA1 in complex with subtilisin BPN'
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
1SUP 1995-11-14 1.6 SUBTILISIN BPN' AT 1.6 ANGSTROMS RESOLUTION: ANALYSIS OF DISCRETE DISORDER AND COMPARISON OF CRYSTAL FORMS
1Y4A 2005-05-17 1.6 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59R/E60S mutant
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
1S01 1990-10-15 1.7 LARGE INCREASES IN GENERAL STABILITY FOR SUBTILISIN BPN(PRIME) THROUGH INCREMENTAL CHANGES IN THE FREE ENERGY OF UNFOLDING
1TM1 2004-11-09 1.7 CRYSTAL STRUCTURE OF THE COMPLEX OF SUBTILISIN BPN' WITH CHYMOTRYPSIN INHIBITOR 2
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
1Y3B 2005-05-17 1.8 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 E60S mutant
1SBH 1995-12-07 1.8 SUBTILISIN BPN' 8397+1 (E.C. 3.4.21.14) MUTANT (M50F, N76D, G169A, Q206C, N218S, K256Y)
1GNS 2002-06-27 1.8 SUBTILISIN BPN'
1AQN 1998-01-14 1.8 SUBTILISIN MUTANT 8324
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
1SUC 1994-01-31 1.8 CALCIUM-INDEPENDENT SUBTILISIN BY DESIGN
1A2Q 1998-04-29 1.8 SUBTILISIN BPN' MUTANT 7186
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
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
1Y33 2005-05-17 1.8 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 T58P mutant
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
1AK9 1997-11-12 1.8 SUBTILISIN MUTANT 8321
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)
1Y3D 2005-05-17 1.8 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 R67A 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
1Y48 2005-05-17 1.84 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 R65A 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
1GNV 2002-06-24 1.9 CALCIUM INDEPENDENT SUBTILISIN BPN' MUTANT
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
1Y4D 2005-05-17 2.0 Crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59R/E60S 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
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
2SNI 1988-09-07 2.1 STRUCTURAL COMPARISON OF TWO SERINE PROTEINASE-PROTEIN INHIBITOR COMPLEXES. EGLIN-C-SUBTILISIN CARLSBERG AND CI-2-SUBTILISIN NOVO
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
1SUA 1998-01-14 2.1 SUBTILISIN BPN'
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)
1SBI 1995-12-07 2.2 SUBTILISIN BPN' 8397 (E.C. 3.4.21.14) MUTANT (M50F, N76D, G169A, Q206C, N218S)
5OX2 2018-01-10 2.24 Crystal structure of thymoligase, a substrate-tailored peptiligase variant
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
1UBN 1999-06-08 2.4 SELENOSUBTILISIN BPN
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