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
1TM5 2004-06-10T00:00:00+0000 1.45 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59A mutant
1TM1 2004-06-10T00:00:00+0000 1.7 CRYSTAL STRUCTURE OF THE COMPLEX OF SUBTILISIN BPN' WITH CHYMOTRYPSIN INHIBITOR 2
1SIB 1993-08-02T00:00:00+0000 2.4 REFINED CRYSTAL STRUCTURES OF SUBTILISIN NOVO IN COMPLEX WITH WILD-TYPE AND TWO MUTANT EGLINS. COMPARISON WITH OTHER SERINE PROTEINASE INHIBITOR COMPLEXES
1TMG 2004-06-10T00:00:00+0000 1.67 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59F mutant
1A2Q 1998-01-08T00:00:00+0000 1.8 SUBTILISIN BPN' MUTANT 7186
2SNI 1988-09-05T00:00:00+0000 2.1 STRUCTURAL COMPARISON OF TWO SERINE PROTEINASE-PROTEIN INHIBITOR COMPLEXES. EGLIN-C-SUBTILISIN CARLSBERG AND CI-2-SUBTILISIN NOVO
1TM3 2004-06-10T00:00:00+0000 1.57 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59k mutant
1TO1 2004-06-11T00:00:00+0000 1.68 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 Y61A mutant
1TM7 2004-06-10T00:00:00+0000 1.59 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59Y mutant
1TO2 2004-06-11T00:00:00+0000 1.3 crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 M59K, in pH 9 cryosoak

Relevant UniProtKB Entries

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