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
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