Thermodynamic identification of stable folding intermediates in the B-subunit of cholera toxin.


Abstract

The structural stability and domain structure of the pentameric B-subunit of cholera toxin have been measured as a function of different perturbants in order to assess the magnitude of the interactions within the B-subunits. For these studies, temperature, guanidine hydrochloride (GuHCl), and pH were used as perturbants, and the effects were measured by high-sensitivity differential scanning calorimetry, isothermal reaction calorimetry, fluorescence spectroscopy, and partial protease digestion. At pH 7.5 and in the absence of any additional perturbants, the thermal unfolding of the B-subunit pentamer is characterized by a single peak in the heat capacity function centered at 77 degrees C and characterized by a delta Hcal of 328 kcal/mol of B-subunit pentamer and delta Hvh/delta Hcal of 0.3. Lowering the pH down to 4 or adding GuHCl up to 2 M results in a decrease of the calorimetric enthalpy with no significant effect on the van't Hoff enthalpy. The transition enthalpy decreases in a sigmoidal fashion with pH, with an inflection point centered at pH 5.3. Isothermal titration calorimetric studies as a function of pH also report a transition centered at pH 5.3 and characterized by an enthalpy change of 27 kcal/mol of B-subunit pentamer at 27 degrees C. Below this pH, the enthalpy change for the unfolding transition is reduced to approximately 100 kcal/mol of B-subunit pentamer. Similar behavior is obtained with GuHCl. In this case, a first transition is observed at 0.5 M GuHCl and a second one at 3 M GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4382, 4383, 4384, 4385, 4386 Extra Details: NaN3(3 mM) was added in the experiment structural stability; unfolding transition; cooperative;,pentameric ring

Submission Details

ID: vij3APEQ

Submitter: Connie Wang

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

Version: 1

Publication Details
Bhakuni V;Xie D;Freire E,Biochemistry (1991) Thermodynamic identification of stable folding intermediates in the B-subunit of cholera toxin. PMID:2036374
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
5LZG 2017-05-31 1.13 Cholera toxin El Tor B-pentamer in complex with inhibitor PC262
5LZJ 2017-05-31 1.2 Cholera toxin El Tor B-pentamer in complex with inhibitor Laura237
3CHB 1998-08-12 1.25 CHOLERA TOXIN B-PENTAMER COMPLEXED WITH GM1 PENTASACCHARIDE
1JR0 2002-05-08 1.3 CHOLERA TOXIN B-PENTAMER WITH LIGAND BMSC-0011
1RDP 2004-10-26 1.35 Cholera Toxin B-Pentamer Complexed With Bivalent Nitrophenol-Galactoside Ligand BV3
1RF2 2004-10-26 1.35 Cholera Toxin B-Pentamer Complexed With Bivalent Nitrophenol-Galactoside Ligand BV4
1RD9 2004-10-26 1.44 Cholera Toxin B-Pentamer Complexed With Bivalent Nitrophenol-Galactoside Ligand BV2
1MD2 2002-12-11 1.45 CHOLERA TOXIN B-PENTAMER WITH DECAVALENT LIGAND BMSC-0013
5ELC 2016-03-30 1.5 Cholera toxin El Tor B-pentamer in complex with Lewis-y
5ELF 2016-03-30 1.55 Cholera toxin El Tor B-pentamer in complex with A-pentasaccharide
1RCV 2004-10-26 1.6 Cholera Toxin B-Pentamer Complexed With Bivalent Nitrophenol-Galactoside Ligand BV1
5ELE 2016-03-30 1.6 Cholera toxin El Tor B-pentamer in complex with A Lewis-y
1S5D 2004-04-06 1.75 Cholera holotoxin with an A-subunit Y30S mutation, Crystal form 2
1S5E 2004-04-06 1.9 Cholera holotoxin, Crystal form 1
3EFX 2008-09-23 1.94 Novel binding site identified in a hybrid between cholera toxin and heat-labile enterotoxin, 1.9A crystal structure reveals the details
1CHP 1996-03-08 2.0 SURPRISING LEADS FOR A CHOLERA TOXIN RECEPTOR BINDING ANTAGONIST; CRYSTALLOGRAPHIC STUDIES OF CTB MUTANTS
1G8Z 2001-07-25 2.0 HIS57ALA MUTANT OF CHOLERA TOXIN B-PENATMER
2CHB 1997-12-03 2.0 CHOLERA TOXIN B-PENTAMER COMPLEXED WITH GM1 PENTASACCHARIDE
1CHQ 1996-03-08 2.1 SURPRISING LEADS FOR A CHOLERA TOXIN RECEPTOR BINDING ANTAGONIST; CRYSTALLOGRAPHIC STUDIES OF CTB MUTANTS
1S5B 2004-04-06 2.13 Cholera holotoxin with an A-subunit Y30S mutation Form 3
1CT1 1997-10-15 2.3 CHOLERA TOXIN B-PENTAMER MUTANT G33R BOUND TO RECEPTOR PENTASACCHARIDE
1XTC 1996-08-01 2.4 CHOLERA TOXIN
2BH1 2005-05-13 2.4 X-ray structure of the general secretion pathway complex of the N- terminal domain of EpsE and the cytosolic domain of EpsL of Vibrio cholerae
1FGB 1996-12-23 2.4 TOXIN
6HSV 2019-03-20 2.45 Engineered higher-order assembly of Cholera Toxin B subunits via the addition of C-terminal parallel coiled-coiled domains
1P9R 2003-10-14 2.5 Crystal Structure of Vibrio cholerae putative NTPase EpsE
1S5C 2004-04-06 2.5 Cholera holotoxin with an A-subunit Y30S mutation, Crystal form 1
1S5F 2004-04-06 2.6 Cholera holotoxin, Crystal form 2
1P9W 2003-10-14 2.7 Crystal Structure of Vibrio cholerae putative NTPase EpsE
4KSR 2013-09-04 4.2 Crystal Structure of the Vibrio cholerae ATPase GspE Hexamer
4KSS 2013-09-04 7.58 Crystal Structure of Vibrio cholerae ATPase GspsE Hexamer

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Type II secretion system protein E P37093 GSPE_VIBCH
97.1 Cholera enterotoxin subunit B P01556 CHTB_VIBCH