Salt-induced formation of the molten globule state of cytochrome c studied by isothermal titration calorimetry.


Abstract

Although the molten globule state has been proposed as a major intermediate of protein folding, it has proven difficult to obtain thermodynamic data characterizing this state. To explore another approach for characterizing the molten globule state, salt-induced formation of the molten globule state of horse cytochrome c at pH 1.8 was studied by isothermal titration calorimetry. By titrating the acid-unfolded cytochrome c with sodium perchlorate, an exothermic reaction was observed. The titration curve obtained from the heat was cooperative and agreed well with the conformational transition curve measured by CD at 222 nm. This result indicated that the salt-induced conformation change is well approximated by a two-state transition between the acid-unfolded and molten globule states. The heat for formation of the molten globule state estimated by isothermal titration calorimetry was consistent with the enthalpy change for unfolding of the sodium perchlorate-stabilized molten globule state at pH 1.8, which was measured by differential scanning calorimetry and CD. These results indicate that the heat of titration largely reflects the enthalpy change of the conformational transition. From these results, we consider that isothermal titration calorimetry will become a useful approach for investigating the molten globule state. Study holds ProTherm entries: 10958, 10959, 10960, 10961, 10962, 10963, 10964, 10965, 10966 Extra Details: compact denatured state; anion binding; protein folding;,thermal unfolding

Submission Details

ID: HaaKTwL93

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:41 p.m.

Version: 1

Publication Details
Hamada D;Kidokoro S;Fukada H;Takahashi K;Goto Y,Proc. Natl. Acad. Sci. U.S.A. (1994) Salt-induced formation of the molten globule state of cytochrome c studied by isothermal titration calorimetry. PMID:7937949
Additional Information

Study Summary

Number of data points 32
Proteins Cytochrome c ; Cytochrome c
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dCp ionic:NaClO4: 100 mM, prot_conc:0.1 mg/ml ; Experimental Assay: Tm ionic:NaClO4: 100 mM, prot_conc:0.1 mg/ml ; Experimental Assay: dHvH ionic:NaClO4: 100 mM, prot_conc:0.1 mg/ml ; Experimental Assay: dCp ionic:NaClO4: 50 mM, prot_conc:0.1 mg/ml ; Experimental Assay: Tm ionic:NaClO4: 50 mM, prot_conc:0.1 mg/ml ; Experimental Assay: dHvH ionic:NaClO4: 50 mM, prot_conc:0.1 mg/ml ; Experimental Assay: dCp prot_conc:0.1 mg/ml, ionic:NaClO4: 20 mM ; Experimental Assay: Tm prot_conc:0.1 mg/ml, ionic:NaClO4: 20 mM ; Experimental Assay: dHvH prot_conc:0.1 mg/ml, ionic:NaClO4: 20 mM ; Experimental Assay: dCp prot_conc:0.1 mg/ml, ionic:NaClO4: 10 mM ; Experimental Assay: Tm prot_conc:0.1 mg/ml, ionic:NaClO4: 10 mM ; Experimental Assay: dHvH prot_conc:0.1 mg/ml, ionic:NaClO4: 10 mM ; Experimental Assay: dCp ionic:NaClO4: 60 mM, prot_conc:5 mg/ml ; Experimental Assay: dHcal ionic:NaClO4: 60 mM ; Experimental Assay: Tm ionic:NaClO4: 60 mM, prot_conc:5 mg/ml ; Experimental Assay: dHvH ionic:NaClO4: 60 mM, prot_conc:5 mg/ml ; Experimental Assay: dCp ionic:NaClO4: 50 mM, prot_conc:5 mg/ml ; Experimental Assay: dHcal ionic:NaClO4: 50 mM ; Experimental Assay: Tm ionic:NaClO4: 50 mM, prot_conc:5 mg/ml ; Experimental Assay: dHvH ionic:NaClO4: 50 mM, prot_conc:5 mg/ml ; Experimental Assay: dCp ionic:NaClO4: 40 mM, prot_conc:5 mg/ml ; Experimental Assay: dHcal ionic:NaClO4: 40 mM ; Experimental Assay: Tm ionic:NaClO4: 40 mM, prot_conc:5 mg/ml ; Experimental Assay: dHvH ionic:NaClO4: 40 mM, prot_conc:5 mg/ml ; Experimental Assay: dCp ionic:NaClO4: 30 mM, prot_conc:5 mg/ml ; Experimental Assay: dHcal ionic:NaClO4: 30 mM ; Experimental Assay: Tm ionic:NaClO4: 30 mM, prot_conc:5 mg/ml ; Experimental Assay: dHvH ionic:NaClO4: 30 mM, prot_conc:5 mg/ml ; Experimental Assay: dCp prot_conc:5 mg/ml, ionic:NaClO4: 20 mM ; Experimental Assay: dHcal ionic:NaClO4: 20 mM ; Experimental Assay: Tm prot_conc:5 mg/ml, ionic:NaClO4: 20 mM ; Experimental Assay: dHvH prot_conc:5 mg/ml, ionic:NaClO4: 20 mM
Libraries Mutations for sequence GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

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
1KTD 2002-01-15T00:00:00+0000 2.4 CRYSTAL STRUCTURE OF CLASS II MHC MOLECULE IEK BOUND TO PIGEON CYTOCHROME C PEPTIDE
2B4Z 2005-09-27T00:00:00+0000 1.5 Crystal structure of cytochrome C from bovine heart at 1.5 A resolution.
2YBB 2011-03-02T00:00:00+0000 19.0 Fitted model for bovine mitochondrial supercomplex I1III2IV1 by single particle cryo-EM (EMD-1876)
3J2T 2012-12-23T00:00:00+0000 9.5 An improved model of the human apoptosome
5C0Z 2015-06-12T00:00:00+0000 1.12 The structure of oxidized rat cytochrome c at 1.13 angstroms resolution
5C9M 2015-06-28T00:00:00+0000 1.36 The structure of oxidized rat cytochrome c (T28A) at 1.362 angstroms resolution.
5DF5 2015-08-26T00:00:00+0000 1.3 The structure of oxidized rat cytochrome c (T28E) at 1.30 angstroms resolution.
5JUY 2016-05-10T00:00:00+0000 4.1 Active human apoptosome with procaspase-9
6FF5 2018-01-03T00:00:00+0000 1.74 X-ray structure of bovine heart cytochrome c at high ionic strength
6N1O 2018-11-09T00:00:00+0000 1.55 Oxidized rat cytochrome c mutant (S47E)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.3 Cytochrome c P00021 CYC_COLLI
92.2 Cytochrome c P00020 CYC_ANAPL
92.3 Cytochrome c P81280 CYC_ALLMI
90.5 Cytochrome c Q52V10 CYC_SAISC
93.3 Cytochrome c P00012 CYC_MIRLE
93.3 Cytochrome c Q52V09 CYC_CEPBA
93.3 Cytochrome c P00013 CYC_MINSC
93.3 Cytochrome c P00014 CYC_MACGI
94.3 Cytochrome c P00011 CYC_CANLF
94.3 Cytochrome c P62898 CYC_RAT
94.3 Cytochrome c P00008 CYC_RABIT
94.3 Cytochrome c P62897 CYC_MOUSE
95.2 Cytochrome c P68098 CYC_LAMGU
95.2 Cytochrome c P68100 CYC_ESCRO
95.2 Cytochrome c P68099 CYC_CAMDR
94.3 Cytochrome c P00007 CYC_HIPAM
97.1 Cytochrome c P62896 CYC_SHEEP
97.1 Cytochrome c P62895 CYC_PIG
97.1 Cytochrome c P62894 CYC_BOVIN
99.0 Cytochrome c P68096 CYC_EQUBU
99.0 Cytochrome c P68097 CYC_EQUAS
100.0 Cytochrome c P00004 CYC_HORSE
90.3 Cytochrome c B4USV4 CYC_OTOGA