Physico-chemical characterization of products of unfolding of cytochrome c by calcium chloride.


Abstract

Cytochrome c (cyt c) denaturation by calcium chloride (CaCl2) and guanidine hydrochloride (GdnHCl) denaturation in presence of low fixed concentrations of CaCl2 has been carried out by UV/Vis spectrophotometry at pH 7.0 and 25 degrees C. The unfolding process was followed by measuring changes in difference molar extinction coefficient around 400 nm and delta epsilon 290. The products of denaturation were further characterized by intrinsic viscosity ([eta]) measurements. It has been observed that the reversible unfolding of cyt c by CaCl2 occurs in two distinct stages or involves three species (or states) namely N<-->X<-->D. Characterization of the native state, N intermediate state, X and the end product, D suggests that (i) During N<-->X only heme is exposed and no secondary structure unfolding occurs so that X state remains as compact as the native state. (ii) Stage X<-->D shows the melting of secondary structure. (iii) The end product corresponds to a random coil. and (iv) Thermodynamic characterization of the end product shows that heme plays an important role in the stability of the protein and removal of heme will lead to the unfolding of cyt c. Mixed denaturation shows a highly cooperative reversible transition between the native and denatured conformation. Analysis of the mixed results shows that (1) Gdn+ does not have any binding site (s) on the native cyt c, (2) there is one binding site for Ca2+ which stabilizes the protein, and (3) the binding constant, ks, is 5 M-1 for Ca2+. Study holds ProTherm entries: 7275 Extra Details: denaturation; cytochome c; guanidine; calcium chloride

Submission Details

ID: 5hvkCBRN4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:33 p.m.

Version: 1

Publication Details
Ahmad Z;Ahmad F,Biochim. Biophys. Acta (1994) Physico-chemical characterization of products of unfolding of cytochrome c by calcium chloride. PMID:8075155
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
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
100.0 Cytochrome c P00004 CYC_HORSE
99.0 Cytochrome c P68097 CYC_EQUAS
99.0 Cytochrome c P68096 CYC_EQUBU
97.1 Cytochrome c P62894 CYC_BOVIN
97.1 Cytochrome c P62895 CYC_PIG
97.1 Cytochrome c P62896 CYC_SHEEP
94.3 Cytochrome c P00007 CYC_HIPAM
95.2 Cytochrome c P68099 CYC_CAMDR
95.2 Cytochrome c P68100 CYC_ESCRO
95.2 Cytochrome c P68098 CYC_LAMGU
94.3 Cytochrome c P62897 CYC_MOUSE
94.3 Cytochrome c P00008 CYC_RABIT
94.3 Cytochrome c P62898 CYC_RAT
94.3 Cytochrome c P00011 CYC_CANLF
93.3 Cytochrome c P00014 CYC_MACGI
93.3 Cytochrome c P00013 CYC_MINSC
93.3 Cytochrome c Q52V09 CYC_CEPBA
93.3 Cytochrome c P00012 CYC_MIRLE
90.5 Cytochrome c Q52V10 CYC_SAISC
92.3 Cytochrome c P81280 CYC_ALLMI
92.2 Cytochrome c P00020 CYC_ANAPL
91.3 Cytochrome c P00021 CYC_COLLI
90.3 Cytochrome c B4USV4 CYC_OTOGA