Equilibrium studies of the effect of difference in sequence homology on the mechanism of denaturation of bovine and horse cytochromes-c.


Abstract

We have carried out equilibrium studies of the effect of the amino acid residue difference in the primary structure of bovine cytochrome-c (b-cyt-c) and horse cyt-c (h-cyt-c) on the mechanism of their folding <--> unfolding processes at pH 6.0 and 25 degrees C. It has been observed that guanidinium chloride (GdmCl)-induced denaturation of b-cyt-c follows a two-state mechanism and that of h-cyt-c is not a two-state process. This conclusion is reached from the coincidence and non-coincidence of GdmCl-induced transition curves of bovine and horse proteins, respectively, monitored by measurements of absorbance at 405, 530 and 695 nm and circular dichroism (CD) at 222, 416 and 405 nm. These measurements on h-cyt-c in the presence of GdmCl in the concentration range 0.75-2.0 M also suggest that the protein retains all the native far-UV CD but has slightly perturbed tertiary interaction. The intermediate in the presence of these low denaturant concentrations does not have the structural characteristics of a molten globule as judged by the 8-Anilino-1-napthalene sulfonic acid (ANS) binding and near-UV CD experiments. We have also carried out thermal denaturation studies of bovine and horse cyts-c in the presence of GdmCl monitored by absorbance at 405 nm and far-UV CD at 222 nm. The heat-induced denaturation measurements in the presence of the denaturant show (1) that denaturation of b-cyt-c is a two-state process and that of h-cyt-c does not follow a two-state mechanism, and (2) that the enthalpy change on denaturation of both proteins strongly depends on GdmCl concentration. Study holds ProTherm entries: 15736, 15737, 15738, 15739, 15740, 15741, 15742, 15743, 15744, 15745, 15746, 15747, 15748, 15749, 15750, 15751, 15752, 15753, 15754, 15755, 15756, 15757, 15758, 15759, 15760, 15761, 15762, 15763, 15764, 15765, 15766, 15767, 15768, 15769, 15770, 15771, 15772, 15773, 15774, 15775, 15776, 15777, 15778, 15779, 15780, 15781, 15782, 15783, 15784, 15785, 15786, 15787 Extra Details: at molar absorption coefficient e = 405 nm. Even though the unit for 'm' is kJ/mol/M, here the unit is given as the authors assigned. Guanidinium chloride unfolding; Thermal unfolding; Protein stability; Circular dichroism; Molten globule

Submission Details

ID: uLQQjxGv

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Moza B;Qureshi SH;Ahmad F,Biochim. Biophys. Acta (2003) Equilibrium studies of the effect of difference in sequence homology on the mechanism of denaturation of bovine and horse cytochromes-c. PMID:12637011
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
1OCD 1997-06-16 CYTOCHROME C (OXIDIZED) FROM EQUUS CABALLUS, NMR, MINIMIZED AVERAGE STRUCTURE
2N3B 2015-10-28 Structure of oxidized horse heart cytochrome c encapsulated in reverse micelles
2N9J 2016-02-17 Solution structure of oxidized human cytochrome c
1AKK 1997-09-17 SOLUTION STRUCTURE OF OXIDIZED HORSE HEART CYTOCHROME C, NMR, MINIMIZED AVERAGE STRUCTURE
5ZKV 2019-05-22 Solution structure of molten globule state of L94G mutant of horse cytochrome-c
1FI9 2000-08-23 SOLUTION STRUCTURE OF THE IMIDAZOLE COMPLEX OF CYTOCHROME C
2FRC 1997-07-29 CYTOCHROME C (REDUCED) FROM EQUUS CABALLUS, NMR, MINIMIZED AVERAGE STRUCTURE
1J3S 2004-05-18 Solution Structure of Reduced Recombinant Human Cytochrome c
1FI7 2000-08-23 Solution structure of the imidazole complex of cytochrome C
1LC2 2003-06-03 Solution Structure Of Reduced Horse Heart Cytochrome c in 30% Acetonitrile Solution, NMR 30 Structures
2N3Y 2016-12-14 NMR structure of the Y48pCMF variant of human cytochrome c in its reduced state
2GIW 1998-12-09 SOLUTION STRUCTURE OF REDUCED HORSE HEART CYTOCHROME C, NMR, 40 STRUCTURES
1LC1 2003-06-03 Solution Structure Of Reduced Horse Heart Cytochrome c in 30% Acetonitrile Solution, NMR Minimized Average Structure
2N9I 2016-02-17 Solution structure of reduced human cytochrome c
1M60 2002-08-07 Solution Structure of Zinc-substituted cytochrome c
1I5T 2001-03-21 SOLUTION STRUCTURE OF CYANOFERRICYTOCHROME C
1GIW 1998-12-09 SOLUTION STRUCTURE OF REDUCED HORSE HEART CYTOCHROME C, NMR, MINIMIZED AVERAGE STRUCTURE
5C0Z 2016-09-21 1.12 The structure of oxidized rat cytochrome c at 1.13 angstroms resolution
5TY3 2017-09-06 1.25 Crystal structure of K72A variant of Human Cytochrome c
5DF5 2016-09-14 1.3 The structure of oxidized rat cytochrome c (T28E) at 1.30 angstroms resolution.
3ZOO 2013-10-23 1.35 Structure of the Y46F mutant of human cytochrome c
5O10 2018-03-28 1.36 Y48H mutant of human cytochrome c
5C9M 2016-09-21 1.36 The structure of oxidized rat cytochrome c (T28A) at 1.362 angstroms resolution.
2B4Z 2005-10-11 1.5 Crystal structure of cytochrome C from bovine heart at 1.5 A resolution.
5EXQ 2016-11-30 1.6 Human cytochrome c Y48H
2AIU 2006-07-18 1.6 Crystal Structure of Mouse Testicular Cytochrome C at 1.6 Angstrom
3ZCF 2013-10-23 1.65 Structure of recombinant human cytochrome c
6FF5 2018-03-21 1.74 X-ray structure of bovine heart cytochrome c at high ionic strength
3O1Y 2012-01-25 1.75 Electron transfer complexes: Experimental mapping of the redox-dependent cytochrome c electrostatic surface
3WUI 2014-07-16 1.8 Dimeric horse cytochrome c formed by refolding from molten globule state
1WEJ 1998-12-09 1.8 IGG1 FAB FRAGMENT (OF E8 ANTIBODY) COMPLEXED WITH HORSE CYTOCHROME C AT 1.8 A RESOLUTION
3WC8 2013-12-11 1.8 Dimeric horse cytochrome c obtained by refolding with desalting method
6DUJ 2019-06-26 1.82 Crystal structure of A51V variant of Human Cytochrome c
3O20 2012-01-25 1.9 Electron transfer complexes:experimental mapping of the Redox-dependent Cytochrome C electrostatic surface
3NWV 2011-03-09 1.9 Human cytochrome c G41S
1HRC 1994-11-01 1.9 HIGH-RESOLUTION THREE-DIMENSIONAL STRUCTURE OF HORSE HEART CYTOCHROME C
2YBB 2011-10-19 19.0 Fitted model for bovine mitochondrial supercomplex I1III2IV1 by single particle cryo-EM (EMD-1876)
5IY5 2017-01-11 2.0 Electron transfer complex of cytochrome c and cytochrome c oxidase at 2.0 angstrom resolution
1CRC 1996-03-08 2.08 CYTOCHROME C AT LOW IONIC STRENGTH
3NBT 2010-07-14 2.1 Crystal structure of trimeric cytochrome c from horse heart
4NFG 2014-09-24 2.11 K13R mutant of horse cytochrome c and yeast cytochrome c peroxidase complex
4RSZ 2015-01-14 2.19 The X-ray structure of the Primary Adduct formed in the Reaction between Cisplatin and Cytochrome c
3NBS 2010-07-14 2.2 Crystal structure of dimeric cytochrome c from horse heart
1KTD 2002-05-01 2.4 CRYSTAL STRUCTURE OF CLASS II MHC MOLECULE IEK BOUND TO PIGEON CYTOCHROME C PEPTIDE
1U75 2004-09-28 2.55 Electron Transfer Complex between Horse Heart Cytochrome c and Zinc-Porphyrin Substituted Cytochrome c Peroxidase
6ECJ 2019-08-14 2.7 Human cytochrome c G41T
2PCB 1993-07-15 2.8 CRYSTAL STRUCTURE OF A COMPLEX BETWEEN ELECTRON TRANSFER PARTNERS, CYTOCHROME C PEROXIDASE AND CYTOCHROME C
3JBT 2015-11-18 3.8 Atomic structure of the Apaf-1 apoptosome
5JUY 2016-10-19 4.1 Active human apoptosome with procaspase-9
5WVE 2017-02-08 4.4 Apaf-1-Caspase-9 holoenzyme
3J2T 2013-04-10 9.5 An improved model of the human apoptosome

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.3 Cytochrome c P21665 CYC_VARVA
90.4 Cytochrome c P00015 CYC2_MOUSE
90.5 Cytochrome c Q640U4 CYC1_XENTR
91.4 Cytochrome c P00022 CYC_CHESE
90.5 Cytochrome c P00019 CYC_STRCA
90.5 Cytochrome c P00018 CYC_DRONO
90.5 Cytochrome c Q5RFH4 CYC_PONAB
90.5 Cytochrome c P99998 CYC_PANTR
90.5 Cytochrome c P99999 CYC_HUMAN
90.5 Cytochrome c Q6WUX8 CYC_GORGO
90.5 Cytochrome c P00017 CYC_APTPA
91.4 Cytochrome c P67882 CYC_MELGA
91.4 Cytochrome c P67881 CYC_CHICK
94.2 Cytochrome c P00020 CYC_ANAPL
91.4 Cytochrome c Q52V08 CYC_MACSY
91.4 Cytochrome c P00002 CYC_MACMU
93.2 Cytochrome c P00021 CYC_COLLI
94.2 Cytochrome c P81280 CYC_ALLMI
91.4 Cytochrome c Q52V10 CYC_SAISC
93.2 Cytochrome c B4USV4 CYC_OTOGA
95.2 Cytochrome c P00013 CYC_MINSC
94.3 Cytochrome c P00014 CYC_MACGI
96.2 Cytochrome c P00012 CYC_MIRLE
96.2 Cytochrome c P00008 CYC_RABIT
96.2 Cytochrome c Q52V09 CYC_CEPBA
100.0 Cytochrome c P00004 CYC_HORSE
97.1 Cytochrome c P00011 CYC_CANLF
97.1 Cytochrome c P62898 CYC_RAT
97.1 Cytochrome c P62897 CYC_MOUSE
99.0 Cytochrome c P68096 CYC_EQUBU
99.0 Cytochrome c P68097 CYC_EQUAS
98.1 Cytochrome c P68098 CYC_LAMGU
98.1 Cytochrome c P68100 CYC_ESCRO
98.1 Cytochrome c P68099 CYC_CAMDR
97.1 Cytochrome c P00007 CYC_HIPAM
100.0 Cytochrome c P62896 CYC_SHEEP
100.0 Cytochrome c P62895 CYC_PIG
100.0 Cytochrome c P62894 CYC_BOVIN