Defining folding and unfolding reactions of apocytochrome b5 using equilibrium and kinetic fluorescence measurements.


Abstract

The refolding and unfolding kinetics of a soluble domain of apocytochrome b5 extending from residue 1 to 104 have been characterized using stopped flow and equilibrium-based fluorescence methods. The isolated apoprotein unfolds reversibly in the presence of GuHCl. From cooperative unfolding curves, the conformational stability (Delta G(uw)), in the absence of denaturant, is estimated to be 11.6 +/- 1.5 kJ mol-1 at 10 degrees C. The stability of apocytochrome b5 is lower than that of the corresponding form of the holoprotein (Delta G approximately 25 kJ mol-1) and exhibits a transition midpoint at 1.6 M GuHCl. Kinetic studies support the concept of a two-state model with both unfolding and refolding rates showing an exponential dependence on denaturant concentration with no evidence of the formation of transient intermediates in either limb of the chevron plot. Apocytochrome b5 is therefore an example of a protein in which both kinetics and equilibria associated with folding are described by a two-state model. The values of mku and mkf obtained from kinetic analysis are an indication of a transition state (mku/meq of 0.29) that resembles the native form by retaining similar solvent accessibility and many of the noncovalent interactions found in the apoprotein. The changes in heat capacity support a transition state that resembles the apoprotein with a value for Delta Cpf of -3.6 kJ mol-1 K-1 estimated for the refolding reaction. From these measurements, a model of refolding that involves the rapid nucleation of hydrophobic residues around Trp26 is suggested as a major event in the formation of the native apoprotein. Study holds ProTherm entries: 5778 Extra Details:

Submission Details

ID: mzFzSK8k3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Manyusa S;Whitford D,Biochemistry (1999) Defining folding and unfolding reactions of apocytochrome b5 using equilibrium and kinetic fluorescence measurements. PMID:10413531
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
2M33 2013-02-06 Solution NMR structure of full-length oxidized microsomal rabbit cytochrome b5
1B5B 1998-06-17 RAT FERROCYTOCHROME B5 B CONFORMATION, NMR, 1 STRUCTURE
1I5U 2001-03-21 SOLUTION STRUCTURE OF CYTOCHROME B5 TRIPLE MUTANT (E48A/E56A/D60A)
1AQA 1997-09-17 SOLUTION STRUCTURE OF REDUCED MICROSOMAL RAT CYTOCHROME B5, NMR, MINIMIZED AVERAGE STRUCTURE
1IET 1997-04-21 APOCYTOCHROME B5, PH 6.2, 298 K, NMR, MINIMIZED AVERAGE STRUCTURE
1DO9 2000-01-05 SOLUTION STRUCTURE OF OXIDIZED MICROSOMAL RABBIT CYTOCHROME B5. FACTORS DETERMINING THE HETEROGENEOUS BINDING OF THE HEME.
1SH4 2004-08-10 Solution structure of oxidized bovine microsomal cytochrome B5 Mutant V45H
2AXX 1998-03-04 THE SOLUTION STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, NMR, 21 STRUCTURES
5XE4 2018-02-14 NMR solution structure of the aromatic mutant H43W H67F cytochrome b5
1IEU 1997-04-21 APOCYTOCHROME B5, PH 6.2, 298 K, NMR, 10 STRUCTURES
1MNY 2002-11-13 Dimethyl propionate ester heme-containing cytochrome b5
1BLV 1998-07-29 SOLUTION STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5 IN THE PRESENCE OF 2 M GUANIDINIUM CHLORIDE: MONITORING THE EARLY STEPS IN PROTEIN UNFOLDING
1AW3 1998-02-04 THE SOLUTION NMR STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, MINIMIZED AVERAGE STRUCTURE
1JEX 2001-07-11 SOLUTION STRUCTURE OF A67V MUTANT OF RAT FERRO CYTOCHROME B5
2I96 2007-09-04 Solution structure of the oxidized microsomal human cytochrome b5
1BFX 1998-08-12 THE SOLUTION NMR STRUCTURE OF THE B FORM OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, MINIMIZED AVERAGE STRUCTURE
1F04 2000-06-21 SOLUTION STRUCTURE OF OXIDIZED BOVINE MICROSOMAL CYTOCHROME B5 MUTANT (E44A, E48A, E56A, D60A) AND ITS INTERACTION WITH CYTOCHROME C
5XEE 2018-02-14 NMR solution structure of the aromatic mutant H43F H67F cytochrome b5
1J0Q 2003-08-12 Solution Structure of Oxidized Bovine Microsomal Cytochrome b5 mutant V61H
1F03 2000-06-21 SOLUTION STRUCTURE OF OXIDIZED BOVINE MICROSOMAL CYTOCHROME B5 MUTANT (E44A, E48A, E56A, D60A) AND ITS INTERACTION WITH CYTOCHROME C
1B5A 1998-06-17 RAT FERROCYTOCHROME B5 A CONFORMATION, NMR, 1 STRUCTURE
1I87 2001-05-16 SOLUTION STRUCTURE OF THE WATER-SOLUBLE FRAGMENT OF RAT HEPATIC APOCYTOCHROME B5
1NX7 2004-06-15 Solution Structure of Oxidized Bovine Microsomal Cytochrome B5
1IB7 2001-04-04 SOLUTION STRUCTURE OF F35Y MUTANT OF RAT FERRO CYTOCHROME B5, A CONFORMATION, ENSEMBLE OF 20 STRUCTURES
1HKO 2003-03-18 NMR structure of bovine cytochrome b5
1I8C 2001-05-16 SOLUTION STRUCTURE OF THE WATER-SOLUBLE FRAGMENT OF RAT HEPATIC APOCYTOCHROME B5
1AXX 1998-03-04 THE SOLUTION STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, NMR, 19 STRUCTURES
3X34 2015-07-15 0.76 Crystal structure of the reduced form of the solubilized domain of porcine cytochrome b5 in form 1 crystal
3X32 2015-07-15 0.83 Crystal structure of the oxidized form of the solubilized domain of porcine cytochrome b5 in form 1 crystal
3X33 2015-07-15 0.93 Crystal structure of the oxidized form of the solubilized domain of porcine cytochrome b5 in form 2 crystal
3X35 2015-07-15 0.95 Crystal structure of the reduced form of the solubilized domain of porcine cytochrome b5 in form 2 crystal
1CYO 1994-11-30 1.5 BOVINE CYTOCHROME B(5)
3OZZ 2011-09-14 1.7 Structure of a cytochrome b5 core-swap mutant
1LQX 2002-09-04 1.8 Crystal structure of V45E mutant of cytochrome b5
1M2I 2003-03-18 1.8 Crystal structure of E44A/E56A mutant of cytochrome b5
1M2M 2003-03-18 1.8 Crystal structure of E44A/E48A/E56A/D60A mutant of cytochrome b5
1M20 2002-09-11 1.8 Crystal Structure of F35Y Mutant of Trypsin-solubilized Fragment of Cytochrome b5
1U9U 2005-02-01 1.86 Crystal structure of F58Y mutant of cytochrome b5
1LR6 2002-09-04 1.9 Crystal structure of V45Y mutant of cytochrome b5
1M59 2003-03-18 1.9 Crystal Structure of P40V Mutant of Trypsin-solubilized Fragment of Cytochrome b5
1EHB 2000-08-09 1.9 CRYSTAL STRUCTURE OF RECOMBINANT TRYPSIN-SOLUBILIZED FRAGMENT OF CYTOCHROME B5
1U9M 2005-02-01 2.0 Crystal structure of F58W mutant of cytochrome b5
1ES1 2000-08-09 2.1 CRYSTAL STRUCTURE OF VAL61HIS MUTANT OF TRYPSIN-SOLUBILIZED FRAGMENT OF CYTOCHROME B5
4HIN 2013-10-16 2.4 2.4A Resolution Structure of Bovine Cytochrome b5 (S71L)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.9 Cytochrome b5 P00168 CYB5_ALOSE
90.2 Cytochrome b5 P56395 CYB5_MOUSE
91.3 Cytochrome b5 P00173 CYB5_RAT
90.2 Cytochrome b5 P00167 CYB5_HUMAN
91.3 Cytochrome b5 P00169 CYB5_RABIT
91.3 Cytochrome b5 P00170 CYB5_HORSE
96.7 Cytochrome b5 P00172 CYB5_PIG
100.0 Cytochrome b5 P00171 CYB5_BOVIN