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
2I96 2006-09-05T00:00:00+0000 0 Solution structure of the oxidized microsomal human cytochrome b5
1DO9 1999-12-20T00:00:00+0000 0 SOLUTION STRUCTURE OF OXIDIZED MICROSOMAL RABBIT CYTOCHROME B5. FACTORS DETERMINING THE HETEROGENEOUS BINDING OF THE HEME.
2M33 2013-01-08T00:00:00+0000 0 Solution NMR structure of full-length oxidized microsomal rabbit cytochrome b5
3X32 2015-01-14T00:00:00+0000 0.83 Crystal structure of the oxidized form of the solubilized domain of porcine cytochrome b5 in form 1 crystal
3X33 2015-01-14T00:00:00+0000 0.93 Crystal structure of the oxidized form of the solubilized domain of porcine cytochrome b5 in form 2 crystal
3X34 2015-01-14T00:00:00+0000 0.76 Crystal structure of the reduced form of the solubilized domain of porcine cytochrome b5 in form 1 crystal
3X35 2015-01-14T00:00:00+0000 0.95 Crystal structure of the reduced form of the solubilized domain of porcine cytochrome b5 in form 2 crystal
1AQA 1997-07-28T00:00:00+0000 0 SOLUTION STRUCTURE OF REDUCED MICROSOMAL RAT CYTOCHROME B5, NMR, MINIMIZED AVERAGE STRUCTURE
1AW3 1997-10-09T00:00:00+0000 0 THE SOLUTION NMR STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, MINIMIZED AVERAGE STRUCTURE
1AXX 1997-10-22T00:00:00+0000 0 THE SOLUTION STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, NMR, 19 STRUCTURES

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