Analysis of folding and unfolding reactions of cytochrome b5.


Abstract

The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b(5) containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibrium reaction may be monitored by changes in absorbance and fluorescence that accompany denaturation of the native protein. Both probes reveal a single cooperative transition with a midpoint at 3 M GuHCl and lead to a value for the protein stability (DeltaG(uw)) of 26.5 kJ mol(-1). This stability is much higher than that reported for the corresponding form of the apoprotein (approximately 7 kJ mol(-1)). Transient changes in fluorescence and absorbance during protein unfolding exhibit biphasic profiles. A fast phase occupying approximately 30% of the total amplitude is observed at high denaturant concentrations and becomes the dominant process within the transition region. The rates associated with each process show a linear dependency on GuHCl concentration, and at zero denaturant concentration the unfolding rates (k(uw)) are 4.5 x 10(-5) s(-1) and 5.2 x 10(-6) s(-1) at 25 degrees C. The pattern of unfolding is not correlated with covalent heterogeneity, since a wide range of variants and site-directed mutants exhibit identical profiles, nor is the unfolding correlated with cis-trans Pro isomerization in the native state. In comparison with the apo form of cytochrome b(5), the kinetics of refolding and unfolding are more complex and exhibit very different transition states. The data support a model for unfolding in which heme-protein interactions give rise to two discernible rates of unfolding. From an analysis of the activation parameters associated with each process it is established that two structurally similar transition states differing by less than 5 kJ mol(-1) exist in the unfolding reaction. Protein refolding exhibits monophasic kinetics but with distinct curvature apparent in plots of ln k(obs) versus denaturant concentration. The data are interpreted in terms of alternative routes for protein folding in which a "fast track" leads to the rapid ordering of structure around Trp26 for refolding while a slower route requires additional reorganization around the hydrophobic core. Study holds ProTherm entries: 5888, 5889 Extra Details:

Submission Details

ID: x5NUVmaD4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

Publication Details
Manyusa S;Mortuza G;Whitford D,Biochemistry (1999) Analysis of folding and unfolding reactions of cytochrome b5. PMID:10572010
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