Enhancing the stability of microsomal cytochrome b5: a rational approach informed by comparative studies with the outer mitochondrial membrane isoform.


Abstract

The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is much less prone to lose heme than the microsomal isoform (Mc b5), with a conserved difference at position 71 (leucine versus serine) playing a major role. We replaced Ser71 in Mc b5 with Leu, with the prediction that it would retard heme loss by diminishing polypeptide expansion accompanying rupture of the histidine to iron bonds. The strategy was partially successful in that it slowed dissociation of heme from its less stable orientation in bMc b5 (B). Heme dissociation from orientation A was accelerated to a similar extent, however, apparently owing to increased binding pocket dynamic mobility related to steric strain. A second mutation (L32I) guided by results of previous comparative studies of Mc and OM b5s diminished the steric strain, but much greater relief was achieved by replacing heme with iron deuteroporphyrin IX (FeDPIX). Indeed, the stability of the Mc(S71L) b5 FeDPIX complex is similar to that of the FeDPIX complex of OM b5. The results suggest that maximizing heme binding pocket compactness in the apo state is a useful general strategy for increasing the stability of engineered or designed proteins. Study holds ProTherm entries: 18945, 18946, 18947, 18948, 18949, 18950, 18951, 18952 Extra Details: Holoprotein apoprotein; cytochrome b5; heme replacement; mutagenesis; stability

Submission Details

ID: RrYU9XKJ

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Sun N;Wang A;Cowley AB;Altuve A;Rivera M;Benson DR,Protein Eng. Des. Sel. (2005) Enhancing the stability of microsomal cytochrome b5: a rational approach informed by comparative studies with the outer mitochondrial membrane isoform. PMID:16246823
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

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.9 Cytochrome b5 P00168 CYB5_ALOSE
97.8 Cytochrome b5 P56395 CYB5_MOUSE
100.0 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
96.4 Cytochrome b5 type B Q9CQX2 CYB5B_MOUSE
100.0 Cytochrome b5 type B P04166 CYB5B_RAT