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
1HKO 2003-03-18 NMR structure of bovine 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
5XEE 2018-02-14 NMR solution structure of the aromatic mutant H43F H67F cytochrome b5
1I5U 2001-03-21 SOLUTION STRUCTURE OF CYTOCHROME B5 TRIPLE MUTANT (E48A/E56A/D60A)
2AXX 1998-03-04 THE SOLUTION STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, NMR, 21 STRUCTURES
1IEU 1997-04-21 APOCYTOCHROME B5, PH 6.2, 298 K, NMR, 10 STRUCTURES
1IB7 2001-04-04 SOLUTION STRUCTURE OF F35Y MUTANT OF RAT FERRO CYTOCHROME B5, A CONFORMATION, ENSEMBLE OF 20 STRUCTURES
2I96 2007-09-04 Solution structure of the oxidized microsomal human cytochrome b5
5XE4 2018-02-14 NMR solution structure of the aromatic mutant H43W H67F cytochrome b5
1DO9 2000-01-05 SOLUTION STRUCTURE OF OXIDIZED MICROSOMAL RABBIT CYTOCHROME B5. FACTORS DETERMINING THE HETEROGENEOUS BINDING OF THE HEME.
2M33 2013-02-06 Solution NMR structure of full-length oxidized microsomal rabbit cytochrome b5
1F03 2000-06-21 SOLUTION STRUCTURE OF OXIDIZED BOVINE MICROSOMAL CYTOCHROME B5 MUTANT (E44A, E48A, E56A, D60A) AND ITS INTERACTION WITH CYTOCHROME C
1NX7 2004-06-15 Solution Structure of Oxidized Bovine Microsomal Cytochrome B5
1I87 2001-05-16 SOLUTION STRUCTURE OF THE WATER-SOLUBLE FRAGMENT OF RAT HEPATIC APOCYTOCHROME B5
1IET 1997-04-21 APOCYTOCHROME B5, PH 6.2, 298 K, NMR, MINIMIZED AVERAGE STRUCTURE
1AQA 1997-09-17 SOLUTION STRUCTURE OF REDUCED MICROSOMAL RAT CYTOCHROME B5, NMR, MINIMIZED AVERAGE STRUCTURE
1SH4 2004-08-10 Solution structure of oxidized bovine microsomal cytochrome B5 Mutant V45H
1JEX 2001-07-11 SOLUTION STRUCTURE OF A67V MUTANT OF RAT FERRO CYTOCHROME B5
1F04 2000-06-21 SOLUTION STRUCTURE OF OXIDIZED BOVINE MICROSOMAL CYTOCHROME B5 MUTANT (E44A, E48A, E56A, D60A) AND ITS INTERACTION WITH CYTOCHROME C
1I8C 2001-05-16 SOLUTION STRUCTURE OF THE WATER-SOLUBLE FRAGMENT OF RAT HEPATIC APOCYTOCHROME B5
1J0Q 2003-08-12 Solution Structure of Oxidized Bovine Microsomal Cytochrome b5 mutant V61H
1BFX 1998-08-12 THE SOLUTION NMR STRUCTURE OF THE B FORM OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, MINIMIZED AVERAGE STRUCTURE
1AW3 1998-02-04 THE SOLUTION NMR STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, MINIMIZED AVERAGE STRUCTURE
1B5A 1998-06-17 RAT FERROCYTOCHROME B5 A CONFORMATION, NMR, 1 STRUCTURE
1MNY 2002-11-13 Dimethyl propionate ester heme-containing cytochrome b5
1B5B 1998-06-17 RAT FERROCYTOCHROME B5 B CONFORMATION, NMR, 1 STRUCTURE
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
4HIL 2013-10-16 1.25 1.25A Resolution Structure of Rat Type B Cytochrome b5
1CYO 1994-11-30 1.5 BOVINE CYTOCHROME B(5)
3OZZ 2011-09-14 1.7 Structure of a cytochrome b5 core-swap mutant
1M2I 2003-03-18 1.8 Crystal structure of E44A/E56A mutant of cytochrome b5
1M20 2002-09-11 1.8 Crystal Structure of F35Y Mutant of Trypsin-solubilized Fragment of Cytochrome b5
1M2M 2003-03-18 1.8 Crystal structure of E44A/E48A/E56A/D60A mutant of cytochrome b5
1EUE 2001-04-04 1.8 RAT OUTER MITOCHONDRIAL MEMBRANE CYTOCHROME B5
1LQX 2002-09-04 1.8 Crystal structure of V45E mutant 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
1LJ0 2002-11-20 2.0 Structure of quintuple mutant of the rat outer mitocondrial cytochrome b5.
1ICC 2001-09-19 2.0 RAT OUTER MITOCHONDRIAL MEMBRANE CYTOCHROME B5
1AWP 1998-10-28 2.0 RAT OUTER MITOCHONDRIAL MEMBRANE CYTOCHROME B5
3MUS 2011-04-13 2.0 2A Resolution Structure of Rat Type B Cytochrome b5
2I89 2006-10-31 2.1 Structure of septuple mutant of Rat Outer Mitochondrial Membrane 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)
1B5M 1997-03-12 2.7 RAT OUTER MITOCHONDRIAL MEMBRANE CYTOCHROME B5

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