The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins.


Abstract

The DNA sequence of bovine microsomal cytochrome b5 has been amplified from a liver cDNA library using a polymerase chain reaction. The amplified cDNA when cloned into plasmids that support the high-level production of cytochrome b5 in E.coli leads to protein overexpression and results in cell colonies bearing a strong red colouration. Using cassette mutagenesis, truncated versions of the cytochrome b5 cDNA have been made that encode the first 90 amino acid residues (Ala1-Lys90), the first 104 amino acids (Ala1-Ser104) and the complete protein (Ala1-Asn133). The location of the overexpressed cytochrome b5 within prokaryotic cells is dependent on the overall length of the protein. Expression of the Ala-Lys90 and Ala1-Ser104 variants leads to a location in the cytoplasmic phase of the bacteria whereas the whole protein, Ala1-Asn133, is found within the bacterial membrane fraction. The last 30 residues of cytochrome b5 therefore contain all of the necessary information to insert the protein into E.coli membranes. The solubility of the Ala1-Ser104 variant permits the solution structure and stability of this protein to be measured using 1- and 2-D 1H-NMR methods and electronic spectroscopy. 1-D NMR studies show that the chemical shifts of the haem and haem ligand resonances of the Ala1-Ser104 variant exhibit only very slight perturbations to their magnetic microenvironments when compared with the tryptic fragment of ferricytochrome b5. These results indicate an arrangement of residues in the haem pocket that is very similar in both the Ala1-Ser104 variant and the tryptic fragment and by 2-D NMR it is shown that this similarity extends to the conformations of the polypeptide backbone and side chains. Electronic spectroscopy of this variant shows absorbance maxima for the Soret peaks at 423 nm (reduced) and 413 nm (oxidized). From absorbance spectra the relative thermal stabilities of the Ala1-Ser104 variant and the tryptic fragment were measured. In the oxidized state the Ala1-Ser104 variant denatures in a single cooperative transition with a midpoint temperature (Tm) of 73 degrees C that is significantly higher than that of 'tryptic' ferricytochrome b5. The reduced form of the protein shows increased transition temperatures (Tm approximately 78 degrees C) reflected in the values of delta Hm, delta Sm and delta(delta G) of 420 kJ/mol, 1096 J/mol/K and 12.38 kJ/mol respectively, estimated for this variant. The increased stability of the Ala1-Ser104 variant and other recombinant forms of cytochrome b5 is correlated with the presence of additional residues at the N- and C-termini.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 9864, 9865, 9866, 9867, 9868, 9869, 9870, 9871, 9872, 9873, 9874, 14514, 14515, 14516, 14517 Extra Details: 1.oxidized,2.fragment Ala7-Lys90 cytochrome b5; expression in Escherichia coli; NMR;,protein denaturation and thermostability

Submission Details

ID: xCwaRSnr3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
Hewson R;Newbold RJ;Whitford D,Protein Eng. (1993) The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins. PMID:8309945
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
1B5A 1998-06-17 RAT FERROCYTOCHROME B5 A CONFORMATION, NMR, 1 STRUCTURE
1IEU 1997-04-21 APOCYTOCHROME B5, PH 6.2, 298 K, NMR, 10 STRUCTURES
1NX7 2004-06-15 Solution Structure of Oxidized Bovine Microsomal Cytochrome B5
1AXX 1998-03-04 THE SOLUTION STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, NMR, 19 STRUCTURES
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
1MNY 2002-11-13 Dimethyl propionate ester heme-containing 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
1JEX 2001-07-11 SOLUTION STRUCTURE OF A67V MUTANT OF RAT FERRO CYTOCHROME B5
1I87 2001-05-16 SOLUTION STRUCTURE OF THE WATER-SOLUBLE FRAGMENT OF RAT HEPATIC APOCYTOCHROME B5
1DO9 2000-01-05 SOLUTION STRUCTURE OF OXIDIZED MICROSOMAL RABBIT CYTOCHROME B5. FACTORS DETERMINING THE HETEROGENEOUS BINDING OF THE HEME.
1B5B 1998-06-17 RAT FERROCYTOCHROME B5 B CONFORMATION, NMR, 1 STRUCTURE
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
1HKO 2003-03-18 NMR structure of bovine cytochrome b5
1I5U 2001-03-21 SOLUTION STRUCTURE OF CYTOCHROME B5 TRIPLE MUTANT (E48A/E56A/D60A)
1I8C 2001-05-16 SOLUTION STRUCTURE OF THE WATER-SOLUBLE FRAGMENT OF RAT HEPATIC APOCYTOCHROME B5
1BFX 1998-08-12 THE SOLUTION NMR STRUCTURE OF THE B FORM OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, MINIMIZED AVERAGE STRUCTURE
2I96 2007-09-04 Solution structure of the oxidized microsomal human cytochrome b5
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
1SH4 2004-08-10 Solution structure of oxidized bovine microsomal cytochrome B5 Mutant V45H
1IB7 2001-04-04 SOLUTION STRUCTURE OF F35Y MUTANT OF RAT FERRO CYTOCHROME B5, A CONFORMATION, ENSEMBLE OF 20 STRUCTURES
1F04 2000-06-21 SOLUTION STRUCTURE OF OXIDIZED BOVINE MICROSOMAL CYTOCHROME B5 MUTANT (E44A, E48A, E56A, D60A) AND ITS INTERACTION WITH CYTOCHROME C
1AW3 1998-02-04 THE SOLUTION NMR STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, MINIMIZED AVERAGE STRUCTURE
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
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
1M2I 2003-03-18 1.8 Crystal structure of E44A/E56A mutant of cytochrome b5
1LQX 2002-09-04 1.8 Crystal structure of V45E 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
1M59 2003-03-18 1.9 Crystal Structure of P40V Mutant of Trypsin-solubilized Fragment of Cytochrome b5
1LR6 2002-09-04 1.9 Crystal structure of V45Y mutant 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