David Whitford

Mutagenesis of residues 27 and 78 modulates heme orientation in cytochrome b5

A comparison of the primary sequences of the heme binding domains of bovine and rat microsomal cytochrome reveal differences at only six residues. These residues must therefore provide the origin for the observed variation in the ratio of the heme orientational isomers, the equilibrium constant of which ranges from ∼9 in the bovine protein to ∼1.6 for rat cytochrome b 5. Residues 7, 20, 21, and 30 are distant from the exposed heme edge whilst Leu27 and Phe78 are located close to different parts of the porphyrin macrocycle. 1H NMR spectra of the heme and heme ligand resonances of a recombinant tobacco cytochrome b 5 extending from Gly1 to Lys89 suggest, in combination with NMR data acquired for other forms of cytochrome b 5 and an inspection of their sequence homology, that the identity of residue 78 influences the relative ratios of heme isomers. The Gly1‐Lys89 domain of tobacco cytochrome b 5 has two equally abundant heme orientational isomers but retains the leucine side chain at position 27 whilst phenylalanine 78 is replaced by tyrosine. A more direct role for residue 78 in modulating the heme ratio is shown by site directed mutagenesis of bovine microsomal cytochrome b 5 where the mutation Phe78>Tyr shifts the equilibrium constant for the heme orientational isomers from 9 to 3.5. Whilst the ratio is clearly shifted towards that exhibited by the rat protein the incomplete transition suggested the involvement of other residues. The mutation of Leu27>Val was shown to result in a slightly smaller change in ratios of each isomer (from 9 to 4.0). Together these results point to the importance of these residues in modulating the ratio of heme isomers.

Purification of the membrane binding domain of cytochrome b5 by immobilised nickel chelate chromatography

The purification of a eukaryotic membrane protein has been achieved using a prokaryotic expression system. Bovine cytochrome b5 is an integral membrane protein (Mr approximately 16500). It comprises of a globular haem containing catalytic domain positioned at the N-terminus of the protein and a hydrophobic membrane binding segment at the C-terminus. The membrane binding domain (MBD) is resistant to purification using conventional strategies that have proved successful in isolating the soluble haem containing fragment. We report here a versatile purification method for the isolation of the MBD involving a gene fusion system. The fusion protein incorporates thioredoxin at the amino terminus and six histidines as the metal affinity binding site followed by cytochrome b5 in a pET expression system. This supports high level expression of cytochrome b5 in E. coli C43(DE3) cells. The fusion protein is effectively solubilised from lysed cells with Triton X-100. A step gradient elution with imidazole under non-denaturing conditions on a His-Bind nickel chelate affinity column, saturated with proteins as a crude cell extract, purified the protein in a single step. Proteolytic digestion of pure fusion protein, with trypsin, yielded the MBD. This fragment was further purified by RP-HPLC to a final yield of approximately 10 mg/l.

Solution structure of α-conotoxin SI

The nuclear magnetic resonance solution structure of α‐conotoxin SI has been determined at pH 4.2. The 36 lowest energy structures show that α‐conotoxin SI exists in a single major solution conformation and is stabilized by six hydrogen bonds. Comparisons are made between the SI solution structure and the solution and crystal structures of α‐conotoxin GI. Surprisingly, a high degree of similarity between the backbone conformations of the GI crystal and the SI solution structures is seen in the region of lowest sequence homology, namely residues Gly‐8 to Ser‐12. This similarity is more surprising when considering that in SI a proline replaces the Arg‐9 found in GI. The correspondence in conformation in this region provides the definitive evidence that it is the loss of the arginine basic charge at residue 9 which determines the differences in toxicity between GI and SI, rather than any changes in conformation induced by the cyclic proline residue.

The thermal stability of the tryptic fragment of bovine microsomal cytochrome b5 and a variant containing six additional residues

Thermally induced denaturation has been measured for both oxidised and reduced forms of the tryptic fragment or bovine microsomal cytochrome b 5 using spectrophotometric methods. In the oxidised state, the tryptic fragment of cytochrome b 5 (Ala7‐Lys90) denatures in a single cooperative transition with a midpoint temperature (T m) of ∼ 67°C (pH 7.0). The reduced form of the tryptic fragment of cytochrome b 5 shows a higher transition temperature of ∼ 73°C at pH 7.0 and this is reflected in the values of ΔH m, ΔS m, and Δ(ΔG) of ∼ 310kJ · mol−1, 900J · mol−1 · K−1 and 5 kJ · mol−1. Increased thermal stability is demonstrated for a variant protein that contains the first 90 amino acid residues of cytochrome b 5. These novel increases in stability are observed in both redox states and result from the presence of six additional residues at the amino‐terminus. The two forms of cytochrome b 5 do not differ significantly in structure with the results suggesting that the reorganisation energy (λ) of the variant protein, as measured indirectly from redox‐linked differences in conformational stability, is small. Consequently the reported subtle differences in reactivity between variants of cytochrome b 5 may result from the presence of additional N‐terminal residues on the surface of the protein.

Solution structure of alpha-conotoxin SI.

The nuclear magnetic resonance solution structure of alpha-conotoxin SI has been determined at pH 4.2. The 36 lowest energy structures show that alpha-conotoxin SI exists in a single major solution conformation and is stabilized by six hydrogen bonds. Comparisons are made between the SI solution structure and the solution and crystal structures of alpha-conotoxin GI. Surprisingly, a high degree of similarity between the backbone conformations of the GI crystal and the SI solution structures is seen in the region of lowest sequence homology, namely residues Gly-8 to Ser-12. This similarity is more surprising when considering that in SI a proline replaces the Arg-9 found in GI. The correspondence in conformation in this region provides the definitive evidence that it is the loss of the arginine basic charge at residue 9 which determines the differences in toxicity between GI and SI, rather than any changes in conformation induced by the cyclic proline residue.