Richard J. Newbold

Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy

Mutations in the gene for guanylate cyclase-activating protein-1 (GCAP1) (GUCA1A) have been associated with autosomal dominant cone dystrophy (COD3). In the present study, a severe disease phenotype in a large white family was initially shown to map to chromosome 6p21.1, the location of GUCA1A. Subsequent single-stranded conformation polymorphism analysis and direct sequencing revealed an A464G transition, causing an E155G substitution within the EF4 domain of GCAP1. Modeling of the protein structure shows that the mutation eliminates a bidentate amino acid side chain essential for Ca2+ binding. This represents the first disease-associated mutation in GCAP1, or any neuron-specific calcium-binding protein within an EF-hand domain, that directly coordinates Ca2+. The functional consequences of this substitution were investigated in an in vitro assay of retinal guanylate cyclase activation. The mutant protein activates the cyclase at low Ca2+ concentrations but fails to inactivate at high Ca2+ concentrations. The overall effect of this would be the constitutive activation of guanylate cyclase in photoreceptors, even at the high Ca2+ concentrations of the dark-adapted state, which may explain the dominant disease phenotype.

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors

The structures of 5‐aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4‐oxosebacic acid and 4,7‐dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P‐side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A‐ and P‐side substrates). The most intriguing result is the novel finding that 4,7‐dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P‐side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A‐side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A‐ and P‐sites is proposed.

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.

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.

Structure of yeast 5-aminolaevulinic acid dehydratase complexed with the inhibitor 5-hydroxylaevulinic acid

The X-ray structure of the enzyme 5-aminolaevulinic acid dehydratase (ALAD) from yeast complexed with the competitive inhibitor 5-hydroxylaevulinic acid has been determined at a resolution of 1.9 A. The structure shows that the inhibitor is bound by a Schiff-base link to one of the invariant active-site lysine residues (Lys263). The inhibitor appears to bind in two well defined conformations and the interactions made by it suggest that it is a very close analogue of the substrate 5-aminolaevulinic acid (ALA).

Characterisation of two genes for guanylate cyclase activator protein (GCAP1 and GCAP2) in the Japanese pufferfish, Fugu rubripes

cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system.