James G. Harman

Allosteric regulation of the cAMP receptor protein

The cyclic AMP receptor protein (CRP) of Escherichia coli is a dimer made up of identical subunits. Each CRP subunit contains a cyclic nucleotide binding pocket and the CRP dimer exhibits negative cooperativity in binding cAMP. In solutions containing cAMP, CRP undergoes sequential conformation changes from the inactive apo-form through the active CRP:(cAMP)(1) complex to the less active CRP:(cAMP)(2) complex depending on the cAMP concentration. Apo-CRP binds DNA with low affinity and no apparent sequence specificity. The CRP:(cAMP)(1) complex exhibits high affinity, sequence-specific DNA binding and interacts with RNA polymerase, whether free in solution or complexed with DNA. The results of genetic, biochemical and biophysical studies have helped to uncover many of the details of cAMP-mediated allosteric control over CRP conformation and activity as a transcription factor. These studies indicate that cAMP binding produces only small, but significant, changes in CRP structure; changes that include subunit realignment and concerted motion of the secondary structure elements within the C-terminal DNA binding domain of each subunit. These adjustments promote CRP surface-patch interaction with RNA polymerase and protrusion of the F-helix to promote CRP site-specific interaction with DNA. Interactions between CRP and RNA polymerase at CRP-dependent promoters produce active ternary transcription complexes.

Sterol specificity of theSaccharomyces cerevisiae ERG6 gene product expressed inEscherichia coli

TheERG6 gene fromSaccharomyces cerevisiae has been functionally expressed inEscherichia coli, for the first time, yielding a protein that catalyzes the bisubstrate transfer reaction whereby the reactive methyl group from (S)-adenosyl-l-methionine is transferred stereoselectively to C-24 of the sterol side chain. The structural requirements of sterol in binding and catalysis were similar to the native protein fromS. cerevisiae. Inhibition of biomethylation was observed with fecosterol and ergosterol which suggests that ergosterol may function in wild-type yeast as a feedback regulator of sterol biosynthesis.

Amino acid substitution at position 99 affects the rate of CRP subunit exchange.

We investigated the characteristics of CRP having amino acid substitutions at position 99. Analysis of amino acid residue proximity to cAMP in molecular dynamics (MD) simulations of the CRP:(cAMP)(2) complex [García, A. E., and Harman, J. G. (1996) Protein Sci. 5, 62-71] showed repositioning of tyrosine 99 (Y99) to interact with the equatorial exocyclic oxygen atom of cAMP. To test the role of Y99 in cAMP-mediated CRP activation, Y99 was substituted with alanine (A) or phenylalanine (F). Cells that contained the WT or mutant forms of CRP induced beta-galactosidase in the presence of cAMP. Purified WT, Y99A, and Y99F CRP showed only a 3- to 4-fold difference in cAMP affinity. There were no apparent differences between the three forms of CRP in cAMP binding cooperativity, in CRP:(cAMP)(1) complex binding to lacP DNA, in the formation of CRP:cAMP:RNAP complexes at lacP, or in CRP efficacy in mediating lacP activity in vitro. The apo-form of Y99A CRP was more sensitive to protease than the apo-form of either WT CRP or Y99F CRP. Whereas the WT or Y99F CRP:(cAMP)(1) complexes were cleaved by protease at hinge-region peptide bonds, the Y99A CRP:(cAMP)(1) complex was cleaved at peptide bonds located at the subunit interface. The rates of subunit exchange for Y99A CRP, both in the apo-form and in a 1:1 complex with cAMP, were significantly greater than that measured for WT CRP. The results of this study show that tyrosine 99 contributes significant structural stability to the CRP dimer, specifically in stabilizing subunit association.

Characterization of the pet operon of Rhodospirillum rubrum

The three genes of the pet operon, coding, respectively, for the Rieske iron-sulfur protein, cytochrome b and cytochrome c1 components of the cytochrome bc1 complex in the photosynthetic bacterium Rhodospirillum rubrum have been sequenced. The amino acid sequences deduced for these three peptides from the nucleotide sequences of the genes have been confirmed, in part, by direct sequencing of portions of the three peptides separated from a sample of the purified, detergent-solubilized complex. These sequences show considerable homology with those previously obtained for the pet operons of other photosynthetic bacteria. Northern blots of R. rubrum mRNA have established that the operon is transcribed as a single polycistronic message, the start site of which has been determined by both primer extension and nuclease protection. Photosynthetic growth of R. rubrum was shown to be inhibited by antimycin A, a specific inhibitor of cytochrome bc1 complexes, and antimycin A-resistant mutants of R. rubrum have been isolated. Preliminary results suggest that it may be possible to express the R. rubrum pet operon in a strain of the photosynthetic bacterium Rhodobacter capsulatus from which the native pet operon has been deleted.

Hyperexpression in Escherichia coli, purification, and characterization of the metallo-β-lactamase of Bacillus cereus 5/B/6

We used site-directed mutagenesis to introduce both a NdeI restriction endonuclease site and an initiator codon at the junction of the leader and structural gene sequences of the metallo-beta-lactamase of Bacillus cereus 5/B/6. This construct allowed us to clone just the beta-lactamase structural gene sequence into an Escherichia coli expression vector. E. coli cells were transformed with the recombinant plasmid, the B. cereus beta-lactamase was expressed, and these E. coli cells were disrupted by sonic oscillation. When the resultant suspensions were clarified by ultracentrifugation, the B. cereus beta-lactamase represented 15% of the total protein in the supernatant. Subsequent gel filtration and ion-exchange chromatography allowed the first reported purification to homogeneity of the B. cereus beta-lactamase from E. coli with an 87% recovery and an overall yield of 17 mg of enzyme per liter of cell culture. The electrophoretic mobilities of the enzyme expressed in and purified from E. coli and the enzyme purified directly from B. cereus were identical in both native and sodium dodecyl sulfate gel electrophoreses. As with the B. cereus enzyme, Km and Vmax (using cephalosporin C as substrate) for the enzyme purified from E. coli were 0.39 mM and 1333 units/mg protein, respectively. Likewise, the Co(II)-reconstituted enzyme purified from E. coli, which retained 29% of the activity of the Zn(II) enzyme, had electronic absorption spectra with maxima at 347, 551, 617, and 646 nm with extinction coefficients of 900, 250, 173, and 150 M-1 cm-1, respectively.