Role for lysine 142 in the excision of adenine from A:G mispairs by MutY DNA glycosylase of Escherichia coli.

MutY participates in the repair of oxidatively damaged DNA by excising adenine from dA:dG and dA:8-oxodG mispairs; this DNA glycosylase can be cross-linked to DNA through Lys-142. We have investigated the properties of a mutant protein in which Lys-142 is replaced by glutamine. Using the rifampicin resistance assay, MutY K142Q was shown to complement the mutY mutator phenotype to the same extent as wild-type MutY. Although MutY K142Q does not form a Schiff base with DNA, it retains in part the catalytic properties of wild-type enzyme. The K142Q mutation selectively impairs processing of DNA containing dA:dG mispairs but not that of substrates containing dA:8-oxodG. Decreased substrate processing is mediated primarily via an increase in K(D) (21.8 nM for MutY vs 298 nM for MutY K142Q). The catalytic constant, measured in single turnover experiments, was not significantly affected. At pH < 6.0, the activity of MutY K142Q on the dA:dG mispair was approximately the same as for wild-type protein, suggesting that a dG(anti) to dG(syn) transition is effected at low pH. The three-dimensional structure of the catalytic domain of MutY K142Q, determined at 1.35 A resolution, shows no significant differences between wild-type and mutant protein, indicating that Lys-142 is not critical for maintaining the conformation of MutY. We conclude that Lys-142 recognizes guanine in the dA:dG mispair, helping position this residue in the syn conformation and facilitating binding of substrate DNA. Lys-142 is not involved in the catalytic steps of base excision.

Characterization of a cross-linked DNA-endonuclease VIII repair complex by electrospray ionization mass spectrometry.

Electrospray mass spectrometry techniques were used to characterize components of the active site in Endonuclease VIII by identifying the amino acid sequence and the binding site for a tryptic peptide derived from Endo VIII in a cross-linked DNA-peptide complex. Endo VIII, a DNA repair enzyme with both glycosylase and lyase activities, was covalently bound to a thymidine glycol-containing oligodeoxynucleotide duplex by converting a transient Schiff base formed during the course of the glycosylase activity to a stable covalent bond by chemical reduction with sodium borohydride. After tryptic digestion of the initial product, the identification of the cross-linked peptide was deduced initially from the molecular mass of the tryptic product obtained by negative ion electrospray mass analysis. Nanospray tandem mass spectrometry (MS/MS) analysis of the tryptic product corroborated the molecular mass of the peptide fragment and verified the point of attachment to the oligomer, but failed to produce sufficient fragmentation to sequence the peptide completely. Direct evidence for the amino acid sequence of the peptide was obtained after enzymatic digestion of the DNA portion of the cross-linked DNA-peptide product and analysis by negative ion nanospray MS/MS. Examination of the ions from collision induced fragmentation disclosed that this substance was the N-terminal tryptic fragment of Endo VIII cross-linked to a portion of the oligomer, and that the N-terminal proline from Endo VIII was covalently bound to the residual deoxyribose moiety at the original location of the thymine glycol in the oligomer.

Prokaryotic translation initiation factor IF3 is an elongated protein consisting of two crystallizable domains.

We show that translation initiation factor IF3 can be split into two fragments of nearly equal size by the Escherichia coli outer membrane protease omptin. Circular dichroism and small-angle neutron scattering show that the two fragments are structured as domains. Each domain is relatively compact, and they are separated by about 45 A in intact IF3. Thus IF3 is an elongated protein that consists of two well-separated domains. We suggest that these two domains are involved in ribosome binding across the cleft of the 30S ribosome. We also report the crystallization of each domain of IF3.

Crystallization and preliminary X-ray diffraction studies of bacterial ribosomal protein L14.

Based on amino-acid sequence homology, it is predicted that ribosomal protein L14 is a member of a recently identified family of structurally related RNA-binding proteins. To verify this, the gene for Bacillus stearothermophilus L14 has been cloned, and the protein has been purified and crystallized. The crystals are in space group C2 with cell dimensions a = 67.0, b = 32.7, c = 49.4 A, and beta = 101.8 degrees, and there is one molecule in the asymmetric unit (V(m) = 2.0 A(3) Da(-1)). They are of high quality, and a native data set has been collected to a resolution of 1.6 A, with an R(merge) of 5.3%.

Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein.

The crystal structure of protein L9 from the Bacillus stearothermophilus ribosome has been determined at 2.8 A resolution using X-ray diffraction methods. This primary RNA-binding protein has a highly elongated and unusual structure consisting of two separated domains joined by a long exposed alpha-helix. Conserved, positively charged and aromatic amino acids on the surfaces of both domains probably represent the sites of specific interactions with 23S rRNA. Comparisons with other prokaryotic L9 sequences show that while the length of the connecting alpha-helix is invariant, the sequence within the exposed central region is not conserved. This suggests that the alpha-helix has an architectural role and serves to fix the relative separation and orientation of the N- and C-terminal domains within the ribosome. The N-terminal domain has structural homology to the smaller ribosomal proteins L7/L12 and L30, and the eukaryotic RNA recognition motif (RRM).

Light Intensity Adaptation and Phycobilisome Composition of Microcystis aeruginosa.

Phycobilisomes isolated from Microcystis aeruginosa grown to midlog at high light (270 microeinsteins per square meter per second) or at low light intensities (40 microeinsteins per square meter per second) were found to be identical. Electron micrographs established that they have a triangular central core apparently consisting of three allophycocyanin trimers surrounded by six rods, each composed of two hexameric phycocyanin molecules. The apparent mass of a phycobilisome obtained by gel filtration is 2.96 x 10(6) daltons. The molar ratio of the phycobiliproteins per phycobilisome is 12 phycocyanin hexamers:9 allophycocyanin trimers. The electron microscopic observations combined with the phycobilisome apparent mass and the phycobiliprotein stoichiometry data indicate that M. aeruginosa phycobilisomes are composed of a triangular central core of three stacks of three allophycocyanin trimers and six rods each containing two phycocyanin hexamers. Adaptation of M. aeruginosa to high light intensity results in a decrease in the number of phycobilisomes per cell with no alteration in phycobilisome composition or structure.

Atypical pulmonary thrombosis caused by a toxic cyanobacterial peptide.

Parenteral injection into mice of a toxic pentapeptide isolated from the cyanobacterium Microcystis aeruginosa induced thrombocytopenia, pulmonary thrombi, and hepatic congestion. The lethality of the toxin was unaffected by several anticoagulants. The acute liver damage that follows injection of the toxin has been attributed to direct action on liver cells but may be due to hypoxemia, heart failure, and shock.

Molecular morphology of cyanobacterial phycobilisomes.

Phycobilisomes were isolated from several cyanobacteria following cell lysis with Triton X-100. They were purified by phosphate precipitation and hydrophobic-interaction chromatography. Their phycobiliprotein compositions were quantitatively determined by application of sets of simultaneous absorbance equations to gel chromatographic separations of the chromoproteins. Phycobilisomes purified from several cyanobacteria had characteristic elution times on agarose gel chromatography. Combining electron microscope observations of phycobilisome structure, phycobiliprotein composition, and agarose gel chromatography estimates of molecular weight permitted the calculation of many details of phycobilisome molecular structure. Complementary chromatic adaptation resulted in a change of phycobilisome composition and structure. The polypeptide compositions of phycobilisomes were examined by sodium dodecyl sulfate-agarose gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The phycobilisomes were composed of phycobilipeptides derived from the constituent phycobiliproteins. Higher molecular-weight phycobilipeptide aggregates were also observed. The dominant forces responsible for the maintenance of phycobilisome structure are concluded to be hydrophobic interactions.

Peridinin-Chlorophyll a Proteins of the Dinoflagellate Amphidinium carterae (Plymouth 450).

The marine dinoflagellate Amphidinium carterae (Plymouth 450) releases several water-soluble peridinin-chlorophyll a proteins after freezethawing. These chromoproteins have a molecular weight of 39.2 x 10(3) and are comprised of noncovalently bound peridinin and chlorophyll a and a nonoligomeric protein. They have distinct isoelectric points and may be resolved into six components by either isoelectric focusing on polyacrylamide gel or ion exchange chromatography. The predominant chromoprotein, which has a pI of 7.5, constitutes about 90% of the extractable peridinin-chlorophyll a protein. It consists of an alanine-rich apoprotein of molecular weight 31.8 x 10(3) stoichiometrically associated with 9 peridinin and 2 chlorophyll a molecules. Additionally, the peridinin-chlorophyll a proteins with pI values of 7.6 and 6.4 were purified and found to have amino acid and chromophore composition essentially identical with the pI 7.5 protein. Peridinin-chlorophyll a protein, pI 7.5, after treatment at alkaline pH was transformed into several more acid pI forms of the protein, strongly suggesting that the naturally occurring proteins are deamidation products of a single protein. Fluorescence excitation and emission spectra demonstrate that light energy absorbed by peridinin induces chlorophyll a fluorescence presumably by intramolecular energy transfer. The peridinin-chlorophyll a proteins presumably function in vivo as photosynthetic light-harvesting pigments.