Biochemical Characterization of Hypothetical Proteins from <i>Helicobacter pylori</i>

<div><p>The functional characterization of Open Reading Frames (ORFs) from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from <i>Helicobacter pylori</i> ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate using derivatized nano-particles, and subsequent identification by mass spectrometry. Candidate genes encoding these proteins were cloned and expressed in <i>Escherichia coli</i>, and the recombinant proteins were purified and characterized biochemically and their biochemical parameters compared with the native ones. These proteins include a guanosine triphosphate (GTP) cyclohydrolase (HP0959), an ATPase (HP1079), an adenosine deaminase (HP0267), a phosphodiesterase (HP1042), an aminopeptidase (HP1037), and new substrates were characterized for a peptidoglycan deacetylase (HP0310). Generally, characterized enzymes were active at acidic to neutral pH (4.0–7.5) with temperature optima ranging from 35 to 55°C, although some exhibited outstanding characteristics.</p></div

Identification of Protein Spots on the 2D gel by LC-MS/MS^a.

a<p>The differentially expressed protein spots on the 2-D gels of ALS were excised and proteins identified by LC-MS/MS.</p>b<p>The length of identified peptide fragments divided by the length of the protein.</p>c<p>Mascot algorithm score; the minimum score is required to have a statistic significance (p<0.05).</p

Comparison of urea-soluble proteins from ALS and non-ALS spinal cords by 2D SDS-PAGE.

<p>(<b>A</b>) Urea-soluble whole tissue lysates were prepared from pooled ALS or non-ALS spinal cords in the RIPA lysis buffer containing 8 M of urea. The first dimension was 18-cm immobilized pH gradient isoelectric focusing (IEF) from pI = 3–11; the second dimension was 10% SDS-PAGE. The gels were stained with Sypro Rubby. High quality spots marked with a–i and a’–i’ were randomly selected as references to normalize the differences between different gels. (<b>B</b>) Differentially expressed protein clusters between ALS and non-ALS spinal cords. The cluster A, B and S from ALS and A’, B’ and S’ from non-ALS were excised from the 2-D gels and subjected to LC-MS/MS protein identification. (<b>C</b>) Western blotting analysis of the protein clusters with anti-GFAP antibody. The urea-soluble whole tissue lysates were resolved by mini-2D SDS-PAGE, transferred to the PVDF membrane and detected with the antibody against GFAP.</p

Kinetic parameters of native pure proteins directly isolated from <i>Helicobacter pylori</i>.

a<p>Abbreviations: bis-<i>p</i>NPP, bis-<i>p</i>-nitrophenyl phosphate; NSAAAPPpNA, N-succinyl-Ala-Ala-Ala-Pro-Phe-<i>p</i>-nitroanilide; PGPApNA, pyroglutamyl-Pro-Arg-p-nitroanilide; SAAApNA, succinyl-Ala-Ala-Ala-<i>p</i>-nitroanilide; PAMCA, Phe-Arg-methylcoumarine amide; SLLVTMCA, succinyl-Leu-Leu-Val-Tyr-methylcoumarine amide.</p>b<p>Kinetic parameters determined at 30°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066605#pone-0066605-g001" target="_blank">Figure 1</a> legend and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066605#s2" target="_blank">Materials and Methods</a>.</p>c<p>Previously reported numbers for the kinetic parameters are obtained from Brenda <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066605#pone.0066605-Scheer1" target="_blank">[26]</a>, and listed in parentheses.</p

Differentially regulated protein acetylation in ALS and non-ALS spinal cords by Western blotting and immunoprecipitation.

<p>(<b>A</b>) Western blotting analysis of total acetylated proteins. The urea-soluble proteins from ALS and non-ALS spinal cords were resolved on SDS-PAGE and followed by Western blotting using the antibody against acetyl-lysine. Arrows indicate bands found in ALS spinal cords, while arrowheads indicate the bands found in non-ALS counterparts. (<b>B</b>) Immunoprecipitation of the acetylated proteins. The soluble protein fractions were immunoprecipitated with the antibody against acetyl-lysine, resolved by SDS-PAGE and stained with Sypro Ruby. The protein bands labelled with U0, U1, … U8 were recovered, digested with trypsin and identified with LC-MS/MS. The proteins that were identified by LC-MS/MS are indicated to the left.</p

Optimal temperature (upper) and pH (lower) for hypothetical proteins from <i>H.</i><i>pylori</i>.

<p>The heat map colors represent the relative percentages of activity (in terms of <i>k_cat</i>) as compared to the maximum (100%) within each enzyme. k_cat values were determined using nonlinear regression to fit the values for initial velocity and substrate concentration to the Michaelis-Menten equation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066605#s2" target="_blank">Materials and Methods</a>. The pH dependence of a reaction was tested in the range of pH 4.0–9.5 at 30°C, and the temperature dependence in the range of 30–70°C at the optimal pH (4.0 for HP0142 and HP0959; 6.0 for HP0179 and HP0310; 7.5 for HP0267; and 4.5 for HP1037). The buffers used were: citrate (pH 4.0–5.0), acetate (pH 5.0–6.0), MES (pH 6.0–7.0), HEPES (pH 7.0–8.0), Tris-HCl (pH 8.0–9.0) and glycine (pH 9.0–9.5), all at 100 mM. Reaction conditions: [E]_o =  0–12 nM, [substrate: HP0310 (<i>p</i>NPacetate and acetylated xylan), HP0267 (adenosine), HP1037 (N-succinyl-Ala-Ala-Ala-Pro-Phe-<i>p</i>-nitroanilide), HP1079 (ATP), HP0959 (GTP), HP1042 (bis-<i>p</i>NPP)] ranging from 0 to 20 mM. Three independent experiments were performed for each parameter and graphs were plotted using mean values.</p

Acetylated Lysine and Peptides of GFAP Identified by LC-MS/MS^a.

a<p>Acetylation was confirmed by MS and MS/MS of the peptide.</p>b<p>Numbering according Genbank accession # P14136; underline indicates the acetylated lysine.</p>c<p>Monoisotopic mass of the neutral peptide.</p>d<p>Mascot algorithm score of each acetylated peptide.</p

Genes tested in this paper.

<p>Genes tested in this paper.</p

Identification of the Acetylated Proteins in ALS Spinal Cord by Immunoprecipitation and LC-MS/MS^a.

a<p>The proteins that were immunoprecipitated with the antibody against acetylated lysine were resolved on SDS-PAGE and stained with Rubby-RED. The protein bands corresponding to the Western blots using the same antibody were excised and identified LC-MS/MS.</p

The acetylated lysine residues in GFAP.

<p>(<b>A</b>) The GFAP sequence with six identified positions for lysine acetylation. Bold sequence, the identified tryptic peptide; underlined K, the acetylated lysine residue. (<b>B</b>) A schematic diagram of human GFAP structure with four α-helical coiled-coil domains (CC1a, CC1b, CC2a, CC2b) and the positions for acetylation (arrows). The acetylated lysines fell into the highly conserved coiled-coil domains.</p