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C.N.R. Short-term Mobility Program 2008 Gabriella Pocsfalvi Proteomic analysis of Sulfolobus solfataricus flagellum Introduction In this project in collaboration with Biomedical Analysis Department, Institute of Medical Physics and Biophysics (IMPB), University of Munster (Germany) we aim to study the motility organelle of the aerobic thermoacidophilic crenarchaeone Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80°C. Most of the archaea have small flagellar filaments (10-14 nm diameter) which are primary responsible for motion but they supposed to have fundamental role also in cell-cell communication, signaling, adherence, nutrient and extracellular DNA uptake as well. Nonetheless flagellation in archaea is widespread, structural and functional characterization of archaeal flagella today is still far less comprehensive than bacterial flagella or pili. The fla operon of S. solfataricus encodes seven open reading frames: five putative accessory proteins (G, F, H, I, J), one structural protein (flaB) and one ORF (SSO2322) of which homolog can only be found in Sulfolobus genomes. S. solfataricus carries only one copy of the flagellin gene and does not show reversed swimming or tumbling. Therefore, it is supposed that the structure of flagella from this organism should be homogenous, in contrast to polymorphic flagella from other archaeal species. Images of flagellar filaments isolated from S. solfataricus have recently been acquired by transmission electron microscopy; and transcriptional regulation of flaB genes in different nutrient conditions has also been reported (Szabo et al., J. Bacterol, 189, 4305, 2006). Archaeal flagellar proteins are secreted via a dedicated flagellar secretion system. Flagellin protein of S. solfataricus have, for example, an unusual “type IV pilin-like” signal peptide which is cleaved by the archaeal homologue (PibD, preflagellin peptidase) of the type IV pilin signal peptidase. Interestingly, the IV pilin-like signal sequences are not only found in flagellin protein but also in the precursors of other 15 extracellular proteins, like the solute (sugar) binding components of ABC transporters. Isolation, structural and functional characterization of the native protein products of fla operon expressed by S. solfataricus, and especially the flagellar bindosome (i.e. all the protein interacting with flagellum and isolated with it), however, are still missing. Therefore, the principal aim of this project was to apply mass spectrometry based proteomics for the exploration of the protein composition of flagellar fraction isolated from S. solfataricus. Preparation of flagella fraction from S. solfataricus P2 for proteomic analysis S. solfataricus P2 grown at 80 °C and pH 3.2 in rich medium (TYS) up to the middle exponentially phase (in 36h). Flagellar filaments were isolated both from the sheared cells (S) and from the culture supernatant (E) by CsCl gradient ultracentrifugation using a modified method of Kalmokoff (J. Bacterol., 1988, 170, 1752-1758). Shearing were performed twice resulting in samples S1 and S2, respectively (Figure 1 a). From the three samples (S1 and S2 sheared and E3 obtained from the culture supernatant) the following five fractions were collected after CsCl gradient centrifugation (Figure 1.a): S1 Flag_SSO_Sheared1 1 C.N.R. Short-term Mobility Program 2008 S2a Flag_SSO_Sheared2a (upper band) S2b Flag_SSO_Sheared2b (lower band) E3a Flag_SSO_Exo3a (upper band) E3b Flag_SSO_Exo3b (lower band) Gabriella Pocsfalvi Protein concentrations were measured by the Fluoroprofile kit (Sigma) and 8 µg of each fraction were loaded on sodium dodecyl sulphate-ployacrylamide gel electrophoresis (SDS-PAGE) (Figure 1.b). 1D-SDS-PAGE based proteomics 1D-SDS-PAGE separation and peptide extraction: Sample (8 μg) was solved in 20 μL buffer (0.125 M Tris-HCl pH 6.8, 4% SDS, 20% Glycerol, 0.2 M DTT, 0.02% bromophenol blue), boiled at 100 °C for 5 min and loaded on 12% polyacrylamide gel according to Laemmli. Selected gel bands were cut manually and in-gel digestion was performed according to Shevchenko using 30 μL 10 mM DTT in 100 mM NH4HCO3 for reduction, 30 μL 55 mM IAA in 100 mM NH4HCO3 for alkylation and 30 μL trypsin and/or chymotrypsin at 6 ng/μL concentration in 5% acetonitrile and 25 mM NH4HCO3 for enzymatic digestion. Samples (10 μL) were analyzed by nanoflow-HPLC-ESIMS/MS. Figure 1. (a) S. solfataricus flagellar samples separated by ultracentrifugation from sheared cells (S1 and S2) and from the extracellular medium (E3). (b) SDS-PAGE of samples obtained after ultracentrifugation: S1, S2a, S2b, E3a and E3b. Gel bands between 27-34.6 kDa were selected for nano-HPLC-ESI-MS/MS IDA analysis. 2 C.N.R. Short-term Mobility Program 2008 Gabriella Pocsfalvi Nano-HPLC-ESI-MS/MS analysis: Tryptic and chymotryptic peptides were analyzed by a hybrid quadrupole time-of-flight (QTOF) instrument, QSTAR Elite (Applied Biosystems, Foster City, CA/Toronto Canada) equipped with a nanoflow electrospray ion source. Pulled silica capillary (360 OD/100 ID, Tip 30 ID) was used as nanoflow tip. Samples (1-10 μL out of 40 μL) was loaded, purified and concentrated on a pre-column PepMap, C18, 5mm length, 300Å, (LCPackings, Sunnyvale, CA USA) at 30 μL/min flow rate. Peptide separation was performed on a capillary column, PepMap, C18, 15cm length, 75μm ID, 300Å (LCPackings, Sunnyvale, CA USA) using solvents A) 2% acetonitrile in 0.1% formic acid (HCOOH) and 0.025% trifluoroacetic acid (TFA) and B) 98% acetonitrile in 0.1% HCOOH and 0.025% TFA at flow rate 300 nL/min by Ultimate 3000 (Dionex, Sunnyvale, CA USA). The following gradient was used: 5-50% B in 30 min, 5098% B in 6 sec. CID experiments were carried out in information dependent analysis (IDA) mode using the Analyst QS 2.0 software. Nitrogen was used as collision gas. Database search was performed by Mascot Server 2.2 program (Matrix Science Ltd, London UK) using the following criteria: database: NCBInr (03-Apr-2008), taxonomy: Sulfolobus solfataricus, type of search: MS/MS Ion Search, enzyme: trypsin with one missed cleavage site allowed, fixed modifications: carbamidomethyl, variable modifications: oxidation on methionie, automatic error tolerant search; peptide tolerance 50 ppm, MS/MS tolerance 0.08 Da. SELDI-TOFMS: SELDI-TOF mass spectra were obtained using PCS-4000 (BioRad) linear LDI instrument on the complex protein samples without separation and prior enzymatic digestion. NP20 protein chips (BioRad) was used and sinapinic acid was applied as matrix. RESULTS SDS-PAGE bands present in the mass range between mass markers at 27 and 34.6 kDa (Figure 1 b) were cut from sample S1, S2a, E3a and E3b, and were analyzed in information dependent analysis mode by nano-HPLC-ESI-MS/MS. Identified proteins are listed in Table 1. Among these proteins we could not identified the putative flagellin B protein (SSO2323) (Figure 2). LAGLDTAIILIAFIITASVLAYVAINMGLFVTQKAKSTINKGEETASTALT LSGSVLYAVNYPLNTRSYWIYFTVSPSSGVSSVELSPTTTAISFTASAEG VTYSNIYKYTLLTVSPSELANVVYANGQYLDLVNQQTSAGQTYVYYP NPYYALLALNYTLYNYYLSTKTPSPIFINSSILSLSSLPSWLKNDNSFTFT LNISGKLVTYYVFVNQTFAFTYPVAGDPLIGSAIAPAGSVIGVILLFGPD LGSHVFQYQTITIQITPNIGSPLTISEYIYQPEGSVSVIG Figure 2. Amino acid sequence of processed putative flagellin B protein (SSO2323) 3 C.N.R. Short-term Mobility Program 2008 Gabriella Pocsfalvi In sample S1 three hypothetical protein have been identified together with a peptidase related protein (SSO2181). The most abundant protein in sample S1 is SSO0881 (Mw. 25020Da) a conserved hypothetical protein similar to yeast and mammalian VPS24, which has been implicated in cell sorting and trafficking. Another putative non characterized protein in the same sample is SSO2749. This protein shows conserved domain of Linocin_M18 bacteriocin protein. Many Grampositive bacteria produce antimicrobial peptides, generally termed bacteriocins. These polypeptides usually has less than 50 amino acid residues long cationic, contain an amphiphilic or hydrophobic region, and often kill their target cells by permeabilising the cell membrane. Antimicrobial peptides with these characteristics are also produced by plants and a wide variety of animals, including humans, and are thus widely distributed in nature. The Linocin_M18 region is found mostly in eubacteria, though homologous sequences have been identified in archaea as well. The best-scoring hit on the sequence of SSO2749 was member pfam04454. The third hypothetical protein, SSO0389 is a TPA surface layer glycoprotein based on homology. In sample S2 subunits of two large protein complexes, thermosome and proteosome were found. Thermosome of Sulfolobus solfataricus, a type II chaperonin composed of eight alpha and eight beta subunits, has been shown to be an RNAbinding protein that participates in ribosomal RNA processing. Proteasomes, on the other hand, are large, organelle-like structure. Their proteolytic active sites are compartmentalized within a central chamber formed by rings of proteins stacked into a cylindrical particle. This is common to energydependent proteases and is postulated to minimize cellular damage as well as facilitate hydrolysis of substrate proteins. Proteasome and thermosome are believe to be compartmentalized in the cytosol, therefore, we have concluded that sample S2 were contaminated due to cell lyses occurred sample defrost prior cell shearing. To remove contamination sample S2 was further purified by CsCl gradient ultracentrifugation. The most abundant protein identified in sample E3 obtained from the culture broth, similarly to sample S1, was SSO0881 hypothetical protein. Amongst other hypothetical proteins, membrane transporter, ABC transporter, ATP binding component, transposons were also identified, and there role in the bindosome of S. solfataricus will be the subject of further studies. 4 C.N.R. Short-term Mobility Program 2008 Gabriella Pocsfalvi Table 1. Proteins identified in samples S1, S2 and E3 by SDS-PAGE nano-HPLC-ESI-MS/MS. Sample S1 S2a 1DE band 1 2 6 7 E3a 1 E3b 2 1 2 3 Locus tag SSO0881 SSO2749 SSO0389 SSO2181 SSO0282 SSO0862 SSO0216 SSO0405 SSO0732 SSO0356 SSO0407 SSO0768 SSO3254 SSO0421 SO0282 SSO0758 SSO0407 SSO0862 SSO073 SSO0286 SSO0345 SSO2450 SSO0736 SSO0216 SSO1514 SSO094 SSO0421 SSO1457 SSO0698 SSO0732 SSO0881 SSO0451 SSO1960 SSO0881 SSO0881 SSO0451 SSO2734 SSO0200 SSO3250 SSO3079 SSO2241 SSO0167 Acc. Num. NCBInr gi|15897772 gi|13816077 gi|13813536 gi|13815479 gi|13813422 gi|13814044 gi|13813351 gi|13813558 gi|13813902 gi|13813501 gi|13813560 gi|13813941 gi|13813362 gi|13813572 gi|13813422 gi|13813928 gi|13813560 gi|13814044 gi|13813906 gi|13813429 gi|13813486 gi|13815753 gi|13813904 gi|13813351 gi|13814743 gi|13814120 gi|13813572 gi|13814682 gi|13813867 gi|13813902 gi|15897772 gi|15897382 gi|15898756 gi|15897772 gi|15897772 gi|15897382 gi|13816059 gi|13813336 gi|13816696 gi|13816494 gi|13815540 gi|13813298 SSO0079 SSO0210 SSO0512 SSO1845 gi|13813212 gi|13813345 gi|13813671 gi|13815103 gi|13814462 gi|13816544 gi|13816671 gi|13814089 gi|13815479 SSO3123 SSO1266 SSO0909 SSO2181 Name Score Mw Mascot hypothetical protein Conserved hypothetical protein Hypothetical protein Peptidase related protein Thermosome beta subunit Thermosome alpha subunit elongation factor 1-alpha PCNA Conserved hypotetical protein Phosphate regulatory protein, putative metE-2 Activator 1, replication factor C, small subunit DNA-directed RNA polymerase, subunit B'' AAA family ATPase Thermosome beta subunit Peptidyl-prolyl cis-trans isomerase, FKBP-type rotamase metE Thermosome alpha subunit Proteasome subunit Conserved hypothetical protein LSU ribosomal protein L1AB TATA binding protein (TBP)-interacting protein, putative Conserved hypothetical protein Elongation factor 1-alpha Conserved hypothetical protein Fibrillarin-like pre-rRNA processing protein AAA family ATPase NAD specific glutamate dehydrogenase SSU ribosomal protein S5AB Conserved hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein Conserved hypothetical protein Membrane transporter BPS2 protein homolog (bps2) Methyl coenzyme M reductase system, component A2 homolog Bacterial-like DNA primase (dnaG) DNA/pantothenate metabolism flavoprotein (dfp) First ORF in transposon ISC1217 First ORF in transposon ISC1217 ABC transporter, ATP binding protein Hypothetical protein Conserved hypothetical protein AAA family ATPase, p60 katanin Peptidase related protein 347 83 46 31 316 189 101 88 76 34 33 32 29 25 418 180 142 120 97 89 79 75 63 57 50 47 46 44 39 34 749 178 44 304 329 153 32 201 42 39 36 33 25020 29523 131899 117402 60387 59695 48573 29333 30232 40360 38340 37774 54173 86578 60387 26700 38340 59695 26639 42681 24512 53283 28095 48573 29968 26520 86578 45538 24200 30232 25020 31896 31687 25020 25020 31896 11874 11084 27496 46490 68610 42541 32 32 32 32 30 29 28 143 25 45382 45892 27584 22942 32315 51345 17612 42278 117402 5 C.N.R. Short-term Mobility Program 2008 Gabriella Pocsfalvi Intact protein analysis in samples S1 and the re-purified S2 by SELDI-TOFMS SELDI-TOFMS was used to analyze samples S1 and S2 after repeated CsCl gradient ultracentrifugation. Figure 3 shows the protein pattern of sample S1. The most abundant protein component of this sample has a molecular mass 24804 Da. This is in agreement with the nanoHPLC-ESI-MS/MS results, and due to the hypothetical protein identified as SSO0881. Sample S2 after repeated step of purification has a similar pattern as S1 (Figure 4). In addition to the high intensity peak at m/z 24908, one can observe a couple of lower intensity peaks at 30-31 kDa as well. These peaks could correspond to structural flagellin proteins (Mw. 31171 Da). Further analyses of this sample, in addition to the newly prepared flagellin samples are in progress. 20000 30000 40000 50000 60000 24904.00 15 uA 10 1050167151_spotB_2.6 5 23288.75 0 20000 30000 40000 50000 60000 Figure 3. SELDI-TOF mass spectrum of Sample S1 on NP20 proteinchip. 20000 30000 40000 50000 60000 24908.44 30 uA 20 1050167151_spotD_3.6 23290.44 10 19992.04 26389.65 31273.82 30136.69 49919.74 0 20000 30000 40000 50000 60000 Figure 4. SELDI-TOF mass spectrum of Sample S2 (S2a and S2b) after repeated step of purification and acquired on NP20 proteinchip. 6