Systematic Identification of the Subproteome of Escherichia coli Cell

Oct 25, 2006 - Systematic Identification of the Subproteome of Escherichia coli Cell Envelope Reveals the Interaction Network of Membrane Proteins and...
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Systematic Identification of the Subproteome of Escherichia coli Cell Envelope Reveals the Interaction Network of Membrane Proteins and Membrane-Associated Peripheral Proteins Chuan-zhong Huang,†,§ Xiang-min Lin,‡,§ Li-na Wu,‡ Dan-feng Zhang,† Dong Liu,† San-ying Wang,† and Xuan-xian Peng*,† Center for Proteomics, Department of Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, People’s Republic of China, and State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China Received May 27, 2006

Membrane proteins of Gram-negative bacteria are key molecules that interface the cells with the environment. Despite recent proteomic identification of numerous oligomer proteins in the Escherichia coli cell envelope, the protein complex of E. coli membrane proteins and their peripherally associated proteins remain ill-defined. In the current study, we systematically analyze the subproteome of E. coli cell envelope enriched in sarcosine-insoluble fraction (SIF) and sarcosine-soluble fraction (SSF) by using proteomic methodologies. One hundred and four proteins out of 184 spots on 2D electrophoresis gels are identified, which includes 31 outer membrane proteins (OMPs). Importantly, our further proteomic studies reveal a number of previously unrecognized membrane-interacting protein complexes, such as the complex consisting of OmpW and fumarate reductase. This established complete proteomic profile of E. coli envelope also sheds new insight into the function(s) of E. coli outer envelope. Keywords: membrane proteins • membrane-associated protein complex • Escherichia coli • OmpW • fumarate reductase

Introduction Gram-negative bacteria have a cell wall composed of two membranes: the outer membrane (OM) and and the inner membrane (IM). The two membranes sandwich a region known as periplasm. Outer membrane proteins (OMPs), between the outmost of the cell and its external natural environment, are important for the response to environmental changes in osmolarity, temperature, drugs, chemicals, and host defense.1-5 Accordingly, studies on OMPs have been increasingly reported. Sodium carbonate and sodium-lauryl sarcosinate (sarcosine) enrichments are the two methods conventionally used in the fractionation/sample-preparation of bacterial OMPs for subproteome analysis (the sodium carbonate method may enrich all membrane proteins including OMPs and inner membrane proteins (IMPs)).6 Recently, these OMPs based on the membrane fraction enriched by the sodium carbonate or sarcosine methods have been characterized in microorganisms including Caulobacter crescentus, Pseudomonas aeruginosa, Helicobacter pylori, Chlorobium tepidum, Francisella tularensis, Synechocystis sp., Pasteurella multocida, Methylococcus capsulatus, showing the identification of 7-41 OMPs.6-13 As for the proteomic * To whom correspondence should be addressed. Dr. Xuanxian Peng, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, People’s Republic of China. Tel., (86)-592-218-7987; fax, (86)-592-218-1015; e-mail, [email protected]. † Xiamen University. ‡ Sun Yat-sen University. § Authors contributed equally.

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Published on Web 10/25/2006

study on the OMPs of Escherichia coli, Molloy identified 26 proteins out of 62 OMPs expressed under the described culture conditions and enriched by the sodium carbonate method.14 Other investigators identified less than 20 OMPs.15-17 Overall, these identified E. coli OMPs only account for a small fraction of total annotated OMPs (to date, a total of 82 OMPs have been annotated by the latest Swiss-Prot database in E. coli), suggesting that 69-75% OMPs were not determined by previous 2-DE proteomic studies based on the sodium carbonate or sarcosine enrichments. More recently, Stenberg18 identified some protein complexes in the E. coli cell envelope by means of the blue native-PAGE in combination with second-dimension SDS-PAGE and mass spectrometry (MS). Their results indicated that soluble proteins might be associated with the bacterial membrane. Other reports also indicated that some soluble metabolic enzymes are observed in the 2-DE gels of bacterial membrane fractions,12,13,19 which may be peripherally associated proteins bound to OMPs/ IMPs.13,20,21 Recently, a membrane-associated protein complex has been shown to play important roles during membrane biosynthesis, antibiotic-resistance, and response to environmental factors in bacteria.22-25 However, to date, the membraneassociated peripheral proteins remain poorly defined. As mentioned earlier, Stenberg18 reported their systematic investigation on membrane protein complexes in E. coli, showing the identification of 34 distinct IMP complexes and 9 different OMP complexes. Stenberg’s study provided plentiful informa10.1021/pr060257h CCC: $33.50

 2006 American Chemical Society

Proteomic Analysis of E. coli Cell Envelope

tion on oligomeric states of membrane proteins, but the identified complex contained few OMPs- or IMPs-associated peripheral protein components. The failure to detect the peripheral protein components in Stenberg’s study is likely due to their method limitation of isolating membrane fraction (the protein-protein interactions within the OMPs- or IMPsassociated protein complexes are fragile and could easily be destroyed if using severe detergents during enrichment). Given that soluble proteins participate in forming membraneassociated protein complexes at the bacterial external envelope, we concurrently analyzed E. coli proteins in sarcosine-soluble fraction (SSF) along with the analysis of OMPs in the sarcosineinsoluble fraction (SIF). Furthermore, one-dimensional nativePAGE and two-dimensional SDS-PAGE were used for the detailed analysis of the protein-protein interaction network consisting of membrane proteins and the interacting peripheral proteins. We interestingly found that a large number of metabolic enzymes were identified in SSF, and a portion of these enzymes were associated with OMPs or IMPs. Importantly, one of our findings suggested that fumarate reductase (Frd) binds to OmpW (an iron-related protein), presumably via the iron-sulfur protein FrdB. This systematic analysis of expression profile of E. coli envelope proteins, including revealed membrane-associated protein complex, also provides a better understanding of protein function(s) of the bacterial outer envelope.

Experimental Procedures Preparation of SIF, SSF, and Membrane Vesicles. E. coli K12 was cultured in LB medium at 37 °C for 10 h as seeds. Fresh cultures were diluted 1:100 in LB medium, and growth was continued for 18 h at 37 °C. Isolation and purification of membrane vesicles, SIF, and SSF were performed as previously described with a few modifications.19,26 The bacterial cells were harvested by centrifugation at 6000g for 10 min at 4 °C and washed with sterile saline three times. The cells were resuspended in 3 vol of sonication buffer (50 mM Tris/HCl, pH 7.4) before being disrupted by intermittent sonic oscillation. Unbroken cells and cellular debris were removed by centrifugation at 12 000g for 10 min. Supernatant was collected and centrifuged at 100 000g for 40 min at 4 °C, and the resulting pellet was resuspended in 0.01 M phosphate-buffered saline (PBS), pH 7.4, and then was used as membrane vesicles for nativePAGE, far-Western blotting, and co-immunoprecipitation (CoIP), or was further applied for preparing of SIF and SSF. For the preparation of SIF and SSF, the pellet was resuspended in 2% sarcosine (pH 7.4) and incubated at room temperature for 20 min, followed by centrifugation at 100 000g for 40 min at 4 °C. The resulting pellet was resuspended in PBS as SIF. The supernatant was further deposited in cold acetone (1:4) at -20 °C for 4 h, and the resulting pellet was resuspended in PBS as SSF by centrifugation at 12 000g for 20 min at 4 °C. Protein quantitation was determined by the Bradford method as previously described.27 For the preparation of membrane vesicles in iron-limit condition, we used the method as mentioned above except for the addition of the ferrous iron chelator 2,2-dipyridyl (DIP) (Sinopharm Chemical Reagent CO., Ltd, China) at 200 µM in LB medium.28 Analysis of SOD Activity. After unbroken cells and cellular debris were removed by centrifugation at 12 000g for 10 min, the samples were divided into supernatant and pellet by centrifugation at 100 000g for 40 min at 4 °C. The SOD-specific

research articles activity of the supernatant and the pellet was determined using the spectrophotometric protocols.29 Second-Dimension Gel Elctrophoresis (2-DE). The electrophoretic separation of proteins was performed essentially as described previously.19 Twenty micrograms of protein was loaded on the first dimensional gel, and isoelectric focising (IEF) was conducted overnight for a total of 10 000 Vh. For the second dimension, the first dimensional gel was equilibrated for 15 min by incubating/rocking in a solution of 0.06 M Tris/ HCI, pH 6.8, 2% SDS, 5% β-mercaptoethanol, and 10% glycine. It was then embedded onto a 12% SDS-PAGE gel according to size. Proteins were visualized by staining with CBB R-250 (Merck, Darmstadt, Germany), and then the wet gels were scanned and analyzed using a GSC-8000 system (Bio-Rad, Hercules, CA). One-Dimensional Native-PAGE and Two-Dimensional SDSPAGE. Analysis of membrane complexes was performed as previously described with a few modifications.18,30 Approximately 150 µg of E. coli membrane proteins was used for each lane of the native-PAGE. Membrane vesicles in PBS were solubilized at 4 °C for 30 min in loading buffer (100 mM TrisCl, 200 mM DTT, 20% glycerol, and 0.2% bromophenol blue) containing 0.5% Triton X-100. Native-PAGE was performed at 80 V for 4 h using a 4% stacking and an 8% separating gel. The SDS-PAGE of the second dimension was performed using a 5% stacking and a 12% separating gel. In-Gel Protein Digestion. The CBB-stained protein spots were cut from the gel and destained three times with 100 µL of 50 mM ammonium bicarbonate/50% acetonitrile (ACN) for 30 min at ambient temperature. The destained gel pieces were completely dried in a SpeedVac vacuum concentrator (Savant Instruments, Farmingdale, NY). Gel pieces were reswollen with 8 µL of 25 mM ammonium bicarbonate containing 10 ng of trypsin at 4 °C for 1 h. After 13.5 h of incubation at 37 °C, the gel pieces were vacuum-dried, 8 µL of 50 mM ammonium bicarbonate was added, and the samples were incubated at 37 °C for 1 h. The supernatant was transferred into another microtube, and 8 µL of 2.5% trifluoroacetyl (TFA)/50% ACN was used for the second step of extraction at 30 °C for 1 h, and the supernatant was combined with the first one. Finally, to extract hydrophobic peptides, 8 µL of 100% ACN was used at room temperature for 1 h, and the supernatant was also combined with the previous supernatants. The combined supernatants were then dried in a SpeedVac and redissolved with 2 µL of 0.5% TFA. MALDI-TOF/MS Analysis. As previously described,31 0.5 µL of the sample solution, along with equivalent matrix solution (R-cyano-4-hydroxycinnamic acid), was mixed and applied onto the MALDI-TOF target for MALDI-TOF/MS analysis. MALDI-TOF spectra were calibrated using trypsin autodigestion peptide signals and matrix ion signals. MALDI analysis was performed using a fuzzy logic feedback control system (Reflex III MALDI-TOF system Bruker, Karlsruhe, Germany) equipped with delayed ion extraction. Peptide masses were searched against the Swiss-Prot database using the MS-Fit program (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) or the NCBI database using the MASCOT program (http://www.matrixscience. com) against E. coli K12 complete genome database in NCBI. Bioinformatics Analysis. The subcellular localization was analyzed by the Swiss-Prot database. The calculation of the grand average of hydropathicity (GRAVY) value for a given protein was determined through the SOSUI database (available Journal of Proteome Research • Vol. 5, No. 12, 2006 3269

research articles at http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html). The proteins exhibiting positive GRAVY values were recognized as hydrophobic. Western Blotting and Far-Western Blotting. Proteins from the gels were transferred to a nitrocellulose (NC) membrane for 1 h at 60 V in transfer buffer (48 mM Tris, 39 mM glycine, and 20% methanol) at 4 °C. The NC membrane was blocked for 2 h with 5% skim milk in TNT buffer (1.211 g of Tris, 8.77 g of NaCl, and 500 µL og Tween-20 in 1 L TNT, pH 7.0) at room temperature. After rinsing three times for 10 min with TNT buffer, the NC membrane was incubated with rabbit antiOmpW at a dilution of 1:5000 in TNT buffer containing 5% skim milk for 1 h at room temperature on a gentle shaker. The membrane was rinsed three times for 10 min with TNT buffer and incubated with goat anti-rabbit-horseradish peroxidase at a dilution of 1:1000 in TNT buffer containing 5% skim milk for 1 h at room temperature. The membrane was then washed with TNT buffer and developed with substrate (dimethylaminoazobenzene) until optimum color developed. The membrane was washed with sterile water, and then far-Western blotting was performed as previously described with a few modifications.32,33 Before the rabbit anti-OmpW was added, the NC membrane was incubated with 5 mg of E. coli K12 membrane proteins in 5 mL of TNT buffer containing 5% skim milk for 24 h at 4 °C. The other procedures are the same as above. Co-IP. Rabbit anti-OmpW was purified with 33% saturated ammonium sulfate and incubated with excess E. coli K12 membrane vesicles for 12 h at 4 °C on a gentle shaker. Then nProtein A Sepharose 4 Fast Flow (Amersham Biosciences Corp.) was added to this mixture and incubated for 12 h at 4 °C on a gentle shaker. The nProtein A Sepharose 4 Fast Flow was collected by centrifugation at 2000g for 3 min and cleaned six times for 10 min with pH 7.0 Tris-HCl buffer followed by incubation in pH 2.4 Gly-HCl buffer for 2 h. After centrifugation at 2000g for 3 min, the supernatant was precipitated by 33% saturated ammonium sulfate at 4 °C for 2 h. The pellet was resuspended in pH 7.0 Tris-HCl buffer and prepared for SDSPAGE.

Results Construct of the Theoretical Map of E. coli OMPs. There were some discrepancies of annotated data of E. coli OMPs between different databases. To circumvent this problem, the theoretical 2-DE maps of OMPs were constructed according to the genome annotation from the Swiss-Prot database to show the distribution of the total predicted 82 proteins on the gel. The y-axis and the x-axis were drawn on a logarithmic scale to represent migration during SDS-PAGE and on a linear scale to imitate protein mobility during IEF, respectively, to simulate protein mobility during 2-DE. As shown in the gel, out of the total 82 OMPs, most (79) were located to the theoretical pI range of 4-9. As for the theoretical molecular mass, most proteins on the 2-DE were located in the range of 14-200 kDa, except for four low-molecular weight protein spots (spots 7982) (Figure 1 in Supporting Information). Of the 82 OMPs in the Swiss-Prot database, 14, 24, 8, and 17 are porins, lipoproteins, TonB-dependent receptor family proteins, and usher proteins, respectively; 40 are integral membrane proteins, and 42 are un-integral membrane proteins. Analysis of GRAVY indicated that only three OMPs were positive (Table 1). Membrane Fraction Is Free of Cytoplasmic Proteins. To show the credibility of the method of isolating the membrane 3270

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Huang et al. Table 1. Summary of the Characteristics of Identified Proteins in 82 OMPs of E. coli

parameter

no. (%) no. (%) no. (%) identified identified identified total by Molloy in this study in total

pI 39-11 MW 1108090-200 Protein classification Porin Lipoprotein TonB-dependent receptor family Usher protein Other Location classification Integral membrane protein Un-integral membrane protein GRAVYa Positive Negative a

79 3

25 0

31 0

36 0

4 54 10 14

0 24 1 0

0 27 4 0

0 32 4 0

14 24 8

5 5 6

6 9 7

6 11 8

17 19

0 9

0 9

0 11

40 42

13 12

13 18

14 22

3 79

0 25

1 30

1 35

The value of GRAVY is calculated without a signal peptide.

fraction used in this study, activity of SOD enzyme was applied to evaluate whether the fraction was contaminated with the cytoplasmic fraction because the SOD enzyme is widely distributed in the cytoplasm. The SOD-specific activity of the soluble fraction and membrane fraction was measured using the spectrophotometric protocols,29 showing 5000U/mg and 7U/mg in the soluble fraction and the membrane fraction, respectively. The result showed that there was strong SOD enzyme activity in the supernatant, but few SOD enzyme activity in the pellet, suggesting that the membrane fraction is free of cytoplasmic components. Identification of Protein Composition in the SSF and SIF Fractions. 1. 2-DE Maps of SSF and SIF. To obtain a complete protein expression profile of E. coli membrane proteins and their associated soluble proteins, both of SSF and SIF were analyzed by 2-DE. Image analysis of the 2-DE gels indicated that, mostly, proteins were focused on the pH range of 4-8, showing 82 spots in the SIF and 117 spots in the SSF (Figure 1). 2. Protein Identification by MALDI-TOF/MS. The proteins on the 2-DE gels were identified by MALDI-TOF/MS. The gel pieces were sliced and washed, reduced and alkylated, digested in-gel with trypsin, extracted from the gel, and subjected to PMF for identification after desalting/concentration. In most cases, mass spectra showed 10 or more peptides that were matched to a Swiss-Prot E. coli database homologue. Peptide masses were searched against the Swiss-Prot database using the MASCOT program (http://www.matrixscience.com). In total, 95.1% of 82 spots (78 spots) in SIF and 90.6% of 117 spots (106 spots) in SSF were identified, representing 42 and 76 proteins, respectively (Table 1 Supporting Information). Of the 76 proteins identified from SSF, 36 (47.4%) were enzymes, in which only 6 were documented to be membrane proteins. Analysis of Identified Proteins. 1. Classification of Identified Proteins According to Cellular Role Categories. In the current study, 104 proteins from 184 spots in both of SIF and SSF were identified and classified as 27 OMPs (6 porins, 9

Proteomic Analysis of E. coli Cell Envelope

research articles

Figure 1. 2-DE maps of a sarcosin-insoluble fraction (A) and a sarcosine-soluble fraction (B). The subcellular location distribution of identified proteins in sarcosine-insoluble fraction (C-1), sarcosine-soluble fraction (C-2), and both fractions (C-3). The fractions were solubilized with lysis buffer and separated on pH 3-9.5 linear IEF gels, followed by SDS-PAGE on a 12% gel. The gels were stained with CBB G250.

lipoproteins, 3 TonB-dependent receptor family, and 9 others), 11 IMPs (2 MFP family and 9 others), 53 cytoplasmic proteins (28 enzymes, 5 membrane-associated proteins, 1 plasmid, 5 ribosomes, 2 chaperonins, and 12 others), 1 periplasmic protein, 1 flagella, and 11 location-unknown proteins (Table 2). In all, 9 OMPs, 2 IMPs, 2 cytoplasmic proteins, and 1 loaction-unkown protein could be synchronously detected in both SIF and SSF. Importantly, proteins belonging to several metabolism pathways including tricarboxylic acid cycle (TAC), glycolysis, and carbohydrate metabolism were defined. Although many proteins of SSF were identified as enzymes, most of them were associated with each other as shown in Table 2. 2. Distribution of Proteins According to Subcellular Localization. Furthermore, subcellular locations of all identified proteins were annotated based on the Swiss-Prot database and TrEMBL database as shown in Table 1 of Supporting Information. In total, 78 and 106 spots were successfully identified in

SIF and SSF, in which a total of 27 OMPs and 11 IMPs were isolated. Of the 78 identified spots in the SIF, 51 (66%), 5 (6%), 9 (12%), and 1 (1%) spots were identified as 22 OMPs, 5 IMPs, 7 cytoplasmic proteins, and 1 periplasmic location, respectively. Of the 106 spots in the SSF, 23 (22%), 12 (11%), 54 (51%), and 1 (1%) spots were identified as 14 OMPs, 8 IMPs, 47 cytoplasmic proteins, and 1 flagella. Totally, of 184 spots identified in the present study, 40%, 34%, 15%, 9%, 1%, and 1% were OMPs, cytoplasmic proteins, unknown location, IMPs, flagella, and periplasmic proteins (Figure 1). 3. Hydrophobicity Analysis. All identified proteins were analyzed using the SOSUI database at http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html, which allows the calculation of the GRAVY value for a given protein as shown in Table 1 of Supporting Information. Positive and negative GRAVY scores indicate hydrophobic and hydrophilic proteins, respectively. The GRAVY values usually vary in the range of (1.2 in mouse Journal of Proteome Research • Vol. 5, No. 12, 2006 3271

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Table 2. Classification of Identified Proteins Based on Accession Names sarcosineinsoluble

Porin

Lipoprotein

TonB-dependent receptor family Others

MFP family Others

OMP OMPC_ECOLI OMPA_ECOLI TSX_ECOLI LAMB_ECOLI OMPF_ECOLI NMPC_ECOLI YBJP_ECOLI YFGL_ECOLI TRAK1_ECOLI SLYB_ECOLI VACJ_ECOLI FECA_ECOLI YNCD_ECOLI BTUB_ECOLI OMPX_ECOLI OSTA_ECOLI OMPW_ECOLI TOLC_ECOLI MIPA_ECOLI FADL_ECOLI OMPT_ECOLI OMPP_ECOLI Imp MDTE_ECOLI ACRA_ECOLI MALK_ECOLI SECD_ECOLI TRAG4_ECOLI

Cytoplasmic Protein Enzyme Tricarboxylic acid cycle

Glycolysis

Carbohydrate metabolism

sarcosinesoluble

OMPC_ECOLI OMPA_ECOLI TSX_ECOLI

Cytoplasmic Protein Carbohydrate metabolism

Other metabolism

PUR9_ECO57 TNAA_ECOLI ASNA_ECOLI UDP_ECOLI

Membrane-associated

EFTU_ECOLI

YBJP_ECOLI YFGL_ECOLI METQ_ECOLI YBHC_ECOLI YFIO_ECOLI SLP_ECOLI

OMPX_ECOLI OSTA_ECOLI OMPW_ECOLI TOLC_ECOLI YAET_ECOLI

MDTE_ECOLI ACRA_ECOLI ATPB_ECOLI ATPA_ECOLI MBHL_ECOLI FABI_ECOLI BAES_ECOLI DHSB_ECOLI

Plasmid Ribosome

Chaperonin Others

RS7_ECOLI CLPB_ECOLI TYPA_ECO27 MREB_ECOLI

sarcosinesoluble

ACKA_ECOLI HLDD_ECOLI TALB_ECOLI DKGA_ECOLI PFLB_ECOLI ASPA_ECOLI DEOB_ECOLI TNAA_ECOLI KBL_ECOLI UPP_ECOLI AHPC_ECOLI MENB_ECOLI FABZ_ECOLI DNAK_ECOLI FTSZ_ECOLI EFTU_ECOLI PTNAB_ECOLI NUOG_ECOLI Q47544_ECOLI RS1_ECOLI RS4_ECOLI RS9_ECOLI RL5_ECOLI CH60_ECOLI TIG_ECOLI EFG_ECOLI YDBC_ECOLI SSPA_ECOLI YDFG_ECOLI SSB_ECOLI YGAU_ECOLI

OMPR_ECOLI MDH_ECOLI Q6S5V7_ECOLI ACON2_ECOLI DHSB_ECOLI ODP1_ECOLI ENO_ECOLI ALF_ECOLI TPIS_ECOLI ADHE_ECOLI GALM_ECOLI 6PGL_ECOLI PTA_ECOLI

liver plasma membrane.34 In the present study, most of the proteins were hydrophilic, and only about 13.5% proteins have positive GRAVY score, including 3 OMPs, 2 IMPs, 5 cytoplasmic proteins, and 4 unknown location proteins. According to the GRAVY values, the hydrophobicity of the OMPs was lower than that of the IMPs.34 OMP Expression by Induction. Of the 82 OMPs annotated from the E. coli K12 genome in the Swiss-Prot database, 13 were expressed by induction (Table 2 of Suppporting Information), in which some may be induced by iron. In our laboratory, FepA, FhuE, CirA, and YbiL could be found in SIF purified from an iron-limited condition, which were not detected in the SIF of a standard LB medium (Figure 2). Molloy reported the detection of AG43, Pal, YeaF, PA1, FhuA, NlpB, FepA, FhuE, CirA, and YbiL in sodium carbonate-insoluble fraction purified from specific mediums.14 Analysis of Membrane Complex. We were interested in understanding whether the non-membrane proteins in SSF and SIF were peripherally associated with membrane proteins. To this end, protein complexes solubilized in 0.5% Triton X-100 3272

sarcosineinsoluble

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YCFP_ECOLI PT1_ECOLI Periplasmic

Periplasmic TORZ_ECOLI Flagella

Flagella Unknown

FLIC_ECOLI Unknown Q8FEW0_ECOL6 YEIN_ECOL DPS_ECOLI Q8VR99_ECOLI Q9R2E3_ECOLI Q8X592_ECO57

YGFZ_ECOLI CDD_ECOLI Q93ES6_ECOLI Q57460_ECOLI YAJQ_ECOLI DPS_ECOLI

were isolated by the native-PAGE. Individual proteins in each complex were consequently resolved into a vertical line through the SDS-PADE gel and then identified by MS. Three lines clearly appeared in the gel (Figure 3A), line 1 complex, line 2 complex, and line 3 complex, and 16 proteins were identified from these complexes, in which 9 had been detected in SIF and SSF (Figure 3A, Table 3). Furthermore, we confirm the presence of OmpW

Figure 2. Comparison of 2-DE maps grown with or without iron limitation. (A) E. coli K-12 grown in conventional LB medium; (B) E. coli K-12 grown in LB medium plus iron chelator 2,2′dipyridyl (DIP). (1) YbiL, (2) FecA, (3) FepA, (4) FhuE, (5) CirA, and (6) FepA

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Proteomic Analysis of E. coli Cell Envelope

Figure 3. Native/SDS-PAGE separation of membrane protein complex with the peripheral associated proteins. Gels were stained with Coomassie blue. The name of proteins identified by mass spectrometry was indicated. (A) Cultured in standard LB medium; (B) cultured in iron-limit LB medium; (C) SDS-PAGE and Western blotting showing the changes in OmpW between standard LB (C1 for SDS-PAGE and C2 for Western blotting) and iron-limited LB (C3 for SDS-PAGE and C4 for Western blotting). Table 3. Summary of Proteins in Membrane Complexes Identified by MALDI-TOF/MS accession name

Swiss-Prot no.

description

ODP1_ECOLI FRDA_ECOLI

P0AFG9 P00363

YDGA_ECOLI FRDB_ECOLI

P77804 P0AC47

OMPW_ECOLI SLP_ECOLI

P0A915 P37194

YBJP_ECOLI

P75818

GLPD_ECOLI

P13035

DCEA_ECOLI METQ_ECOLI

P69908 P28635

ATPA_ECOLI ATPB_ECOLI DADA_ECOLI

P0ABB0 P0ABB4 P0A6J5

PYRD_ECOLI

P0A7E1

Complex 1 Pyruvate dehydrogenase Fumarate reductase flavoprotein subunit (membrane-bound) Protein YdgA precursor Fumarate reductase iron-sulfur protein (membrane-bound) OmpW precursor Outer membrane protein slp precursor Putative lipoprotein ybjP precursor Complex 2 Aerobic glycerol-3-phosphate dehydrogenase Glutamate decarboxylase alpha D-methionine-binding lipoprotein metQ precursor Complex 3 ATP synthase alpha chain ATP synthase beta chain D-amino acid dehydrogenase small subunit Dihydroorotate dehydrogenase

RL1_ECOLI RL9_ECOLI

P0A7L0 P0A7R1

50S ribosomal protein L1 50S ribosomal protein L9

in line 1 complex using antibody, due to the relatively low score of this protein identified by MS (Figure 4B). Line 1 complex included seven proteins, in which OmpW, Slp, and YbjP are OMPs; YdgA is an IMP and may function in

MW (kDa)

Mascot score

subcellular localization

99 66

62 63

cytoplasmic unknown

55 27

82 75

IM unknown

23 21

22 57

OM OM

19

58

OM

57

77

cytoplasmic

53 29

73 109

cytoplasmic OM

55 50 47

115 159 118

37

176

25 15

63 71

IM IM IM, peripheral membrane peripheral membrane cytoplasmic cytoplasmic

the periplasmic space as a dimer;18 and Frd flavoprotein subunit (FrdA), Frd iron-sulfur protein (FrdB), and pyruvate dehydrogenase (Odp1) are not membrane proteins annotated from the Swiss-Prot database (a report indicated that Frd was an integral Journal of Proteome Research • Vol. 5, No. 12, 2006 3273

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Figure 4. Identification of protein-protein interaction in membrane fraction. (A) The location of FrdB and OmpW in the native/ SDS-PAGE gel; (B) identification of OmpW by Western blotting using anti-OmpW as the primary antibody; (C) identification of the FrdB-OmpW complex by far-Western blotting. The farWestern blotting was followed by the Western blotting for OmpW identification. After Western blotting, the NC membrane was incubated with E. coli K12 membrane proteins and then incubated with anti-OmpW. (D) Co-immunoprecipitation using antiOmpW. Gels were stained with Coomassie blue. The name of proteins identified by mass spectrometry was indicated.

membrane protein).35 Frd catalyzes the last step of anaerobic respiration, and Odp1 catalyzes oxidative decarboxylation of pyruvate to form acetyl-CoA. Frd consists of two water-soluble subunits, the flavoprotein (66 kDa) subunit (FrdA), containing a covalently linked flavin adenine dinucleotide, and iron-sulfur protein (27 kDa) subunit (FrdB), containing three different iron-sulfur clusters ([2Fe-2S], [4Fe-4S], and [3Fe-4S]) and two membrane anchor subunits (15 and 13 kDa).35 OmpW functions as an ion channel in planar lipid bilayers,36,37 responding to NaCl38,39 and iron,40 and Slp is a lipoprotein which stabilizes the outer membrane during carbon starvation or stationary phase. Interestingly, the five proteins above are related to glycolysis or iron metabolism, in which FrdB, a subunit of Frd functioning as a key enzyme of glycolysis, is an iron-sulfur protein. The function of the other two proteins of YdgA and YbjP is not known. Furthermore, a far-Western blotting technique was applied to investigate whether OmpW could be bound with the other proteins in the complex. Our result indicated that the bait protein is FrdB when OmpW was used as the prey protein (Figure 4C). It is a very interesting finding because FrdB is an iron-sulfur protein and OmpW is regulated by iron.40 Further, Co-IP indicated that OmpW, FrdB, and Odp1 could be complexed (Figure 4D). Undoubtedly, Frd is complexed with OmpW through its FrdB subunit, and this complex includes Odp1. Line 2 complex included D-methionine-binding lipoprotein (MetQ), and aerobic glycerol-3-phosphate dehydrogenase (GlpD) and glutamate decarboxylase alpha (DceA), respectively, from OM and cytoplasm. MetQ is a component of a D-methionine permease. GlpD and DceA convert glycerol 3-phosphate to dihydroxyacetone and glutamate to γ-aminobutyrate, respectively. Line 3 complex consisted of four IMPs or peripheral membrane proteins, ATP synthase alpha chain (AtpA), ATP synthase beta chain (AtpB), D-amino acid dehydrogenase small subunit 3274

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(DadA), and dehydroorotate dehydrogenase (PyrD), and two cytoplasimic proteins ribosomal proteins, 50S ribosomal protein L1 (RL1) and 50S ribosomal protein L9 (RL9). Both AtpA and AtpB are subunits of ATP synthase, and both RL1 and RL9 are ribosomal proteins. PyrD is necessary to pyrimidine synthesis and, therefore, essential to the production of DNA and RNA, and DadA shows broad substrate specificity, catalyzes the oxidative deamination of most D-amino acids, and also plays a role in alanine metabolism. Therefore, complex 3 is related to amino acid and nuclear acid metabolism. Furthermore, membrane vesicles were isolated from the bacteria cultured in iron-limit medium because OmpW was related to iron at the nuclear acid level.40 Here, we present Western blotting result showing down-regulated OmpW in ironlimited LB as shown in Figure 3C. We interestingly found that there were significant changes in protein locations, although three lines still existed (Figure 3B). Out of the seven proteins in line 1 complex, Slp and YbjP moved to line 2; Odp1, FrdA, FrdB, and OmpW disappeared from the gel; and only YdgA kept at the same location. In line 3 complex, RL1 and RL9 disappeared. The comparative analysis of the two maps indicated that two complexes, YdgA (it is indeed oligomeric)18 and OmpW multicomponent complex (besides Odp1, Frd, and OmpW, Slp and YbjP may be involved in it because the location of the two proteins together moved into the direction of lower MW when Odp1, Frd, and OmpW disappeared) were included in line 1; RL1-RL9 complex, and AtpA-AtpB complex (DadA may be supposed in it) were determined in line 3 complex.

Discussion We are interested in the reason the numbers of OMPs practically identified on 2-DE is far less than annotated ones from the databases. Indeed, several reports have investigated the OM proteome in E. coli, in which 25 out of 82 annotated OMPs from the Swiss-Prot database as the highest detection results was reported by Molloy (62 OMPs were annotated in 2000 under the described culture conditions, and the two proteins of AG43 alpha chain and beta chain were included in AG43_ECOLI entry of the Swiss-Prot databases now).14 In the current study, OMPs were detected in both SSF and SIF. In total, 31 OMPs were identified, in which 26 (4 induction OMPs were included) and 14 were, respectively, from SIF and SSF, and 9 were shared by both fractions, suggesting that sarcosine extraction is an efficient method for OMP isolation which can make SIF enrich OMPs. We constructed a theoretical map of OMPs annotated from the Swiss-Prot database, showing the distribution of 82 OMPs at pI and MW. Among the 82 OMPs, 36 have been identified by the combination data of 2-DE subproteomics, in which 5 were determined only by Molloy and 11 by the present study, while 20 were detected by both. Furthermore, we analyzed the effect of pI, MW, and protein classification on the detection of OMPs as shown Table 1, indicating that OMPs could not have been detected when their pI is more than 9, and MW more than 90. In this regard, 20 proteins may appear to be difficult to separate and display using 2-DE gels because of the limitation of standard 2-DE separation. Among the other 62 proteins, 36 (58.1%) have been identified between Molloy and us. Furthermore, there was significant difference between sodium carbonate purification by Molloy and sarcosine enrichment by us, showing more TonB-dependent receptor family proteins in the sodium carbonate purification and more lipoproteins in sarcosine enrichment. All of TonB-dependent receptor family proteins were

Proteomic Analysis of E. coli Cell Envelope

identified, whereas all of usher proteins were not detected when Molloy’s and our data are combined. We listed all of these undetected proteins as shown in Table 2 (Supporting Information) and summarized the result in Table 1, indicating that the MWs of usher proteins are all greater than 80 kDa with pI’s less than 6. Furthermore, 35% integral membrane proteins and 52.4% un-integral membrane proteins were identified, showing significant difference between them. These results indicate that the characteristics of OMPs result in their detection, suggesting that solvent buffer and voltage are closely related to OMP resolution in 2-DE gel. Meanwhile, the abundance and IEF solution of OMPs may be related to the detection.13 Therefore, only the development of novel OMP-enrichment methods may provide a basic solution in the detection of the majority of OMPs by 2-DE proteomics. An interesting finding is that, for the first time, many enzymes were characterized in the membrane fraction of a prokaryotic cell. Recently, a report has shown that the same result was obtained in live plasma membrane (PM) of a rat, a eukaryotic cell, indicating that about 40% of the annotated proteins of the mouse liver PM represent enzyme subunits with a broad spectrum of catalytic activities including receptor-like protein-tyrosine phosphatase, RPTP, apoptotic proteaseactivating factor 1, and lipoxygenase.34 In the current study, approximately 47% enzymes were also determined in SSF, but the majority of enzymes belonged to energy metabolism system, which may be a difference between prokaryotic and eukaryotic cells. For the first time, our results provide the protein expression profile of SSF components, which benefits our understanding of inner membrane functions. Moreover, the protein complexes of OMPs, IMPs, and cytoplasmic proteins, including metabolic enzymes and protein synthesis components, were documented by two-dimensional native-SDS-polyacrylamide gels. Protein complexes in three lines were determined, indicating OMPs and IMPs with peripherally associated proteins could appear in line. It has been supposed that many soluble proteins are also tethered to the membranes through lipid moieties, hydrophobic patches, or charge interactions or in membrane protein complexes.18 The interaction among OMPs, IMPs, and the peripherally associated proteins is very important in some biological functions. Bacterial type III secretion machines for protein delivery into host cells are an interesting complex of OMPs and IMPs, which forms a needle-like structure and permits cytoplasmic proteins into host cells.41 Moreover, that OMPs are regulated by twocomponent systems is a cooperation function of OMP, IMP, and cytoplasmic proteins. OmpC and OmpF are controlled by EnvZ/OmpR, in which EnvZ is IMP and OmpR is a cytoplasmic protein.42 However, the knowledge about direct proteinprotein interactions between membrane proteins and the peripherally associated proteins remains poorly understood, which may be partly due to the limitation of the membrane protein complex isolation method. Indeed, purification of protein complexes in an intact form is largely dependent on the solubilization conditions used and can differ for various complexes.18 Reports have indicated that carbonate treatment and ultracentrifugation could remove some of the loosely associated proteins from the OM.13,14 In the current study, a mild detergent and simple procedure were applied to prepare the membrane vesicles for the complexes of OMPs or IMPs with their loosely associated proteins. Of the 16 proteins identified, 43.8% are loosely associated proteins. Many associated proteins could be detected in each of the three line complexes analyzed,

research articles which were not obtained in Stenberg’s report,18 suggesting that more protein complexes could be achieved by optimization of the solubilization conditions. In these complexes, Frd is composed of four subunits of FrdABCD.35 We identified FrdA (66 kDa) and FrdB (27 kDa). Another protein, corresponding in molecular mass to the membrane anchor subunit FrdC (15 kDa), was in the same complex but could not be identified. Since the complex was not abundant and the MW of FrdD is only 13 kDa, no protein spots corresponding to the FrdD were detected on the Coomassie-stained gel. The same situation occurred in the study reported by Stenberg et al.18 They indicated that an intact oligomer could be identified even if parts of its subunits were absent. Therefore, we suggest that the Frd is intact. Furthermore, we provided a direct proof using far-Western blotting that the Frd could bind with OmpW through FrdB. Further evidence indicated that this complex might include Odp1, Frd, OmpW, Slp, and YbjP. Because both OmpW and FrdB are iron-related proteins, their function may be assigned. However, the significance of the interaction waits investigation. In addition, RL1-RL9, AtpA-AtpB (DadA may be included), and oligomeric YdgA complexes were suggested, in which the RL1-RL9 complex was related to iron metabolism. In conclusion, in the current study, a profile of the membrane fraction from centrifugation at 100 000g for 40 min at 4 °C was characterized by the analysis of SIF and SSF with the use of proteomic methodologies. We present a first reference map of SSF, discribe the OMPs in the fraction by 2-DE proteomic methodologies, and characterize the interaction network between OMPs, IMPs, and the peripherally associated proteins. Importantly, the complex of Frd, a key enzyme of glycolysis in periplasm, with OmpW, an ion channel, was identified in this study. This complex was characterized to function in iron metabolism. Our results will facilitate the identification of new membrane protein complexes, especially the complexes of OMPs with IMPs and the peripherally associated proteins. The revealed membrane-associated protein complex also provides better understanding of protein function(s) of bacterial outer envelope. Abbreviations: OM, outer membrane; IM, inner membrane; OMPs, outer membrane proteins; IMPs, inner membrane proteins; SSF, sarcosine-soluble; fraction; SIF, sarcosineinsoluble fraction; Frd, fumarate reductase; Co-IP, co-immunoprecipitation; DIP, 2, 2-dipyridyl; GRAVY, grand average of hydropathicity; TAC, tricarboxylic acid cycle; Odp1, pyruvate dehydrogenase; MetQ, D-methionine-binding lipoprotein; GlpD, aerobic glycerol-3-phosphate dehydrogenase; DceA, glutamate decarboxylase alpha; AtpA, ATP synthase alpha chain; AtpB, ATP synthase beta chain; DadA, D-amino acid dehydrogenase small subunit; PyrD, dihydroorotate dehydrogenase; RL1, 50S ribosomal protein L1; RL9, 50S ribosomal protein L9.

Acknowledgment. We thank Dr. Q. Y. Zhang, Branch of Brucker Comp., China, for her help on partial MS analysis. This work was sponsored by grants from NSFC Project 30530610, 973 project 2006CB101807, and Guangzhou Key Project 2006Z3E0251. Supporting Information Available: Supplementary Figure 1, theoretical map of 2-D gel separation of the E. coli OMP subproteome based on predicted pI and molecular masses; supplementary Table 1, summary of proteins in sarcosin-insoluble and sarcosine-soluble fractions identified by MALDI-TOF/MS; supplementary Table 2, classification and characteristics of OMPs. This material is available free of charge via the Internet at http://pubs.acs.org. Journal of Proteome Research • Vol. 5, No. 12, 2006 3275

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