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We purified and characterized a prokaryotic argonaute (pAgo) (KjMP) and its associated protein (KjAA) from a bacterium Kordia jejudonensis. The two proteins present as a complex were revealed by the copurification of KjAA with His-tagged KjMP by Ni-NTA affinity column. The KjAA/KjMP complex was a heterodimer evaluated from the molecular weight estimated using size exclusion chromatography. The pAgo complex presented a guide-dependent target DNA cleavage. RNA was the preferred guide; however, DNA also functioned, albeit weakly. Additionally, 5'-phosphorylate or non-phosphorylated guide was equally effective. The purified complex exhibited nonspecific nuclease activity on dsDNA and ssDNA. This is the first study to report that short pAgo and its associated protein form a complex, which has a nucleic acid-guided target recognition and cleavage.

The Pseudomonas aeruginosa lipoprotein LbcA was discovered because it copurified with and promoted the activity of CtpA, a carboxyl-terminal processing protease (CTP) required for type III secretion system function, and for virulence in a mouse model of acute pneumonia. In this study we explored the role of LbcA by determining its effect on the proteome and its participation in protein complexes. lbcA and ctpA null mutations had strikingly similar effects on the proteome, suggesting that facilitating CtpA might be the most impactful role of LbcA in the bacterial cell. Independent complexes containing LbcA and CtpA, or LbcA and substrate, were isolated from P. aeruginosa cells, indicating that LbcA facilitates proteolysis by recruiting the protease and its substrates independently. An unbiased examination of proteins that copurified with LbcA revealed an enrichment for proteins associated with the cell wall. One of these copurification partners was found to be a new CtpA substrate, and the first substrate that is not a peptidoglycan hydrolase. Many of the other LbcA copurification partners are known or predicted peptidoglycan hydrolases. However, some of these LbcA copurification partners were not cleaved by CtpA, and an in vitro assay revealed that while CtpA and all of its substrates bound to LbcA directly, these non-substrates did not. Subsequent experiments suggested that the non substrates might co-purify with LbcA by participating in multi-enzyme complexes containing LbcA-binding CtpA substrates.IMPORTANCECarboxyl-terminal processing proteases (CTPs) are widely conserved and associated with the virulence of several bacteria, including CtpA in Pseudomonas aeruginosa. CtpA copurifies with the uncharacterized lipoprotein, LbcA. This study shows that the most impactful role of LbcA might be to promote CtpA-dependent proteolysis, and that it achieves this as a scaffold for CtpA and its substrates. It also reveals that LbcA copurification partners are enriched for cell wall-associated proteins, one of which is a novel CtpA substrate. Some of the other LbcA copurification partners are not cleaved by CtpA, but might copurify with LbcA because they participate in multi-enzyme complexes containing CtpA substrates. These findings are important, given the links between CTPs, their associated proteins, peptidoglycan remodeling, and virulence.

International audience; Pseudomonas aeruginosa is one of the most important nosocomial bacteria emerging as a highly multidrug-resistant pathogen. P. aeruginosa produces two siderophores including pyochelin to fulfill its need for iron during infections. We know that both outer and inner membrane proteins FptA and FptX are involved in the ferri-pyochelin uptake, but this process requires increasing molecular and biochemical knowledge. Here, using bacterial two-hybrid and copurification assays we identified the fatty acid coenzyme-A ligase FadD1 as a novel interacting partner of the inner membrane transporter FptX, and found that FadD1 may play a role in pyochelin production. We managed to purify the FadD1-FptX inner membrane complex and obtained low-resolution 3D models, opening the way for future high-resolution structures.

Complex biosynthetic pathways are required for the assembly and insertion of iron-sulfur (Fe-S) cluster cofactors. Each of the four cluster biogenesis systems that have been discovered requires at least one ATPase. Generally, the function of nucleotide hydrolysis in Fe-S cluster biogenesis is understudied. For example, the cytosolic Fe-S cluster assembly (CIA) pathway is proposed to begin with a scaffold, which assembles nascent Fe-S clusters destined for cytosolic and nuclear enzymes. This scaffold, comprised of Nbp35 and Cfd1 in yeast, possesses an ATPase site that is necessary for CIA function, but the role of nucleotide hydrolysis is poorly understood. Herein, we describe the in vitro methods that have been developed to uncover how the ATPase site of the scaffold regulates interaction with one of its partner proteins, Dre2. We describe a qualitative affinity copurification assay and a quantitative assay for evaluating the dissociation constant for the scaffold-partner protein complex. Finally, we describe kinetic methods to measure the kcat and KM values for ATP hydrolysis by the scaffold-partner protein complex and the execution of the ATPase assays in an anaerobic environment. These methods could be applied to study other ATPases to advance our mechanistic understanding of nucleotide hydrolases involved in metallocluster biogenesis.

Funding: JEL thanks the Royal Society for a University Research Fellowship and the Wellcome Trust for the Q-band EPR spectrometer (099149/Z/12/Z). The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modelling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo. Postprint

The regulatory 6S-1 and 6S-2 RNAs of B. subtilis bind to the housekeeping RNA polymerase holoenzyme (σ A -RNAP) with submicromolar affinity. We observed copurification of endogenous 6S RNAs from a published B. subtilis strain expressing a His-tagged RNAP. Such 6S RNA contaminations in σA -RNAP preparations reduce the fraction of enzymes that are accessible for binding to DNA promoters. In addition, this leads to background RNA synthesis by σ A-RNAP utilizing copurified 6S RNA as template for the synthesis of short abortive transcripts termed product RNAs (pRNAs). To avoid this problem we constructed a B. subtilis strain expressing His-tagged RNAP but carrying deletions of the two 6S RNA genes. The His-tagged, 6S RNA-free σA-RNAP holoenzyme can be prepared with sufficient purity and activity by a single affinity step. We also report expression and separate purification of B. subtilis σA that can be added to the His-tagged RNAP to maximize the amount of holoenzyme and, by inference, in vitro transcription activity.

Measuring various biochemical and cellular components in the blood is a routine procedure in clinical practice. Human serum contains hundreds of diverse proteins secreted from all cells and tissues in healthy and diseased states. Moreover, some serum proteins have specific strong interactions with other blood components, but most interactions are probably weak and transient. One of the serum proteins is butyrylcholinesterase (BChE), an enzyme existing mainly as a glycosylated soluble tetramer that plays an important role in the metabolism of many drugs. Our results suggest that BChE interacts with plasma proteins and forms much larger complexes than predicted from the molecular weight of the BChE tetramer. To investigate and isolate such complexes, we developed a two-step strategy to find specific protein–protein interactions by combining native size-exclusion chromatography (SEC) with affinity chromatography with the resin that specifically binds BChE. Second, to confirm protein complexes′ specificity, we fractionated blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation with anti-BChE monoclonal antibodies. The proteins coisolated in complexes with BChE were identified by mass spectroscopy. These binding studies revealed that BChE interacts with a number of proteins in the human serum. Some of these interactions seem to be more stable than transient. BChE copurification with ApoA-I and the density of some fractions containing BChE corresponding to high-density lipoprotein cholesterol (HDL) during ultracentrifugation suggest its interactions with HDL. Moreover, we observed lower BChE plasma activity in individuals with severely reduced HDL levels (≤20 mg/dL). The presented two-step methodology for determination of the BChE interactions can facilitate further analysis of such complexes, especially from the brain tissue, where BChE could be involved in the pathogenesis and progression of AD.

Thiamine monophosphatase (ThMPase) is an enzyme of thiamine metabolism in animals whose molecular nature has still to be elucidated. In this study we have achieved a 714-fold purification of a soluble enzyme possessing ThMPase activity from a chicken liver extract. In addition to ThMPase, acid phosphatase activity was traced during purification. Both activities proved to have coincident elution profiles at all chromatographic steps implying the same enzyme involved. The molecular weight of the enzyme was 18 kDa as estimated by gel filtration. Along with ThMP and p-nitrophenyl phosphate, the purified enzyme was capable of hydrolyzing flavin mononucleotide as well as phosphotyrosine. Subcellular distribution of ThMPase activity was also explored indicating its cytosolic localization. The results of the present work imply the involvement of low molecular weight acid phosphatase in thiamine metabolism in the chicken liver.

Contaminating proteins have been identified by “shotgun” proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni2+-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni2+ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.

We report the selection of DNA-encoded small molecule libraries against protein targets within the cytosol and on the surface of live cells. The approach relies on generation of a covalent linkage of the DNA to protein targets by affinity labeling. This cross-linking event enables subsequent copurification by a tag on the recombinant protein. To access targets within cells, a cyclic cell-penetrating peptide is appended to DNA-encoded libraries for delivery across the cell membrane. As this approach assesses binding of DELs to targets in live cells, it provides a strategy for selection of DELs against challenging targets that cannot be expressed and purified as active.

Fundacao para a Ciencia e a Tecnologia (FCT) through grants PTDC/FIS-NAN/ 0117/2014 and PTDC/BIA-MIC/31645/2017. project UID/Multi/04378/2019 (Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO), funded by FCT/MCTES; project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular), funded by FEDER through COMPETE2020-Programa Operacional Competitividade e Internacionalizacao (POCI); national funds through FCT; by project ONEIDA (LISBOA-01-0145-FEDER-016417), cofunded by FEEI (Fundos Europeus Estruturais e de Investimento) from the Programa Operacional Regional Lisboa 2020; and by national funds from FCT. Funding was also provided by European Society of Clinical Microbiology and Infectious Diseases research grant 2015, awarded to R.G.S. B.V.G., T.A.F., and I.R.G. were supported by fellowships SFRH/BD/131623/2017 respectively. J.S.D. acknowledges the National NMR Network (PTNMR) and Infrastructure Project ROTEIRO/0031/2013-PINFRA/22161/2016 (cofinanced by FEDER through COMPETE 2020, POCI, PORL, and FCT through PIDDAC). Glutamate amidation, a secondary modification of the peptidoglycan, was first identified in Staphylococcus aureus. It is catalyzed by the protein products of the murT and gatD genes, which are conserved and colocalized in the genomes of most sequenced Gram-positive bacterial species. The MurT-GatD complex is required for cell viability, full resistance to β-lactam antibiotics, and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. Great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex. In this work, we demonstrate through the use of nonstructural approaches the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, in the MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Copurification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that the MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence of the effect of substitutions at specific residues in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth. publishersversion published

Članovi proteinske porodice MATH-BTB sudjeluju u ubikvitinskom putu degradacije proteina kao adaptori E3 ligaze ovisne o kulinu 3 i odgovorni su za specifičnost prema ciljanom supstratu. U uročnjaku (Arabidopsis thaliana L.) postoji šest gena ove obitelji (BPM 1-6). Protein BPM1 uz domene MATH i BTB sadrži i konzerviranu domenu SPOP koja zajedno s domenom BTB sudjeluje u stvaranju proteinskih interakcija s kulinom 3. Domena MATH sudjeluje u selektivnim interakcijama s proteinima ciljanim za proteasomsku razgradnju. Takve interakcije utvrđene su između proteina BPM1 i nekih transkripcijskih faktora poput HB6 koji ima ulogu u odgovorima reguliranim apscizinskom kiselinom u biljnim stanicama. Preliminarna istraživanja su pokazala interakciju proteina BPM1 s proteinima DMS3 i RDM1 koji sudjeluju u metilaciji DNA posredovanoj molekulama RNA. U ovom radu interakcije proteinskih domena BPM1 s transkripcijskim faktorom HB6 i proteinima DMS3 odnosno RDM1 su analizirane metodom kopurifikacije „jedan na jedan“. Konstruirani su odgovarajući ekspresijski plazmidi, optimizirani su postupci poticanja ekspresije i pročišćavanja za svaki pojedini protein ili varijantu proteina te su eksprimirani i pročišćeni rekombinantni proteini iz kemijski transformiranih bakterijskih stanica. Interakcija proteina je provjerena hibridizacijom po Westernu i metodom SDS-PAGE. Potvrđena je interakcija proteina BPM1 i DMS3 posredovana domenom MATH te interakcija BPM1 s RDM1 koja nije specifično posredovana niti jednom pojedinom domenom. Interakcija proteina BPM1 i HB6 nije dokazana uslijed degradacije proteina HB6 . Members of MATH-BTB protein family partake as adaptors of cullin 3 dependant E3 ligase in the ubiquitin proteasome pathway and are responsible for specificity towards a substrate. In Arabidopsis thaliana there are 6 genes from this family (BPM 1-6). BPM1 protein along MATH and BTB domains, contains conserved SPOP domain which together with BTB domain partakes in forming of protein interactions with cullin 3. MATH domain has a role in selective interactions with proteins which are targeted for proteasomal degradation. This kind of interactions are established between BPM1 protein and some transcriptional factors like HB6 which has an important role in abscisic acid dependant response in plant cells. Preliminary research have shown an interaction between BPM1 protein and DMS3 and RDM1 proteins which partake in a DNA methylation directed by RNA molecules. Here interactions between BPM1 protein, HB6 transcription factor and proteins DMS3 and RDM1 were analized with „one to one“ pull down method. Adequate plasmid constructs were made, conditions for induced expression and purification for each protein and protein variant were optimised and recombinant proteins were expressed and purified from chemically transformed bacterial strains. Protein-protein interactions were proven by Western blot and SDS-PAGE. Interaction between BPM1 and DMS3 was confirmed and it is primarily mediated by MATH domain. Positive interaction between BPM1 and RDM1 didn't show preference towards any BPM1 domain. BPM1 and HB6 interaction wasn't confirmed due to HB6 degradation.

Host cell proteins (HCPs) are considered a critical quality attribute and are linked to safety and efficacy of biotherapeutic products. Researchers have identified 10 HCPs in Chinese hamster ovary (CHO) that exhibit common characteristics of product association, coelution, and age-dependent expression and therefore are "difficult to remove" during downstream purification. These include cathepsin D, clusterin, galectin-3-binding protein, G-protein coupled receptor 56, lipoprotein lipase, metalloproteinase inhibitor, nidogen-1 secreted protein acidic and rich in cysteine (SPARC), sulfated glycoprotein, and insulin-like growth factor-2 RNA-binding protein. While the levels of HCPs in the investigated biosimilars were within the acceptable range of

Expression, purification, and functional reconstitution of mammalian ion channels are often challenging. Heterologous expression of mammalian channels in bacteria can be advantageous due to unrelated protein environment and the lack of risk of copurification of endogenous proteins, e.g., accessory channel subunits that can influence the channel activity. Also, direct recording of channel activity could be challenging due to their intracellular localization like in the case of mitochondrial channels. The activity of purified channels can be characterized at the single-molecule level by electrophysiological techniques, such as planar lipid bilayers (PLB). In this work, we describe a simple approach to accomplish PLB recording of the activity of single renal outer medullary potassium channels ROMK expressed in E. coli. We focused on the ROMK2 isoform that is present at low levels in the mitochondria and can be responsible for mitoKATP activity. We screened for the best construct to express the codon-optimized ROMK proteins with a 6xHis tag for protein purification. The strategy involved the use of optimal styrene-maleic acid (SMA) copolymer, which forms so-called polymer nanodiscs, to solubilize and purify ROMK-containing SMA lipid particles (SMALPs), which were amenable for fusion with PLB. Reconstituted ROMK channels exhibited ion selectivity, rectification, and pharmacological properties, which are in agreement with previous work on ROMK channels.

Comprehensive analysis of post-translational modifications (PTMs) often depends on the purification of modified peptides prior to LC-MS/MS. The implementation of these enrichment methods requires thorough knowledge of the experimental conditions to achieve optimal selectivity and sensitivity. In this regard, large-scale analysis of lysine acetylation, a key PTM for multiple cellular processes, makes use of monoclonal pan-antibodies designed against this moiety. We report that the immuno-purification of lysine-acetylated peptides is hampered by the copurification of lysine carbamylated peptides, a frequent urea artifact. This specific interaction can be explained by the similar chemical structures of lysine acetylation and lysine carbamylation. As an alternative, we propose a sample preparation protocol based on sodium deoxycholate that eliminates these artifacts and dramatically improves the selectivity and sensitivity of this immuno-purification assay.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.

The CRISPR-Cas system is the prokaryotic immune response that destroys invading foreign nucleic acids. Based on the architecture and distinct mechanism of targeting, the CRISPR-Cas system is classified into six types (I-VI). The Csm complex belongs to the type III system and consists of five subunits (Cas10 and Csm2-5) and a crRNA. The Csm complex targets RNA and RNA-dependent single-strand DNA. Here, we present a protocol for in vitro reconstitution of a Csm complex from a hyperthermophilic archaeon Thermococcus onnurineus NA1 (ToCsm complex). The method consists of coexpression and copurification of the subunits, in vitro synthesis of the crRNA and assembly of the ToCsm complex. Purification with heat treatment and affinity and size exclusion chromatography resulted in homogeneous Cas10/Csm4 and Csm2/Csm5 binary complexes, while in vitro transcription with the T7 promoter enabled synthesis of the crRNA. Addition of each component in the presence of the crRNA with a molar ratio of Cas10/Csm4:Csm3:Csm2/Csm5:crRNA=1:3:2:1 yielded an assembled functional Csm complex. This protocol for reconstitution of the Csm complex is presumably applicable to other thermostable effector complexes, which would allow biochemical, structural, or functional studies of the CRISPR-Cas type III/A system in vitro.

After injecting their genome into the bacterial host cell, bacteriophages need to convert the host metabolism toward efficient phage production. For this, specific proteins have evolved which interact with key host proteins to inhibit, activate or redirect the function of these proteins. Since 70% of the currently annotated phage genes are hypothetical proteins of unknown function, the identification and characterization of these phage proteins involved in host-phage protein-protein interactions remains challenging. Here, we describe a method to identify phage proteins involved in host-phage protein-protein interactions using a combination of affinity purifications and mass spectrometry analyses. A bacterial strain is engineered in which a bacterial target protein is fused to a Strep-tag® II at the C-terminal end. This strain is infected with a specific bacteriophage, followed by an affinity purification of the tagged protein which allows the copurification of all bacterial and phage specific interacting proteins. After SDS-PAGE analysis and an in-gel trypsin digestion, the purified interacting proteins are identified by mass spectrometry analysis. The identification of phage proteins involved in interactions provides first hints toward the elucidation of the biological function of these proteins.

One mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 from Myxococcus xanthus DK1622 for characterization. The M. xanthus DK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination of in vivo and in vitro assays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping of M. xanthus DK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a “universal” MLP for generating functional hybrid NRPSs. IMPORTANCE MbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP from Myxococcus xanthus DK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a “universal” MLP during the construction of functional hybrid NRPSs.

A stochastic approach of copurification of the protease Cathepsin L that results in product fragmentation during purification processing and storage is presented. Cathepsin L was identified using mass spectroscopy, characterization of proteolytic activity, and comparison with fragmentation patterns observed using recombinant Cathepsin L. Cathepsin L existed in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaved a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography were systematically evaluated. Our data demonstrates that copurification of Cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Lastly, Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of Cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove Cathepsin L but resulted in significant product loss, while anion exchange chromatography operated in flow-through mode does not efficiently remove Cathepsin L. Mixed mode chromatography, using Capto™ adhere in this example, provides robust clearance over wide process parameter range (pH 7.7 ± 0.3 and 100 ± 50 mM NaCl), making it an ideal technique to clear Cathepsin L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2732, 2019.

Bacterial cells dispose of numerous strategies to regulate gene expression. Small regulatory RNAs (sRNA) are pervasive molecules that allow gene expression regulation with exquisite precision. These molecules can bind mRNAs and negatively or positively modify their stability and interfere with translation. However, many features of sRNAs render identification of new targets or RNA interacting partners increasingly complex. In this chapter, we present a detailed procedure of MAPS, an in vivo technique based on the copurification of any type of RNA bound to an MS2-tagged sRNA. By focusing on the interaction between two RNAs rather than the outcome of this interaction, MAPS has proven useful in identifying unprecedented sRNA-RNA interactions. Below, we describe how to prepare MAPS samples and how to analyze RNA sequencing data files to determine enrichment ratios of different RNAs in an experimental condition vs a control condition. MAPS can be applied to most sRNAs of Escherichia coli and Salmonella spp., and can be easily optimized to more distant bacterial species.

We report on the characterization of Tic56, a unique component of the recently identified 1-MD translocon at the inner envelope membrane of chloroplasts (TIC) in Arabidopsis (Arabidopsis thaliana) comprising Tic20, Tic100, and Tic214. We isolated Tic56 by copurification with Tandem Affinity Purification-tagged Toc159 in the absence of precursor protein, indicating spontaneous and translocation-independent formation of the translocon at the outer envelope membrane of chloroplasts (TOC) and TIC supercomplexes. Tic56 mutant plants have an albino phenotype and are unable to grow without an external carbon source. Using specific enrichment of protein amino termini, we analyzed the tic56-1 and plastid protein import2 (toc159) mutants to assess the in vivo import capacity of plastids in mutants of an outer and inner envelope component of the anticipated TOC-TIC supercomplex. In both mutants, we observed processing of several import substrates belonging to various pathways. Our results suggest that despite the severe developmental defects, protein import into Tic56-deficient plastids is functional to a considerable degree, indicating the existence of alternative translocases at the inner envelope membrane.

Host cell proteins (HCPs) are process-related impurities present in biopharmaceuticals and are generally considered to be critical quality attributes. Changes in a biopharmaceutical production process may result in qualitative shifts in the HCP population. These shifts are not necessarily detectable when overall HCP levels are measured with traditional approaches such as enzyme-linked immunosorbent assays (ELISAs). Thus, the development of techniques that complement the ELISA's functionality is desirable. Here, a mass spectrometry (MS)-based approach for the analysis of HCP populations in biopharmaceuticals is presented. It consists of (i) the generation of exclusion lists that represent the masses of the active pharmaceutical ingredient (API), (ii) the compilation of inclusion lists based on an HCP catalog derived from the analysis of protein A-purified samples, and (iii) the analysis of purified biopharmaceuticals using the generated exclusion and inclusion lists. With this approach, it was possible to increase sensitivity for HCP detection compared with a standard liquid chromatography tandem MS (LC-MS/MS) run. The workflow was successfully implemented in a comparability exercise assessing HCP populations in drug substance samples before and after a process change. Furthermore, the results suggest that size can be an important factor in the copurification of HCPs and API.

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from Δ cydX mutant strains have reduced oxidase activity in vitro . Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that has been implicated in multiple biological processes including immunity, tumor progression, and vascular biology. However, current protocols for purifying HRG from plasma result in the copurification of contaminating proteins and raise questions over the validity of biological activities ascribed to HRG. In this study, we describe a two-step protocol for the large-scale purification of HRG from human plasma using a combination of metal affinity and ion exchange chromatography. The protocol employs a rapid and simple strategy to isolate highly purified HRG that minimizes proteolytic cleavage of the protein. The purification of HRG was assessed at each stage by measuring the amount of HRG immunoreactive protein using a specific monoclonal antibody against total protein, and demonstrated ~1,000-fold purification with an overall yield of ~32%. Mass spectrometry analysis demonstrated that plasma-derived HRG was free of contaminating proteins and gel electrophoresis showed it to have minimal proteolytic degradation. Characterization of protein by physical method showed that the protein exists as a single, monodisperse species. In contrast to the previous studies of HRG purified by different methods, HRG purified using the new procedure demonstrated a reduced profile of functions. Although the HRG retained binding to heparin and phosphatidic acid, it did not interact with necrotic cells or other cellular lipids. These data demonstrate that HRG does not exhibit the broad interactive properties that have been reported previously, suggesting that copurification of HRG-binding partners or other impurities are responsible for some of the reported functional properties. The findings in this study demonstrate that the new purification procedure can provide a ready source of pure HRG to assess ligand specificity and biological function of this important plasma protein.

HIV-1 incorporates a large array of host proteins into virions. Determining the host protein composition in HIV virions has technical difficulties, including copurification of microvesicles. We developed an alternative purification technique using cholesterol that differentially modulates the density of virions and microvesicles (density modification, DM) allowing for high-yield virion purification that is essential for tandem mass spectrometric and quantitative proteomic (iTRAQ) analysis. DM purified virions were analyzed using iTRAQ and validated against Optiprep (60% iodixanol) purified virions. We were able to characterize host protein incorporation in DM-purified HIV particles derived from CD4+ T-cell lines; we compared this data set to a reprocessed data set of monocyte-derived macrophages (MDM) derived HIV-1 using the same bioinformatics pipeline. Seventy-nine clustered proteins were shared between the MDM derived and T-cell derived data set. These clusters included an extensive collection of actin isoforms, HLA proteins, chaperones, and a handful of other proteins, many of which have previously been documented to interact with viral proteins. Other proteins of note were ERM proteins, the dynamin domain containing protein EH4, a phosphodiesterase, and cyclophilin A. As these proteins are incorporated in virions produced in both cell types, we hypothesize that these proteins may have direct interactions with viral proteins or may be important in the viral life cycle. Additionally, identified common set proteins are predicted to interact with1000 related human proteins. Many of these secondary interacting proteins are reported to be incorporated into virions, including ERM proteins and adhesion molecules. Thus, only a few direct interactions between host and viral proteins may dictate the host protein composition in virions. Ultimately, interaction and expression differences in host proteins between cell types may drive virion phenotypic diversity, despite conserved viral protein-host protein interactions between cell types.

Nucleolar isolation allows exhaustive characterization of the nucleolar content. Centrifugation-based protocols are not adapted to isolation of nucleoli directly from a plant tissue because of copurification of cellular debris. We describe here a method that allows the purification of nucleoli using fluorescent-activated cell sorting from Arabidopsis thaliana leaves. This approach requires the expression of a specific nucleolar protein such as fibrillarin fused to green fluorescent protein in planta.

The bacterium Escherichia coli remains the leading host for protein expression in large quantity for the purpose of crystallization or other biochemical studies. However, expression of multicomponent protein complexes remains a challenge, and is often laborious and time-consuming. Here we developed a method named EcoExpress, which allows efficient construction of plasmids to express individual protein with user-defined epitope-tag, followed by one-pot assembly of a single vector to express the entire protein complex for copurification. A versatile set of vectors was designed to provide various choices to control the expression of a protein with different promoters, and to accept different number of components for coexpression. Using EcoExpress, we demonstrated that each subunit within a protein complex could be expressed individually or simultaneously, and the entire complex could be copurified. In addition, to overcome the decreased assembly efficiency with the increasing number of components, a novel oligonucleotides blocking method was designed and tested. EcoExpress provides the scientific community with a toolbox to rapidly investigate the function of protein complexes.

The members of the cation diffusion facilitator (CDF) family transport heavy metal ions and play an important function in zinc ion homeostasis of the cell. A recent structure of an Escherichia coli CDF transporter protein YiiP has revealed its dimeric nature and autoregulatory zinc transport mechanism. Here, we report the cloning and heterologous production of four different CDF transporters, two each from the pathogenic mesophilic bacterium Salmonella typhimurium and from the hyperthermophilic bacterium Aquifex aeolicus, in E. coli host cells. STM0758 of S. typhimurium was able to restore resistance to zinc ions when tested by complementation assays in the zinc-sensitive GG48 strain. Furthermore, copurification of bicistronically produced STM0758 and cross-linking experiments with the purified protein have revealed its possible oligomeric nature. The interaction between heavy metal ions and Aq_2073 of A. aeolicus was investigated by titration calorimetry. The entropy-driven, high-affinity binding of two Cd2+ and two Zn2+ per protein monomer with Kd values of around 100 nm and 1 μm, respectively, was observed. In addition, at least one more Zn2+ can be bound per monomer with low affinity. This low-affinity site is likely to possess a functional role contributing to Zn2+ transport across membranes.

Voltage-gated K(+) channels composed of Kv7.2 and Kv7.3 are the predominant contributors to the M-current, which plays a key role in controlling neuronal activity. Various lines of evidence have indicated that Kv7.2 and Kv7.3 form a heteromeric channel. However, the subunit stoichiometry and arrangement within this putative heteromer are so far unknown. Here, we have addressed this question using atomic force microscopy imaging of complexes between isolated Kv7.2/Kv7.3 channels and antibodies to epitope tags on the two subunits, Myc on Kv7.2 and HA on Kv7.3. Initially, tsA 201 cells were transiently transfected with equal amounts of cDNA for the two subunits. The heteromer was isolated through binding of either tag to immunoaffinity beads and then decorated with antibodies to the other tag. In both cases, the distribution of angles between pairs of bound antibodies had two peaks, at around 90° and around 180°, and in both cases the 90° peak was about double the size of the 180° peak. These results indicate that the Kv7.2/Kv7.3 heteromer generated by cells expressing approximately equal amounts of the two subunits assembles as a tetramer with a predominantly 2:2 subunit stoichiometry and with a random subunit arrangement. When the DNA ratio for the two subunits was varied, copurification experiments indicated that the subunit stoichiometry was variable and not fixed at 2:2. Hence, there are no constraints on either the subunit stoichiometry or the subunit arrangement.

Three mechanistically different sample extraction methodologies, namely, silica spin columns, phenol-chloroform, and an automated magnetic capture of polymer-complexed DNA (via an Automate Express instrument), were compared for their abilities to purify nucleic acids from blood culture fluids for use in TaqMan assays for detection of Staphylococcus aureus. The extracts from silica columns required 100- to 1000-fold dilutions to sufficiently reduce the powerful PCR inhibitory effects of the anticoagulant sodium polyanetholsulfonate, a common additive in blood culture media. In contrast, samples extracted by either phenol-chloroform or the Automate Express instrument required little or no dilution, respectively, allowing for an approximate 100-fold improvement in assay sensitivity. Analysis of 60 blood culture bottles indicated that these latter two methodologies could be used to detect lower numbers of pathogens and that a growing S. aureus culture could be detected 2 hours earlier than when using silica columns. Of the three tested methodologies, the Automate Express instrument had the shortest time to result, requiring only approximately 80 minutes to process 12 samples. These findings highlight the importance of considering the mechanism when selecting a DNA extraction methodology, given that certain PCR inhibitors act in a similar fashion to DNA in certain chemical environments, resulting in copurification, whereas other methodologies use different chemistries that have advantages during the DNA purification of certain types of samples.

A synthetic de novo designed heterodimeric coiled-coil was used to copurify two target fluorescent proteins, Venus and enhanced cyan fluorescent protein (ECFP). The coiled-coil consists of two 21-amino acid repetitive sequences, (EIAALEK)3 and (KIAALKE)3, named E3 and K3, respectively. These sequences were fused to the C-termini of ECFP or Venus followed by either a strep- or a his-tag, respectively, for affinity purification. Mixed lysates of Venus-K3 and ECFP-E3 were subjected to consecutive affinity purification and showed highly specific association between the coiled-coil pair by SDS-PAGE, gel filtration, isothermal titration calorimetry (ITC), and fluorescence resonance energy transfer (FRET). The tagged proteins eluted as heterodimers at the concentrations tested. FRET analysis further showed that the coiled-coil pair was stable in buffers commonly used for protein purification, including those containing high salt concentration and detergent. This study shows that the E3/K3 pair is very well suited for the copurification of two target proteins expressed in vivo because of its high specificity: it forms exclusively heterodimers in solution, it does not interact with any cellular proteins and it is stable under different buffer conditions.

GspB is a serine-rich glycoprotein adhesin of Streptococcus gordonii that is exported to the bacterial surface by the accessory Sec system. This dedicated export pathway is comprised of seven components (SecA2, SecY2, and five accessory Sec proteins [Asp1 to Asp5]). The latter proteins have no known homologs beyond the Asps of other species. Asp1 to Asp3 are absolutely required for export of the substrate GspB, but their roles in this process are unknown. Using copurification analysis and far-Western blotting, we found that Asp2 and Asp3 could individually bind the serine-rich repeat (SRR) domains of GspB. Deletion of both SRR regions of GspB led to a decrease in its export, suggesting that binding of the Asps to the SRR regions is important for GspB transport by the accessory Sec system. The Asps also bound a heterologous substrate for the accessory Sec system containing a slow-folding MalE variant, but they did not bind wild-type MalE. The combined results indicate that the Asps may recognize the export substrate through preferential interactions with its unstructured or unfolded regions. Glycosylation of the SRR domains on GspB prevented Asp binding, suggesting that binding of the Asps to the preprotein occurs prior to its full glycosylation. Together, these findings suggest that Asp2 and Asp3 are likely to function in part as chaperones in the early phase of GspB transport.

Deubiquitinating enzymes (DUBs) have emerged as essential players in a myriad of cellular processes, yet the regulation of DUB function remains largely unknown. While some DUBs rely on the formation of complex for regulation of enzymatic activity, many DUBs utilize interdomain interactions to regulate catalysis. Here we report the biochemical characterization of a multidomain deubiquitinating enzyme, Ubp15, from Saccharomyces cerevisiae. Steady-state kinetic investigation showed that Ubp15 is a highly active DUB. We identified active-site residues that are required for catalysis. We have also identified key residues on Ubp15 required for ubiquitin binding and catalysis. We further demonstrated that Ubp15's enzymatic activity is regulated by the N- and C-terminal domains that flank the catalytic core domain. Moreover, we demonstrated that Ubp15 physically interacts with a WD40 repeat-containing protein, Cdh1, by copurification experiments. Interestingly, unlike other DUBs that specifically interact with WD40 repeat-containing proteins, Cdh1 does not function in stimulating Ubp15's activity. The possible cellular function of Ubp15 in cell cycle regulation is discussed in view of the specific interaction between Ubp15 and Cdh1, an activator of the anaphase-promoting complex/cyclosome (APC/C).

Sucrose density gradient-enriched membrane preparations and membrane fraction enrichment through affinity purification techniques are commonly used in proteomic analysis. However, published proteomic profiles characterized by the above methods show the presence of nuclear proteins in addition to membrane proteins. While shuttling of nuclear proteins across cellular compartments and their transient residency at membrane interfaces could explain some of these observations, the presence of nuclear proteins in proteomic profiles generated with crude and enriched membranes could be the result of nonspecific contamination of nuclear debris during cell fractionation procedures. We hypothesized that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients. Using sucrose gradient-enriched plasma membranes from breast cancer cell lines derived from the MCF-7 cell line, we provide experimental evidence to indicate that micronuclei are present in fresh preparations of plasma membranes. The origin of these micronuclei was traced to budding of nuclei in intact cells. Furthermore, mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.

This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9α-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in Escherichia coli. E. coli cell cultures, expressing both KshA and KshB, converted 4-androstene-3,17-dione (AD) into 9α-hydroxy-4-AD (9OHAD) with a >60% molar yield over 48 h of incubation. Coexpression and copurification were critical to successfully obtain pure and active KSH. Biochemical analysis revealed that the flavoprotein KshB is an NADH-dependent reductase using flavin adenine dinucleotide as a cofactor. Reconstitution experiments confirmed that KshA, KshB, and NADH are essential for KSH activity with steroid substrates. KSH hydroxylation activity was inhibited by several divalent metal ions, especially by zinc. The reconstituted KSH displayed subtle steroid substrate specificity; a range of 3-ketosteroids, i.e., 5α-Η, 5β-Η, Δ1, and Δ4 steroids, could act as KSH substrates, provided that they had a short side chain. The formation of 9OHAD from AD by KSH was confirmed by liquid chromatography-mass spectrometry analysis and by the specific enzymatic conversion of 9OHAD into 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione using 3-ketosteroid Δ1-dehydrogenase. Only a single KSH is encoded in the genome of the human pathogen Mycobacterium tuberculosis H37Rv, shown to be important for survival in macrophages. Since no human KSH homolog exists, the M. tuberculosis enzyme may provide a novel target for treatment of tuberculosis. Detailed knowledge about the biochemical properties of KSH thus is highly relevant in the research fields of biotechnology and medicine.

The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA-binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop - lacZ fusion in mutant strains defective for either mopA , mopB , or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression.

Adenylate cyclase activity in human thyroid homogenates was studied after stimulation with thyrotropin (TSH) and thyroid stimulating antibodies (TSAb). The results show: 1) TSAb prepared from different patients with Graves' disease show different adenylate cyclase activation patterns and a lag phase is frequently observed. 2) TSH and TSAb appear to cause mutually inhibitory activation of thyroid adenylate cyclase. 3) The maximal adenylate cyclase activation is higher with TSH than with TSAb, but this could possibly be due to contamination of TSAb preparations with an adenylate cyclase inhibitor. 4) There is no absolute copurification of TSH sensitive and TSAb sensitive adenylate cyclase in various subcellular fractions of thyroid homogenate. 5) Incubation of thyroid homogenate with cortisol cause a dose dependent decrease in the adenylate cyclase response to TSAb whereas the response to TSH is either increased or unchanged. The results indicate that TSH and TSAb activate thyroid adenylate cyclase through different pathways in the plasma membrane.

Except for apyrases, ATPases generally target only the gamma-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10,000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

[NiFe] hydrogenases catalyze the reversible conversion of H2 into protons and electrons. The reaction takes place at the active site, which is composed of a nickel and an iron atom and three diatomic ligands, two cyanides and one carbon monoxide, bound to the iron. The NiFe(CN-)2CO cofactor is synthesized by an intricate posttranslational maturation process, which is mediated by a set of six conserved Hyp proteins. Depending on the cellular location and the physiological function, additional auxiliary proteins are involved in hydrogenase biosynthesis. Here we present evidence that the auxiliary proteins HoxL and HoxV assist in assembly of the Fe(CN-)2CO moiety. This unit was identified as a cofactor intermediate of the oxygen-tolerant membrane-bound [NiFe] hydrogenase (MBH) in the beta-proteobacterium Ralstonia eutropha H16. Both HoxL and HoxV proved to be essential for H2-oxidizing activity and MBH-driven growth on H2. Copurification studies revealed that HoxL and HoxV directly interact with the hydrogenase apoprotein. HoxV forms complexes with HoxL and HypC, a HoxL paralogue that is essential for cofactor assembly. These observations suggest that HoxL acts as a specific chaperone assisting the transfer of the Fe(CN-)2CO cofactor intermediate from the Hyp machinery to the MBH. This shuttle also involves the scaffold protein HoxV. Indeed, infrared spectroscopy and metal analysis identified for the first time a non-redox-active Fe(CN-)2CO intermediate coordinated to HoxV.

Quantitative analysis of tandem-affinity purified cross-linked ( x ) protein complexes (QTAX) is a powerful technique for the identification of protein interactions, including weak and/or transient components. Here, we apply a QTAX-based tag-team mass spectrometry strategy coupled with protein network analysis to acquire a comprehensive and detailed assessment of the protein interaction network of the yeast 26S proteasome. We have determined that the proteasome network is composed of at least 471 proteins, significantly more than the total number of proteins identified by previous reports using proteasome subunits as baits. Validation of the selected proteasome-interacting proteins by reverse copurification and immunoblotting experiments with and without cross-linking, further demonstrates the power of the QTAX strategy for capturing protein interactions of all natures. In addition, >80% of the identified interactions have been confirmed by existing data using protein network analysis. Moreover, evidence obtained through network analysis links the proteasome to protein complexes associated with diverse cellular functions. This work presents the most complete analysis of the proteasome interaction network to date, providing an inclusive set of physical interaction data consistent with physiological roles for the proteasome that have been suggested primarily through genetic analyses. Moreover, the methodology described here is a general proteomic tool for the comprehensive study of protein interaction networks.

The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane [OM] proteins OmcA, MtrB, and MtrC; OM-associated protein MtrA; periplasmic protein CctA; and cytoplasmic membrane protein CymA) were characterized by protein purification, analytical ultracentrifugation, and cross-linking methods. Five of these proteins are heme proteins, OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa), and CymA (21 kDa), and can be visualized after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by heme staining. We show for the first time that MtrC, MtrA, and MtrB form a 198-kDa complex with a 1:1:1 stoichiometry. These proteins copurify through anion-exchange chromatography, and the purified complex has the ability to reduce multiple forms of Fe(III) and Mn(IV). Additionally, MtrA fractionates with the OM through sucrose density gradient ultracentrifugation, and MtrA comigrates with MtrB in native gels. Protein cross-linking of whole cells with 1% formaldehyde show new heme bands of 160, 151, 136, and 59 kDa. Using antibodies to detect each protein separately, heme proteins OmcA and MtrC were shown to cross-link, yielding the 160-kDa band. Consistent with copurification results, MtrB cross-links with MtrA, forming high-molecular-mass bands of approximately 151 and 136 kDa.

The proteins encoded by the A56R and K2L genes of vaccinia virus form a heterodimer (A56/K2) and have a fusion regulatory role as deletion or mutation of either causes infected cells to form large syncytia spontaneously. Here, we showed that syncytia formation is dependent on proteins of the recently described entry fusion complex (EFC), which are also required for virus-cell fusion and low-pH-triggered cell-cell fusion. This finding led us to consider that A56/K2 might prevent fusion by direct or indirect interaction with the EFC. To test this hypothesis, we made a panel of recombinant vaccinia viruses that have a tandem affinity purification tag attached to A56, K2, or the A28 EFC protein. Interaction between A56/K2 and the EFC was demonstrated by their copurification from detergent-treated lysates of infected cells and identification by mass spectrometry or Western blotting. In addition, a purified soluble transmembrane-deleted form of A56/K2 was shown to interact with the EFC. Tagged A56 did not interact with the EFC in the absence of K2, nor did tagged K2 interact with the EFC in the absence of A56. The finding that both A56 and K2 are required for efficient binding to the EFC fits well with prior experiments showing that mutation of either A56 or K2 results in spontaneous fusion of infected cells. Because A56 and K2 are located on the surface of infected cells, they are in position to interact with the EFC of released progeny virions and prevent back-fusion and syncytia formation.

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Substrates are targeted to the Tat pathway by signal peptides containing a pair of consecutive arginine residues. The membrane proteins TatA, TatB and TatC are the essential components of this pathway in Escherichia coli. The complexes that these proteins form at native levels of expression have been investigated by the use of affinity tag-coding sequences fused to chromosomal tat genes. Distinct TatA and TatBC complexes were identified using size-exclusion chromatography and shown to have apparent molecular masses of ∼ 700 and 500 kDa, respectively. Following in vivo expression, the Tat substrate protein SufI was found to copurify with the TatBC, but not the TatA, complex. This binding required the SufI signal peptide. Substitution of the twin-arginine residues in the SufI signal peptide by either twin lysine or twin alanine residues abolished export. However, both variant SufI proteins still copurified with the TatBC complex. These data show that the twin-arginine residues of the Tat consensus motif are not essential for binding of precursor to the TatBC complex but are required for the successful entry of the precursor into the transport cycle. The effect on substrate binding of single amino acid substitutions in TatC that affect Tat transport were studied using TatC variants Phe94Ala, Glu103Ala, Glu103Arg and Asp211Ala. Only variant Glu103Arg showed reduced copurification of SufI with TatBC. The transport defects associated with the other TatC variants do not, therefore, arise from an inability to bind substrate proteins.

The E and G subunits of the yeast V-ATPase are believed to be part of the peripheral or stator stalk(s) responsible for physically and functionally linking the peripheral V1 sector, responsible for ATP hydrolysis, to the membrane V0 sector, containing the proton pore. The E and G subunits interact tightly and specifically, both on a far Western blot of yeast vacuolar proteins and in the yeast two-hybrid assay. Amino acids 13-79 of the E subunit are critical for the E-G two-hybrid interaction. Different tagged versions of the G subunit were expressed in a diploid cell, and affinity purification of cytosolic V1 sectors via a FLAG-tagged G subunit resulted in copurification of a Myc-tagged G subunit, implying more than one G subunit was present in each V1 complex. Similarly, hemagglutinin-tagged E subunit was able to affinity-purify V1 sectors containing an untagged version of the E subunit from heterozygous diploid cells, suggesting that more than one E subunit is present. Overexpression of the subunit G results in a destabilization of subunit E similar to that seen in the complete absence of subunit G (Tomashek, J. J., Graham, L. A., Hutchins, M. U., Stevens, T. H., and Klionsky, D. J. (1997) J. Biol. Chem. 272, 26787-26793). These results are consistent with recent models showing at least two peripheral stalks connecting the V1 and V0 sectors of the V-ATPase and would allow both stalks to be based on an EG dimer.

The core oligosaccharide component of the lipopolysaccharide can be subdivided into inner and outer core regions. In Escherichia coli , the inner core consists of two 3- deoxy - d - manno- octulosonic acid and three glycero - manno -heptose residues. The HldE protein participates in the biosynthesis of ADP- glycero - manno -heptose precursors used in the assembly of the inner core. HldE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HldE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HldE2 domain). We have employed the structure of the E. coli ribokinase as a template to model the HldE1 domain and predict critical amino acids required for enzyme activity. Mutation of these residues renders the protein inactive as determined in vivo by functional complementation analysis. However, these mutations did not affect the secondary or tertiary structure of purified HldE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HldE and mutant HldE1 proteins with amino acid substitutions in the predicted ATP binding site caused a dominant negative phenotype as revealed by increased bacterial sensitivity to novobiocin. Copurification experiments demonstrated that HldE and HldE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HldE1 protein. Altogether, our data support the notions that the HldE functional unit is a dimer and that structural components present in each HldE1 monomer are required for enzymatic activity.

Light-responsive bioconjugates are synthesized by a two-step protocol calling first for cotelomerization (chain-transfer polymerization) of N-isopropylacrylamide and N-acryloxysuccinimide. The desired bioligand (biotin) is used in modified form as chain-transfer agent in this step. As a consequence, 100% of the produced bioconjugates carry this group. In a second step, the cotelomers (bioconjugates) are rendered photoresponsive by linking a chromophore ((3-aminopropyloxy)azobenzene) group to the N-acryloxysuccinimide side chains. The resulting structures show a critical solution temperature in pure water of 16°C when the azo groups in the side chains are predominately in the (stable) trans state. Irradiation with UV light (330 nm) switches the azo group into the more hydrophilic cis state, and the critical solution temperature rises to 18°C. Irradiation with visible light (> 440 nm) switches the group back to the trans state. Adjusting the temperature to an intermediate level, the bioconjugates are used to demonstrate the concept of photo affinity precipitation, i.e., the specific capture and recovery by light-induced precipitation of a target molecule (avidin) from a serum-containing cell-culture supernatant. The avidin was obtained in highly purified form; no nonspecific copurification of protein impurities was observable. © 2005 Wiley Periodicals, Inc.

Mobile light-harvesting complex II (LHCII) is implicated in the regulation of excitation energy distribution between Photosystem I (PSI) and Photosystem II (PSII) during state transitions. To investigate how LHCII interacts with PSI during state transitions, PSI was isolated from Arabidopsis thaliana plants treated with PSII or PSI light. The PSI preparations were made using digitonin. Chemical cross-linking using dithio-bis(succinimidylpropionate) followed by diagonal electrophoresis and immunoblotting showed that the docking site of LHCII (Lhcb1) on PSI is comprised of the PSI-H, -L, and -I subunits. This was confirmed by the lack of energy transfer from LHCII to PSI in the digitonin-PSI isolated from plants lacking PSI-H and -L. Digitonin-PSI was purified further to obtain an LHCII.PSI complex, and two to three times more LHCII was associated with PSI in the wild type in State 2 than in State 1. Lhcb1 was also associated with PSI from plants lacking PSI-K, but PSI from PSI-H, -L, or -O mutants contained only about 30% of Lhcb1 compared with the wild type. Surprisingly, a significant fraction of the LHCII bound to PSI in State 2 was not phosphorylated. Cross-linking prior to sucrose gradient purification resulted in copurification of phosphorylated LHCII in the wild type, but not with PSI from the PSI-H, -L, and -O mutants. The data suggest that migration of LHCII during state transitions cannot be explained sufficiently by different affinity of phosphorylated and unphosphorylated LHCII for PSI but is likely to involve structural changes in thylakoid organization.

During intraerythrocytic infection, Plasmodium falciparum parasites crystallize toxic heme released during hemoglobin catabolism. The proposed mechanism of quinoline inhibition of crystal growth is either by a surface binding or a substrate sequestration mechanism. The kinetics of heme crystal growth was examined in this work using a new high-throughput crystal growth determination assay based on the differential solubility of free vs. crystalline FP in basic solutions. Chloroquine ( ic 50 =4.3 μM) and quinidine ( ic 50 =1.5 μM) showed a previously not recognized reversible inhibition of FP crystal growth. This inhibition decreased by increasing amounts of heme crystal seed, but not by greater amounts of FP substrate. Crystal growth decreases as pH rises from 4.0 to 6.0, except for a partial local maxima reversal from pH 5.0 to 5.5 that coincides with increased FP solubility. The new crystal growth determination assay enabled a partial screen of existing clinical drugs. Nitrogen heterocycle cytochrome P450 inhibitors also reversibly blocked FP crystal growth, including the azole antifungal drugs clotrimazole ( ic 50 =12.9 μM), econazole ( ic 50 =19.7 μM), ketoconazole ( ic 50 =6.5 μM), and miconazole ( ic 50 =21.4 μM). Fluconazole did not inhibit. Both subcellular fractionation of parasites treated with subinhibitory concentrations of ketoconazole and in vitro hemozoin growth assays demonstrated copurification of hemozoin and ketoconazole. The chemical diversity of existing CYP inhibitor libraries that bind FP presents new opportunities for the discovery of antimalarial drugs that block FP crystal growth by a surface binding mechanism and possibly interfere with other FP-sensitive Plasmodium pathways.

We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae. Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials. The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs. Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome. In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNA's translational status within the cell. The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs.