N-terminal His-tag Research Articles

The role of TerW protein in the tellurite resistance of uropathogenic Escherichia coli

The ter operon, which is found on a large conjugative plasmid, pTE53, from the uropathogenic strain Escherichia coli KL53, mediates heavy metal ion resistance to tellurium compounds. Here we present the function of the terW gene, which is transcribed from its own promoter in the opposite direction to the terZABCDE loci and encodes a protein of 155 amino acids. A TerW protein containing an N-terminal His-tag was overexpressed, purified and analysed for the in vitro DNA-binding. Green fluorescent protein fusions, a fluorimetric assay, nonradioactive gel retardation and capillary electrophoresis footprinting were used to examine the interaction between the bioinformatically-predicted DNA-binding region and TerW. Here we show that TerW binds specifically to the potential promoter region of the terZABCDE genes, which are responsible for the tellurite resistance of the Escherichia coli host cells. Our results suggest that the transcriptional regulation of ter genes is operon-like.

E2 Conjugating Enzyme Selectivity and Requirements for Function of the E3 Ubiquitin Ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2∼Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2∼Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2∼Ub conjugate is derived from other molecular determinants.

Processivity and Subcellular Localization of Glycogen Synthase Depend on a Non-catalytic High Affinity Glycogen-binding Site

Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS.

Designed Coiled Coils Promote Folding of a Recombinant Bacterial Collagen

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.

Activation of F-Actin Binding Capacity of Ezrin: Synergism of PIP2 Interaction and Phosphorylation

Ezrin is a membrane-cytoskeleton linker protein that can bind F-actin in its active conformation. Several means of regulation of ezrin's activity have been described including phosphorylation of Thr-567 and binding of L-α-phosphatidylinositol-4,5-bisphosphate (PIP2). However, the relative contributions of these events toward activation of the protein and their potential interdependence are not known. We developed an assay based on solid-supported membranes, to which different ezrin mutants (ezrin T567A (inactive mutant), wild-type, and T567D (active pseudophosphorylated mutant)) were bound, that enabled us to analyze the influence of phosphorylation and PIP2 binding on ezrin's activation state in vitro. The lipid bilayers employed contained either DOGS-NTA-Ni to bind the proteins via an N-terminal His-tag, or PIP2, to which ezrin binds via specific binding sites located in the N-terminal region of the protein. Quantitative analysis of the binding behavior of all three proteins to the two different receptor lipids revealed that all three bind with high affinity and specificity to the two receptor lipids. Fluorescence microscopy on ezrin-decorated solid-supported membranes showed that, dependent on the mode of binding and the phosphorylation state, ezrin is capable of binding actin filaments. A clear synergism between phosphorylation and the receptor lipid PIP2 was observed, suggesting a conformational switch from the dormant to the active, F-actin binding state by recognition of PIP2, which is enhanced by the phosphorylation.

Activation of Colicin M by the FkpA Prolyl Cis-Trans Isomerase/Chaperone

Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA.

Expression of Human Globular Adiponectin-Glucagon-Like Peptide-1 Analog Fusion Protein and Its Assay of Glucose-Lowering Effect In Vivo

In this study, human globular adiponectin-glucagon-like peptide-1 analog (gAd-GLP-1-A) fusion protein was expressed and its glucose-lowering effect was measured in vivo. We constructed a prokaryotic expression vector PET28a-gAd-GLP-1-A and transformed the vector into Escherichia coli BL21 (DE3). A recombinant fusion protein of about 25KD was expressed from BL21 (DE3) cells after isopropylthio-β-D-galactoside induction. This protein was N-terminal His-tagged gAd-GLP-1-A fusion protein. Most of the protein was expressed in inclusion body. The fusion protein in inclusion body was purified by using High-Affinity Nickel Iminodiacetic Acid Resin and refolded in urea gradient refolding buffer. The refolded protein was incubated with enterokinase to remove the N-terminal His-tag. The fusion protein without His-tag is gAd-GLP-1-A fusion protein, which exhibited significant glucose-lowering effect in diabetic mice.

Identification and Characterization of a Novel-type Ferric Siderophore Reductase from a Gram-positive Extremophile

Iron limitation is one major constraint of microbial life, and a plethora of microbes use siderophores for high affinity iron acquisition. Because specific enzymes for reductive iron release in gram-positives are not known, we searched Firmicute genomes and found a novel association pattern of putative ferric siderophore reductases and uptake genes. The reductase from the schizokinen-producing alkaliphile Bacillus halodurans was found to cluster with a ferric citrate-hydroxamate uptake system and to catalyze iron release efficiently from Fe[III]-dicitrate, Fe[III]-schizokinen, Fe[III]-aerobactin, and ferrichrome. The gene was hence named fchR for ferric citrate and hydroxamate reductase. The tightly bound [2Fe-2S] cofactor of FchR was identified by UV-visible, EPR, CD spectroscopy, and mass spectrometry. Iron release kinetics were determined with several substrates by using ferredoxin as electron donor. Catalytic efficiencies were strongly enhanced in the presence of an iron-sulfur scaffold protein scavenging the released ferrous iron. Competitive inhibition of FchR was observed with Ga(III)-charged siderophores with K(i) values in the micromolar range. The principal catalytic mechanism was found to couple increasing K(m) and K(D) values of substrate binding with increasing k(cat) values, resulting in high catalytic efficiencies over a wide redox range. Physiologically, a chromosomal fchR deletion led to strongly impaired growth during iron limitation even in the presence of ferric siderophores. Inductively coupled plasma-MS analysis of ΔfchR revealed intracellular iron accumulation, indicating that the ferric substrates were not efficiently metabolized. We further show that FchR can be efficiently inhibited by redox-inert siderophore mimics in vivo, suggesting that substrate-specific ferric siderophore reductases may present future targets for microbial pathogen control.

Targeted protein pullout from human tissue samples using competitive elution

One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, IMAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.

Molecular characterization and structure determination of human ADAMTSL4

The thrombospondin type 1 repeat (TSR) is an ancient extracellular protein domain that is commonly found in invertebrate and vertebrate proteins. The ADAMTSL4 protein, also known as TSRC1, belongs to the TSR superfamily and has multiple thrombospondin repeats, most of which are clustered at the C-terminus. It has been reported that some TSP1 domain-containing proteins, e.g. thrombospondin 1 and thrombospondin 2, could induce apoptosis of endothelial cells but it is not clear how these proteins operate in death pathways. Our recent work shows that mutations in ADAMTSL4 are responsible for autosomal-recessive isolated ectopia lentis, i.e. abnormal positioning of the lens of the eye and affect the development of the zonular fiber. However, little is known about the function of ADAMTSL4. To shed more light on the function of the ADMTSL4 and its roles in different tissues we extended our work to carry out molecular characterization of this gene and its variants. We have obtained cDNA clones encoding the full ADAMTSL4 protein and its truncated isoform. Both are subcloned into the pET28a vector for expression in E. coli. In anticipation of possible expression challenges in this host, e.g. formation of inclusion bodies or lack of expression, we subcloned each fragment into the yeast vector, pYES2. The gene in both cases has been fused in frame with a region encoding an N-terminal His-Tag to facilitate the purification of the recombinant protein. DNA analysis indicates that each fragment has been correctly cloned into the pYES2 vector. Each construct was transformed into Saccharomyces cerevisiae for protein expression. Our preliminary analysis indicates that one of the genes is expressed in S. cerevisiae but at a very low level. Work to optimize the expression of ADAMTSL4 in yeast as well as in E. coli is in progress. We also extracted the seven domains of the ADAMTSL4 using PCR. Each domain was subcloned into the E. coli overexpression vector, pET28a. Expression studies of all the constructs have shown that the seven domains have been successfully overexpressed in E. coli. The overexpression was confirmed using Western blot techniques. The recombinant protein of each domain is purified for NMR and x-ray studies.

Characterisation of PduS, the pdu Metabolosome Corrin Reductase, and Evidence of Substructural Organisation within the Bacterial Microcompartment

PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu) operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS) has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(III)alamin to cob(II)alamin and cob(II)alamin to cob(I)alamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

The small intestinal BB Na(+)/H(+) antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P(3)) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His(6) proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P(2) and PI(3,4,5)P(3) bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na(+)/H(+) exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P(2) and PI(3,4,5)P(3) binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P(2) and PI(3,4,5)P(3) binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P(2) or PI(3,4,5)P(3) binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr(501)-Arg(512) and Arg(520)-Arg(552)) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.

Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several “hot spots,” such as Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.

Probing the Conformation of a Prion Protein Fibril with Hydrogen Exchange

A fragment of the prion protein, PrP(89-143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR. Although single amide resolution is afforded by NMR measurements, HXMS is well suited to the study of natural prions because it does not require labeling with NMR active isotopes. Thus, natural prions obtained from infected animals can be compared with model systems such as PrP(89-143, P101L) studied here. In our study, we find two segments of sequence that display a high level of protection from exchange, residues 102-109 and 117-136. In addition, there is a region that displays exchange behavior consistent with the presence of a conformationally heterogeneous turn. We discuss our data with respect to several structural models proposed for infectious PrP aggregates and highlight HXMS as one of the few techniques well suited to studying natural prions.

Nucleobindin 1 Is a Calcium-regulated Guanine Nucleotide Dissociation Inhibitor of Gαi1

Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent calcium cations. Calcium binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α subunits. We show that dimeric calcium-free sNUCB1 binds to expressed Gα(i1) and that calcium binding inhibits the interaction. The binding of sNUCB1 to Gα(i1) inhibits its basal rate of GDP release and slows its rate and extent of GTPγS uptake. Additionally, our tissue culture experiments show that sNUCB1 prevents receptor-mediated Gα(i)-dependent inhibition of adenylyl cyclase. Thus, we conclude that sNUCB1 is a calcium-dependent guanine nucleotide dissociation inhibitor (GDI) for Gα(i1). To our knowledge, sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. We also show that the mechanism of GDI activity of sNUCB1 is unique and does not arise from the consensus GoLoco motif found in RGS proteins. We propose that cytoplasmic NUCB1 might function to regulate heterotrimeric G protein trafficking and G protein-coupled receptor-mediated signal transduction pathways.

Versatility of Trigger Factor Interactions with Ribosome-Nascent Chain Complexes

Trigger factor (TF) is the first molecular chaperone that interacts with nascent chains emerging from bacterial ribosomes. TF is a modular protein, consisting of an N-terminal ribosome binding domain, a PPIase domain, and a C-terminal domain, all of which participate in polypeptide binding. To directly monitor the interactions of TF with nascent polypeptide chains, TF variants were site-specifically labeled with an environmentally sensitive NBD fluorophore. We found a marked increase in TF-NBD fluorescence during translation of firefly luciferase (Luc) chains, which expose substantial regions of hydrophobicity, but not with nascent chains lacking extensive hydrophobic segments. TF remained associated with Luc nascent chains for 111 +/- 7 s, much longer than it remained bound to the ribosomes (t((1/2)) approximately 10-14 s). Thus, multiple TF molecules can bind per nascent chain during translation. The Escherichia coli cytosolic proteome was classified into predicted weak and strong interactors for TF, based on the occurrence of continuous hydrophobic segments in the primary sequence. The residence time of TF on the nascent chain generally correlated with the presence of hydrophobic regions and the capacity of nascent chains to bury hydrophobicity. Interestingly, TF bound the signal sequence of a secretory protein, pOmpA, but not the hydrophobic signal anchor sequence of the inner membrane protein FtsQ. On the other hand, proteins lacking linear hydrophobic segments also recruited TF, suggesting that TF can recognize hydrophobic surface features discontinuous in sequence. Moreover, TF retained significant affinity for the folded domain of the positively charged, ribosomal protein S7, indicative of an alternative mode of TF action. Thus, unlike other chaperones, TF appears to employ multiple mechanisms to interact with a wide range of substrate proteins.

Structural and Enzymatic Insights into the ATP Binding and Autophosphorylation Mechanism of a Sensor Histidine Kinase

DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions. To analyze the ATP-binding features of HPK7 HKs and dissect their mechanism of autophosphorylation at the molecular level, we have studied DesK in complex with ATP using high resolution structural approaches in combination with biochemical studies. We report the first crystal structure of an HK in complex with its natural nucleotidic substrate. The general fold of the ATP-binding domain of DesK is conserved, compared with well studied members of other families. Yet, DesK displays a far more compact structure at the ATP-binding pocket: the ATP lid loop is much shorter with no secondary structural organization and becomes ordered upon ATP loading. Sequence conservation mapping onto the molecular surface, semi-flexible protein-protein docking simulations, and structure-based point mutagenesis allow us to propose a specific domain-domain geometry during autophosphorylation catalysis. Supporting our hypotheses, we have been able to trap an autophosphorylating intermediate state, by protein engineering at the predicted domain-domain interaction surface.

Peroxisomal Plant 3-Ketoacyl-CoA Thiolase Structure and Activity Are Regulated by a Sensitive Redox Switch

The breakdown of fatty acids, performed by the beta-oxidation cycle, is crucial for plant germination and sustainability. beta-Oxidation involves four enzymatic reactions. The final step, in which a two-carbon unit is cleaved from the fatty acid, is performed by a 3-ketoacyl-CoA thiolase (KAT). The shortened fatty acid may then pass through the cycle again (until reaching acetoacetyl-CoA) or be directed to a different cellular function. Crystal structures of KAT from Arabidopsis thaliana and Helianthus annuus have been solved to 1.5 and 1.8 A resolution, respectively. Their dimeric structures are very similar and exhibit a typical thiolase-like fold; dimer formation and active site conformation appear in an open, active, reduced state. Using an interdisciplinary approach, we confirmed the potential of plant KATs to be regulated by the redox environment in the peroxisome within a physiological range. In addition, co-immunoprecipitation studies suggest an interaction between KAT and the multifunctional protein that is responsible for the preceding two steps in beta-oxidation, which would allow a route for substrate channeling. We suggest a model for this complex based on the bacterial system.

Expression and single-step purification of mercury transporter (merT) from Cupriavidus metallidurans in E. coli

The mercury transporter, merT, from Cupriavidus metallidurans was cloned into pRSET-C and expressed in various E. coli hosts. Expression of merT gene failed in common expression hosts like E. coli BL21(DE3), E. coli BL21(DE3)pLysS and E. coli GJ1158 due to expression induced toxicity. The protein was successfully expressed in E. coli C43(DE3) as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 detergent. The detergent solubilized protein with N-terminal His-tag was purified in a single-step by immobilized metal affinity chromatography with a yield of 8 mg l(-1).

Mass Spectrometry Following Mild Enzymatic Digestion Reveals Phosphorylation of Recombinant Proteins in Escherichia coli Through Mechanisms Involving Direct Nucleotide Binding

A straightforward method using mild enzymatic digestions combined with MALDI mass spectrometry (MS) was used to enhance determination of the multiple phosphorylation sites of a set of recombinant nucleotide-binding proteins in Escherichia coli, including kinases and cystathionine beta-synthase (CBS) domain containing proteins. The protein kinases reveal abundant phosphorylations in the kinase domains and relatively low phosphogluconoylation (258 Da) at the N-terminal His-tag. In contrast, the CBS domain-containing proteins possess a highly conserved phosphorylation in vivo at Ser-2 of the His-tag. Multistage MS/MS and selected reaction monitoring established that the CBS domain proteins also contain a combined modification of gluconoylation (178 Da) and phosphorylation (80 Da) at two different sites, instead of an isobaric phosphogluconoylation (258 Da) event at the N-terminus. Functional analysis of 20 recombinant proteins as identified by mass spectrometry has shown the phosphorylation at the N-terminal His-tag is relevant to nucleotide binding and phosphotransfer reaction catalyzed by a serine protein kinase.