Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis sigma/anti-sigma factor protein complexes

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the sigma factor/anti-sigma factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of sigma factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems. (C) 2010 Elsevier Inc. All rights reserved.

High-resolution methyl edited GFT NMR experiments for protein resonance assignments and structure determination

Three-dimensional (3D) structure determination of proteins is benefitted by long-range distance constraints comprising the methyl groups, which constitute the hydrophobic core of proteins. However, in methyl groups (of Ala, Ile, Leu, Met, Thr and Val) there is a significant overlap of C-13 and H-1 chemical shifts. Such overlap can be resolved using the recently proposed (3,2)D HCCH-COSY, a G-matrix Fourier transform (GFT) NMR based experiment, which facilitates editing of methyl groups into distinct spectral regions by combining their C-13 chemical shifts with that of the neighboring, directly attached, C-13 nucleus. Using this principle, we present three GFT experiments: (a) (4,3)D NOESY-HCCH, (b) (4,3)D H-1-TOCSY-HCCH and (c) (4,3)D C-13-TOCSY-HCCH. These experiments provide unique 4D spectral information rapidly with high sensitivity and resolution for side-chain resonance assignments and NOE analysis of methyl groups. This is exemplified by (4,3)D NOESY-HCCH data acquired for 17.9 kDa non-deuterated cytosolic human J-protein co-chaperone, which provided crucial long-range distance constraints for its 3D structure determination.

PeptideMine - A webserver for the design of peptides for protein-peptide binding studies derived from protein-protein interactomes

Background: Signal transduction events often involve transient, yet specific, interactions between structurally conserved protein domains and polypeptide sequences in target proteins. The identification and validation of these associating domains is crucial to understand signal transduction pathways that modulate different cellular or developmental processes. Bioinformatics strategies to extract and integrate information from diverse sources have been shown to facilitate the experimental design to understand complex biological events. These methods, primarily based on information from high-throughput experiments, have also led to the identification of new connections thus providing hypothetical models for cellular events. Such models, in turn, provide a framework for directing experimental efforts for validating the predicted molecular rationale for complex cellular processes. In this context, it is envisaged that the rational design of peptides for protein-peptide binding studies could substantially facilitate the experimental strategies to evaluate a predicted interaction. This rational design procedure involves the integration of protein-protein interaction data, gene ontology, physico-chemical calculations, domain-domain interaction data and information on functional sites or critical residues. Results: Here we describe an integrated approach called ``PeptideMine'' for the identification of peptides based on specific functional patterns present in the sequence of an interacting protein. This approach based on sequence searches in the interacting sequence space has been developed into a webserver, which can be used for the identification and analysis of peptides, peptide homologues or functional patterns from the interacting sequence space of a protein. To further facilitate experimental validation, the PeptideMine webserver also provides a list of physico-chemical parameters corresponding to the peptide to determine the feasibility of using the peptide for in vitro biochemical or biophysical studies. Conclusions: The strategy described here involves the integration of data and tools to identify potential interacting partners for a protein and design criteria for peptides based on desired biochemical properties. Alongside the search for interacting protein sequences using three different search programs, the server also provides the biochemical characteristics of candidate peptides to prune peptide sequences based on features that are most suited for a given experiment. The PeptideMine server is available at the URL: http://caps.ncbs.res.in/peptidemine

X-ray and molecular-dynamics studies on Mycobacterium leprae single-stranded DNA-binding protein and comparison with other eubacterial SSB structures

The crystal structures of two forms of Mycobacterium leprae single-stranded DNA-binding protein (SSB) have been determined at 2.05 and 2.8 A resolution. Comparison of these structures with the structures of other eubacterial SSBs indicates considerable variation in their quaternary association, although the DNA-binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but could be related to variation in protein stability. Molecular-dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype Escherichia coli SSB and the individual subunits of both proteins. Together, the X-ray studies and molecular-dynamics simulations yield information on the relatively rigid and flexible regions of the molecule and on the effect of oligomerization on flexibility. The simulations provide insight into the changes in subunit structure on oligomerization. They also provide insight into the stability and time evolution of the hydrogen bonds/water bridges that connect the two pairs of monomers in the tetramer.

Chloroquine Inhibits Heme-Dependent Protein Synthesis in Plasmodium falciparum

A cell-free protein-synthesizing system has been reconstituted using the S-30 fraction or ribosomes and the S-100 fraction from Plasmodium falciparum. Addition of heme in vitro stimulates cell-free protein synthesis strikingly. Chloroquine inhibits the heme-dependent protein synthesis in the parasite lysate. The drug has also been found to inhibit parasite protein synthesis in situ at therapeutic concentrations soon after addition to parasite cultures. Ribosomes as well as the S-100 fraction isolated from such chloroquine-treated cultures are defective in protein synthesis. Addition of hemin plus glucose 6-phosphate or high concentrations of GTP, cAMP, and an active preparation of eIF-2 to the parasite cell-free system restores protein synthesis to a significant extent in chloroquine-treated cultures. Under conditions of inhibition of protein synthesis in situ by chloroquine in the culture, the parasite eukaryotic initiation factor 2-alpha- (eIF-2-alpha) is phosphorylated in the parasite lysate to a greater extent than that observed in the control culture. Addition of hemin in vitro suppresses this phosphorylation. eIF-2-alpha kinase activity is present in the parasite lysate and is not a contaminant derived from the human erythrocytes used to culture the parasite. The heme-chloroquine interactive effects can also be demonstrated with purified eIF-2-alpha kinase from rabbit reticulocyte lysate. It is proposed that chloroquine inhibits heme-dependent protein synthesis in the parasite and this is an early event mediating the growth-inhibitory effects of the drug.

Early pregnancy termination in rats immunized with denatured chicken riboflavin carrier protein

Immunoneutralization of the maternal riboflavin carrier protein in the pregnant rat with antibodies to chicken egg vitamin carrier has earlier been shown to terminate their pregnancies. In order to understand the nature of the epitopic conformations capable of eliciting antibodies bioneutralizing the endogenous riboflavin carrier protein in the pregnant rat, we compared pregnancy progression in the fertile rodents following active immunization with either the native, SDS-denatured, reduced-carboxymethylated or SDS-treated reduced carboxymethylated avian egg white riboflavin carrier protein. The data revealed that despite the total antibody titers being higher in the animals immunized with the native protein, the antibodies elicited against the denatured avian vitamin carrier exhibited relatively better potencies to bioneutralize the endogenous maternal protein as evidenced by higher rates of early fetal resorption.

Effect of amino acid substitutions at the subunit interface on the stabiity and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.

1-Anilino-8-naphthalene sulfonate (ANS) binding to proteins revisited: Investigation of dye binding to molten globule states by electrospray ionization mass spectrometry

The binding of 1-anilino-8-naphthalene-sulfonic acid to globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESIMS). Mass spectra of apomyoglobin recorded in the pH range 2−7 establish that maximal ANS binding is observed at pH 4.0. As many as seven distinct species may be observed in the gas phase which correspond to protein molecules containing one to six molecules of bound ANS. At neutral pH only a single molecule of ANS is bound. In the case of cytochrome c, maximal binding is observed at pH 4.0, with five molecules being bound. Binding is suppressed at neutral pH. In both cases ESIMS demonstrates maximal ANS binding at pH values where the proteins have been reported to exist in molten globule states. ANS binding is not observed for lysozyme, which has a tightly folded structure over the entire pH range. Reduction of disulfide bonds in lysozyme leads to the detection of ANS-bound species at neutral pH. Binding is suppressed at low pH due to complete unfolding of the reduced protein. The results suggest that ESIMS may provide a convenient method of probing the stoichiometry and distribution of dye complexes with molten protein globules

Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica

A phosphorylcholine-binding protein from the hemolymph of the snail Achatina fulica was purified to near homogeneity using a Sepharose phenylphosphorylcholine affinity column. The protein bound to the affinity column was eluted with 5 mM phosphorylcholine as a single symmetrical peak. The purified protein (400 Kda) contained 35–40% carbohydrate. On SDS-PAGE the protein separated into two bands of 20 and 24 Kda, and had a pI of 5.9. On immunodiffusion, antiserum to the snail phosphorylcholine binding protein did not cross-react against other phosphorylcholine binding proteins, like rat serum phosphorylcholine-binding protein (PCBP), limulus C-reactive protein (CRP), or human CRP. On pretreatment of the snail hemolymph with this antiserum, the hemagglutination titer of the hemolymph was markedly decreased. The purified snail phosphorylcholine binding protein agglutinated rabbit erythrocytes in the absence of divalent cation (Ca+2) but trace amount of Ca+2 increased its binding. The strongest inhibitor of the agglutination reaction was lactose, followed by melibiose and 2-deoxygalactose. The relationships of the snail phosphorylcholine binding protein to other hemolymph agglutinins and to CRPs are discussed in light of common phylogeny.

Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein

To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.

A simplified method for the purification of riboflavin-binding protein from hen's egg yolk

A simple and efficient procedure for the purification of the riboflavin-binding protein from hen's egg yolk is described. This method involves the removal by exclusion of lipoproteins and subsequent fractionation of soluble yolk proteins held on a DEAE-cellulose column by a salt gradient which is followed by purification by gel filtration on Sephadex G-100. The protein thus isolated is homogeneous by various physicoehemical, immunological, and functional criteria.

Riboflavin carrier protein from carp (C. carpio) eggs: comparison with avian riboflavin carrier protein

A protein exhibiting immunological cross-reactivity with the chicken egg-white riboflavin carrier protein was detected by radioimmunoassay in the eggs and serum of the fresh water fish Cyprinus carpio and subsequently purified to homogeneity by use of affinity chromatography. Fish riboflavin carrier protein resembled chicken riboflavin carrier protein with respect to most of its physicochemical characteristics. The major epitopes of chicken riboflavin carrier protein were shown to be conserved in the fish protein as probed with monoclonal antibodies to the avian vitamin carrier.

Quantitative and compositional changes in myelin of undernourished and protein malnourished rat brains

Effects of undernutrition and protein malnutrition on the quantitative and qualitative changes in myelin isolated from rat brain at 3 and 8 weeks of age were investigated. Undernutrition during suckling period was induced by increasing the litter size, and continued from the 3rd to the 8th week by limited food intake, or the rats were rehabilitated with adequate food. Protein malnutrition was induced by feeding the lactating dams 5% protein diet as against 25% protein diet in controls. The protein malnourished rats were rehabilitated from the 3rd to the 8th week with the normal 25% protein diet. Undernutrition produced 16% and 35% reductions in the myelin content at 3 and 8 weeks of age, respectively, and was only partially restored on rehabilitation. Protein malnutrition caused more drastic reduction of 27% in the myelin content at 3 weeks, which was also partially restored on rehabilitation. The specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase was not affected by undernutrition, whereas protein malnutrition caused a 25% reduction at 3 weeks, which was totally reversed by rehabilitation. Undernutrition had not altered the relative composition of myelin proteins, but protein malnutrition resulted in a significant reduction in the proteolipid protein at 3 weeks of age, which could be reversed by rehabilitation.