Protein microarrays: a chance to study microorganisms?

Within the last 5 years, protein microarrays have been developed and applied to multiple approaches: identification of protein–protein interactions or protein–small molecule interactions, cancer profiling, detection of microorganisms and toxins, and identification of antibodies due to allergens, autoantigens, and pathogens. Protein microarrays are small size (typically in the microscopy slide format) planar analytical devices with probes arranged in high density to provide the ability to screen several hundred to thousand known substrates (e.g., proteins, peptides, antibodies) simultaneously. Due to their small size, only minute amounts of spotted probes and analytes (e.g., serum) are needed; this is a particularly important feature, for these are limited or expensive. In this review, different types of protein microarrays are reviewed: protein microarrays (PMAs), with spotted proteins or peptides; antibody microarrays (AMAs), with spotted antibodies or antibody fragments (e.g., scFv); reverse phase protein microarrays (RPMAs), a special form of PMA where crude protein mixtures (e.g., cell lysates, fractions) are spotted; and nonprotein microarrays (NPMAs) where macromolecules other than proteins and nucleic acids (e.g., carbohydrates, monosaccharides, lipopolysaccharides) are spotted. In this study, exemplary experiments for all types of protein arrays are discussed wherever applicable with regard to investigations of microorganisms.     

Systems for the detection and analysis of protein–protein interactions

The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review.K. Kuroda, M. Kato and J. Mima contributed equally to this work.     

In situ protein microarrays capable of real-time kinetics analysis based on surface plasmon resonance imaging

In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein–protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (∼3–7 × 10⁻¹³ M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein–protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields.     

Application of the random coil index to studying protein flexibility

Protein flexibility lies at the heart of many protein–ligand binding events and enzymatic activities. However, the experimental measurement of protein motions is often difficult, tedious and error-prone. As a result, there is a considerable interest in developing simpler and faster ways of quantifying protein flexibility. Recently, we described a method, called Random Coil Index (RCI), which appears to be able to quantitatively estimate model-free order parameters and flexibility in protein structural ensembles using only backbone chemical shifts. Because of its potential utility, we have undertaken a more detailed investigation of the RCI method in an attempt to ascertain its underlying principles, its general utility, its sensitivity to chemical shift errors, its sensitivity to data completeness, its applicability to other proteins, and its general strengths and weaknesses. Overall, we find that the RCI method is very robust and that it represents a useful addition to traditional methods of studying protein flexibility. We have implemented many of the findings and refinements reported here into a web server that allows facile, automated predictions of model-free order parameters, MD RMSF and NMR RMSD values directly from backbone ¹H, ¹³C and ¹⁵N chemical shift assignments. The server is available at . Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An interrupted beta-propeller and protein disorder: structural bioinformatics insights into the N-terminus of alsin

Defects in the human ALS2 gene, which encodes the 1,657-amino-acid residue protein alsin, are linked to several related motor neuron diseases. We created a structural model for the N-terminal 690-residue region of alsin through comparative modelling based on regulator of chromosome condensation 1 (RCC1). We propose that this alsin region contains seven RCC1-like repeats in a seven-bladed beta-propeller structure. The propeller is formed by a double clasp arrangement containing two segments (residues 1–218 and residues 525–690). The 306-residue insert region, predicted to lie within blade 5 and to be largely disordered, is poorly conserved across species. Surface patches of evolutionary conservation probably indicate locations of binding sites. Both disease-causing missense mutations—Cys157Tyr and Gly540Glu—are buried in the propeller and likely to be structurally disruptive. This study aids design of experimental studies by highlighting the importance of construct length, will enhance interpretation of protein–protein interactions, and enable rational site-directed mutagenesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

XH/<Emphasis Type="Italic">π</Emphasis> interactions with the <Emphasis Type="Italic">π</Emphasis> system of porphyrin ring in porphyrin-containing proteins

Searching structures of porphyrin-containing proteins from the Protein Data Bank revealed that the π system of every porphyrin ring is involved in XH/π interactions, with most of the porphyrins having several interactions. Both five-membered pyrrole rings and six-membered chelate rings are involved in XH/π interactions; the number of interactions with five-membered rings is larger than the number of interactions with six-membered rings. We found interactions with C–H and N–H groups as hydrogen-atom donors; however, the number of CH/π interactions is much larger than the number of NH/π interactions. The amino acids involved in the interactions show a high conservation score. Our results that every porphyrin is involved in XH/π interactions and that amino acids involved in these interactions are highly conserved demonstrate that XH/π interactions play an important role in porphyrin–protein stability. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterizing weak protein–protein complexes by NMR residual dipolar couplings

Protein–protein interactions occur with a wide range of affinities from tight complexes characterized by femtomolar dissociation constants to weak, and more transient, complexes of millimolar affinity. Many of the weak and transiently formed protein–protein complexes have escaped characterization due to the difficulties in obtaining experimental parameters that report on the complexes alone without contributions from the unbound, free proteins. Here, we review recent developments for characterizing the structures of weak protein–protein complexes using nuclear magnetic resonance spectroscopy with special emphasis on the utility of residual dipolar couplings.     

Arabidopsis DDB1a and DDB1b are critical for embryo development

DNA DAMAGED BINDING PROTEIN 1 (DDB1) is a highly conserved protein of around 125 kDa. It serves as a substrate adaptor subunit to a CUL4-based E3 ubiquitin ligase within the ubiquitin proteasome pathway. However, based on a set of three beta-propellers, the protein is able to mediate various protein–protein interactions, suggesting that it participates in many developmental and physiological processes in the plant. Arabidopsis encodes for two closely related DDB1 proteins, named DDB1a and DDB1b. While loss-of DDB1a does not severely affect development, loss-of DDB1b has been reported to result in an embryo lethal phenotype. Here we describe two novel ddb1b T-DNA insertion mutants that are not embryo lethal, which we utilized as genetic tools to dissect DDB1b from DDB1a function. Information generated by these studies showed that the C-terminal part of the DDB1 proteins is critical for specific protein–protein interactions. In addition, we demonstrated that DDB1a, like DDB1b, is critical for embryo development, and that both proteins have distinct functions in whole plant development.     

Structural determination of biomolecular interfaces by nuclear magnetic resonance of proteins with reduced proton density

Protein interactions are important for understanding many molecular mechanisms underlying cellular processes. So far, interfaces between interacting proteins have been characterized by NMR spectroscopy mostly by using chemical shift perturbations and cross-saturation via intermolecular cross-relaxation. Although powerful, these techniques cannot provide unambiguous estimates of intermolecular distances between interacting proteins. Here, we present an alternative approach, called REDSPRINT (REDduced/Standard PRoton density INTerface identification), to map protein interfaces with greater accuracy by using multiple NMR probes. Our approach is based on monitoring the cross-relaxation from a source protein (or from an arbitrary ligand that need not be a protein) with high proton density to a target protein (or other biomolecule) with low proton density by using isotope-filtered nuclear Overhauser spectroscopy (NOESY). This methodology uses different isotropic labeling for the source and target proteins to identify the source-target interface and also determine the proton density of the source protein at the interface for protein-protein or protein-ligand docking. Simulation indicates significant gains in sensitivity because of the resultant relaxation properties, and the utility of this technique, including a method for direct determination of the protein interface, is demonstrated for two different protein–protein complexes.     

Recognizing protein–protein interfaces with empirical potentials and reduced amino acid alphabets

Background  

In structural genomics, an important goal is the detection and classification of protein–protein interactions, given the structures of the interacting partners. We have developed empirical energy functions to identify native structures of protein–protein complexes among sets of decoy structures. To understand the role of amino acid diversity, we parameterized a series of functions, using a hierarchy of amino acid alphabets of increasing complexity, with 2, 3, 4, 6, and 20 amino acid groups. Compared to previous work, we used the simplest possible functional form, with residue–residue interactions and a stepwise distance-dependence. We used increased computational ressources, however, constructing 290,000 decoys for 219 protein–protein complexes, with a realistic docking protocol where the protein partners are flexible and interact through a molecular mechanics energy function. The energy parameters were optimized to correctly assign as many native complexes as possible. To resolve the multiple minimum problem in parameter space, over 64000 starting parameter guesses were tried for each energy function. The optimized functions were tested by cross validation on subsets of our native and decoy structures, by blind tests on series of native and decoy structures available on the Web, and on models for 13 complexes submitted to the CAPRI structure prediction experiment.     

Enhanced crystal packing due to solvent reorganization through reductive methylation of lysine residues in oxidoreductase from <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Protein crystallization is in part driven by the changes in the entropy of the system, but opinions differ as to whether the solute (protein) or solvent (water) molecules make more of a contribution to the overall entropic change. Methylation of lysine residues in proteins has been used to enhance protein crystallization. We investigated using molecular dynamics simulations with explicit solvent molecules, the behavior of several native proteins and their methylated counterparts chosen from an earlier large-scale study. Methylated lysines are capable of making a variety of interactions including H-bonds with protein residues and solvent. We demonstrate that methylation on the lysine slightly increases its side chain conformational entropy by about 3.5 J mol⁻¹ K⁻¹. Analysis of the radial and spatial distributions of the water molecules around the methylated lysine surface in oxidoreductase from Streptococcus pneumoniae revealed a larger sphere of water molecules with low entropy, as compared with solvent associated with unmethylated lysine. If methylated lysine were to make interactions at the protein–protein interface, the low-entropy water molecules associated with methylated lysines would be released, resulting in a gain of entropy. We show that this gain more than compensates for the loss of protein entropy. Therefore, we propose that lysine methylation favors the formation of crystals through solvent entropic gain.     

Amino acid contacts in proteins adapted to different temperatures: hydrophobic interactions and surface charges play a key role

Thermophiles, mesophiles, and psychrophiles have different amino acid frequencies in their proteins, probably because of the way the species adapt to very different temperatures in their environment. In this paper, we analyse how contacts between sidechains vary between homologous proteins from species that are adapted to different temperatures, but displaying relatively high sequence similarity. We investigate whether specific contacts between amino acids sidechains is a key factor in thermostabilisation in proteins. The dataset was divided into two subsets with optimal growth temperatures from 0–40 and 35–102°C. Comparison of homologues was made between low-temperature species and high-temperature species within each subset. We found that unspecific interactions like hydrophobic interactions in the core and solvent interactions and entropic effects at the surface, appear to be more important factors than specific contact types like salt bridges and aromatic clusters. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA binding and oxidative DNA damage induced by a quercetin copper(II) complex: potential mechanism of its antitumor properties

The interaction of a quercetin copper(II) complex with DNA was investigated using UV–vis spectra, fluorescence measurement, viscosity measurement, agarose gel electrophoresis, and thiobarbituric acid reactive substances assay. The results indicate that the quercetin copper(II) complex can promote the cleavage of plasmid DNA, producing single and double DNA strand breaks, and intercalate into the stacked base pairs of DNA. Moreover, the complex can induce oxidative DNA damage involving generation of reactive oxygen species such as H₂O₂ and Cu(I)OOH. In addition, the cytotoxicity experiments carried out with A549 cells confirmed its apoptosis-inducing activity. And we also demonstrate that the levels of survivin protein expression in A549 cells decreased, and that relative activity of caspase-3 increased significantly after treatment with the complex. So our results suggest that the antitumor mechanism of the quercetin copper(II) complex involves not only its oxidative DNA damage with generation of reactive oxygen species but also its specific interaction with DNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protein structural changes induced by glutathione-coated CdS quantum dots as revealed by Trp phosphorescence

We evaluated the potential of tryptophan (Trp) phosphorescence spectroscopy for investigating conformational states of proteins involved in interaction with nanoparticles. Characterization of protein–nanoparticle interaction is crucial in assessing biological hazards related to use of nanoparticles. We synthesized glutathione-coated CdS quantum dots (GSH-CdS), which exhibited an absorption peak at 366 nm, indicative of 2.4 nm core size. Chemical analysis of purified GSH-CdS suggested an average molecular formula of GSH₁₈S₅₆Cd₆₀. Investigations were conducted on model proteins varying in terms of isoelectric point, degree of burial of the Trp probe, and quaternary structure. GSH-CdS fluorescence measurements showed improvement in nanoparticle quantum yield induced by protein interaction. Trp phosphorescence was used to examine the possible perturbations in the protein native fold induced by GSH-CdS. Phosphorescence lifetime measurements highlighted significant conformational changes in some proteins. Despite their small size, GSH-CdS appeared to interact with more than one protein molecule. Rough determination of the affinity of GSH-CdS for proteins was derived from the change in phosphorescence lifetime at increasing nanoparticle concentrations. The estimated affinities were comparable to those observed for specific protein–ligand interactions and suggest that protein–nanoparticle interaction may have a biological impact.     

Mapping protein–protein interaction by 13C′-detected heteronuclear NMR spectroscopy

The copper-mediated protein–protein interaction between yeast Atx1 and Ccc2 has been examined by protonless heteronuclear NMR and compared with the already available ¹H–¹⁵N HSQC information. The observed chemical shift variations are analyzed with respect to the actual solution structure, available through intermolecular NOEs. The advantage of using the CON-IPAP spectrum with respect to the ¹H–¹⁵N HSQC resides in the increased number of signals observed, including those of prolines. CBCACO-IPAP experiments allow us to focus on the interaction region and on side-chain carbonyls, while a newly designed CEN-IPAP experiment on side-chains of lysines. An attempt is made to rationalize the chemical shift variations on the basis of the structural data involving the interface between the proteins and the nearby regions. It is here proposed that protonless ¹³C direct-detection NMR is a useful complement to ¹H based NMR spectroscopy for monitoring protein–protein and protein–ligand interactions. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at     

Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr–Purcell–Meiboom–Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k _ex = 500–2000 s⁻¹ were typically observed in APO-rMUP for residues located adjacent to a β-turn comprising residues 83–87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local ‘breathing’ motions rather than a concerted global conformational change. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biochemical characterization of the minichromosome maintenance protein from the archaeon Thermoplasma acidophilum

Minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. Studies have shown that the MCM complex from the thermoacidophilic euryarchaeon Thermoplasma acidophilum (TaMCM) has some properties not reported in other archaeal MCM helicases. Here, the biochemical properties of the TaMCM are studied. The protein binds single-stranded DNA, has DNA-dependent ATPase activity and ATP-dependent 3′ → 5′ helicase activity. The optimal helicase conditions with regard to temperature, pH and salinity are similar to the intracellular conditions in T. acidophilum. It is also found that about 1,000 molecules of TaMCM are present per actively growing cell. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning and expression of three peptide-linked β2-microglobulin molecules in <Emphasis Type="Italic">Escherichia coli</Emphasis> with an isocaudamer technique

The application of the peptide-linked β2-microglobulin (β2m) strategy is limited in some cases due to the incompatibility between the sequences of the peptides and the restriction sites of the plasmid vectors. An isocaudamer technique was adapted to overcome this restriction. Three peptide-linked β2m genes, HBc_18–27-hβ2m gene, OVA_257–264-mβ2m gene and HER2/neu_369–377-mβ2m gene, were inserted into the pET28a vectors with this technique. The corresponding proteins were expressed in Escherichia coli with yields of over 50 mg/l culture and purities of over 80%. This strategy facilitates the construction of peptide-linked β2m molecules and will simplify the preparation of major histocompatibility complex-peptide complexes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Leaf-punch method to prepare a large number of PCR templates from plants for SNP analysis

DNA preparation is indispensable for genotyping by DNA polymorphism analysis, and that for a large number of plants is laborious. In the present study, a small leaf disk of rice, 1–2 mm in diameter, punched by a mini cork borer was found to be directly usable as a PCR template. DNA fragments <300 bp were amplified efficiently. Leaf disks of 1–1.5 mm in diameter were better than those of 2 mm for a small volume of reaction mixture. Multiplex PCR was possible with four or eight primer pairs using the small leaf disk as a template. Leaf disks of Arabidopsis, Lotus, wheat, soybean, tomato, Chinese cabbage, and melon were also good PCR templates. This method for preparation of PCR templates, named the leaf-punch method, was applicable to SNP analysis of a large number of plants by dot-blot-SNP analysis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.