Protein-protein conjugation on a lectin matrix.

An efficient method of protein-protein conjugation yielding primarily monoconjugates is described. A glycoprotein enzyme, invertase, was ‘spaced-out’ on a succinyl concanavalin A sepharose matrix and reacted with 1% glutaraldehyde. The excess glutaraldehyde was washed out and a second, non-glycoprotein, enzyme, urease, was reacted with the ‘activated’ invertase. The column was washed till the washings were free of enzymatic activity. On elution with α-methyl glucoside both enzymes were detected in the eluate. Resolution on Sepharose 6B revealed that the eluted invertase was completely conjugated to urease. The molecular size of the conjugate suggested that it was a monoconjugate. The glutaraldehyde treated enzyme retained its immunological reactivity in the conjugate. This method of protein-protein conjugation is applicable if one of the two involved proteins is a glycoprotein.     

Protein-protein interactions in membranes

In this article we review the current status of our understanding of membrane mediated interactions from theory and experiment. Phenomenological mean field and molecular models will be discussed and compared to recent experimental results from dynamical neutron scattering and atomic force microscopy.     

Protein-protein interactions in plants

The study of protein-protein interactions (PPIs) is essential to uncover unknown functions of proteins at the molecular level and to gain insight into complex cellular networks. Affinity purification and mass spectrometry (AP-MS), yeast two-hybrid, imaging approaches and numerous diverse databases have been developed as strategies to analyze PPIs. The past decade has seen an increase in the number of identified proteins with the development of MS and large-scale proteome analyses. Consequently, the false-positive protein identification rate has also increased. Therefore, the general consensus is to confirm PPI data using one or more independent approaches for an accurate evaluation. Furthermore, identifying minor PPIs is fundamental for understanding the functions of transient interactions and low-abundance proteins. Besides establishing PPI methodologies, we are now seeing the development of new methods and/or improvements in existing methods, which involve identifying minor proteins by MS, multidimensional protein identification technology or OFFGEL electrophoresis analyses, one-shot analysis with a long column or filter-aided sample preparation methods. These advanced techniques should allow thousands of proteins to be identified, whereas in-depth proteomic methods should permit the identification of transient binding or PPIs with weak affinity. Here, the current status of PPI analysis is reviewed and some advanced techniques are discussed briefly along with future challenges for plant proteomics.     

Protein-protein interactions in human disease

Many human diseases are the result of abnormal protein-protein interactions involving endogenous proteins, proteins from pathogens or both. The inhibition of these aberrant associations is of obvious clinical significance. Because of the diverse nature of protein-protein interactions, however, the successful design of therapeutics requires detailed knowledge of each system at a molecular and atomic level. Several recent studies have identified and/or characterised specific interactions from various disease systems, including cervical cancer, bacterial infection, leukaemia and neurodegenerative disease. A range of approaches are being developed to generate inhibitors of protein-protein interactions that may form useful therapeutics for human disease.     

Protein-protein interactions in signaling cascades

The process of signal transduction is dependent on specific protein-protein interactions. In many cases these interactions are mediated by modular protein domains that confer specific binding activity to the proteins in which they are found. Rapid progress has been made in the biochemical characterization of binding interactions, the identification of binding partners, and determination of the three-dimensional structures of binding modules and their ligands. The resulting information establishes the logical framework for our current understanding of the signal transduction machinery. In this overview a variety of protein interaction modules are discussed, and issues relating to binding specificity and the significance of a particular interaction are considered.     

Protein-protein interactions required during translation

Protein synthesis requires the involvement of numerous accessory factors that assist the ribosome in translation initiation, elongation, and termination. Extensive protein-protein and protein-RNA interactions are required to bring together the accessory factors, tRNAs, ribosomes, and mRNA into a productive complex and these interactions undergo dynamic alterations during each step of the translation initiation process. Initiation represents the most complex aspect of translation, requiring more accessory proteins, called initiation factors, than either elongation or termination. Not surprisingly, initiation is most often the rate-limiting step of translation and, as such, most (but not all) examples of translational regulation involve the regulation of protein-protein or protein-RNA interactions of the initiation complex. In this review, we focus on those interactions required for efficient translation initiation and how such interactions are regulated by developmental or environmental signals.     

Protein-protein interactions that regulate the energy stress activation of sigma(B) in Bacillus subtilis

Sigma(B) is an alternative sigma factor that controls the general stress response in Bacillus subtilis. In the absence of stress, sigma(B) is negatively regulated by anti-sigma factor RsbW. RsbW is also a protein kinase which can phosphorylate RsbV. When cells are stressed, RsbW binds to unphosphorylated RsbV, produced from the phosphorylated form of RsbV by two phosphatases (RsbU and RsbP) which are activated by stress. We now report the values of the K(m) for ATP and the K(i) for ADP of RsbW (0.9 and 0.19 mM, respectively), which reinforce the idea that the kinase activity of RsbW is directly regulated in vivo by the ratio of these nucleotides. RsbW, purified as a dimer, forms complexes with RsbV and sigma(B) with different stoichiometries, i.e., RsbW(2)-RsbV(2) and RsbW(2)-sigma(B)(1). As determined by surface plasmon resonance, the dissociation constants of the RsbW-RsbV and RsbW-sigma(B) interactions were found to be similar (63 and 92 nM, respectively). Nonetheless, an analysis of the complexes by nondenaturing polyacrylamide gel electrophoresis in competition assays suggested that the affinity of RsbW(2) for RsbV is much higher than that for sigma(B). The intracellular concentrations of RsbV, RsbW (as a monomer), and sigma(B) measured before stress were similar (1.5, 2.6, and 0.9 micro M, respectively). After ethanol stress they all increased. The increase was greatest for RsbV, whose concentration reached 13 micro M, while those of RsbW (as a monomer) and sigma(B) reached 11.8 and 4.9 micro M, respectively. We conclude that the higher affinity of RsbW for RsbV than for sigma(B), rather than a difference in the concentrations of RsbV and sigma(B), is the driving force that is responsible for the switch of RsbW to unphosphorylated RsbV.     

Protein-protein interactions in concentrated electrolyte solutions

Protein-protein interactions were measured for ovalbumin and for lysozyme in aqueous salt solutions. Protein-protein interactions are correlated with a proposed potential of mean force equal to the free energy to desolvate the protein surface that is made inaccessible to the solvent due to the protein-protein interaction. This energy is calculated from the surface free energy of the protein that is determined from protein-salt preferential-interaction parameter measurements. In classical salting-out behavior, the protein-salt preferential interaction is unfavorable. Because addition of salt raises the surface free energy of the protein according to the surface-tension increment of the salt, protein-protein attraction increases, leading to a reduction in solubility. When the surface chemistry of proteins is altered by binding of a specific ion, salting-in is observed when the interactions between (kosmotrope) ion-protein complexes are more repulsive than those between the uncomplexed proteins. However, salting-out is observed when interactions between (chaotrope) ion-protein complexes are more attractive than those of the uncomplexed proteins.     

Protein-protein interactions in intracellular membrane fusion

The fusion of intracellular vesicles with their target membranes is an essential feature of the compartmental structure of eukaryotic cells. This process requires proteins that dictate the targeting of a vesicle to the correct cellular location, mediate bilayer fusion and, in some systems, regulate the precise time at which fusion occurs. Recent biophysical and structural studies of these proteins have begun to provide a foundation for understanding their functions at a molecular level.     

NMDA受体信号复合体中蛋白质的相互作用

谷氨酸能兴奋性突触的突触后密集区(postsynaptic density,PSD)包含多种受体蛋白、骨架蛋白和信号蛋白,它们通过分子中特定的结构域相互识别并动态地结合,形成多个信号复合体,参与突触后受体功能的调节及其下游特异性信号转导通路的激活。其中,NMDA受体信号复合体中蛋白质-蛋白质的相互作用及其调控机制的阐明,对于深入了解神经发育、突触可塑性、兴奋性毒性等生理病理的分子机制有重要意义。     

Protein-protein interactions in an alphavirus membrane.

Using homobifunctional chemical cross-linkers with various span distances, we have determined the near-neighbor associations and planar organization of the E1 and E2 envelope glycoproteins which compose the icosahedral surface of Sindbis virus. We have found that E1-E2 heterodimers, which form the virus protomeric units, exist in two conformationally distinct forms, reflecting their nonequivalent positions in the icosahedron. Three of these heterodimers form the trimeric morphologic units (capsomeres) which are held together by central E1-E1 interactions. In addition, we present data which suggest that E2-E2 interactions organize the capsomeres into pentameric and hexameric geometric units and that E1-E1 interactions between capsomeres maintain the icosahedral lattice in mature virions.     

Protein-protein interactions in the synaptonemal complex.

In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II.     

Protein-protein interactions in pathogen recognition by plants

Protein-protein interactions have emerged as key determinants of whether plant encounters with pathogens result in disease or successful plant defense. Genetic interactions between plant resistance genes and pathogen avirulence genes enable pathogen recognition by plants and activate plant defense. These gene-for-gene interactions in some cases have been shown to involve direct interactions of the products of the genes, and have indicated plant intracellular localization for certain avirulence proteins. Incomplete specificity of some of the interactions in laboratory assays suggests that additional proteins might be required to confer specificity in the plant. In many cases, resistance and avirulence protein interactions have not been demonstrable, and in some cases, other plant components that interact with avirulence proteins have been found. Investigation to date has relied heavily on biochemical and cytological methods including in vitrobinding assays and immunoprecipitation, as well as genetic tools such as the yeast two-hybrid system. Observations so far, however, point to the likely requirement for multiple, interdependent protein associations in pathogen recognition, for which these techniques can be insufficient. This article reviews the protein-protein interactions that have been described in pathogen recognition by plants, and provides examples of how rapid future progress will hinge on the adoption of new and developing technologies.     

Protein-protein interactions: better by the dozen

A combined reanalysis of the two largest yeast protein-protein interaction studies to date provides a large consolidated data set, with a level of accuracy matching the reliability of small-scale experiments.     

Protein-protein interactions in the archaeal core replisome

Most of the core components of the archaeal chromosomal DNA replication apparatus share significant protein sequence similarity with eukaryotic replication factors, making the Archaea an excellent model system for understanding the biology of chromosome replication in eukaryotes. The present review summarizes current knowledge of how the core components of the archaeal chromosome replication apparatus interact with one another to perform their essential functions.     

Protein-protein interactions among Helicobacter pylori cag proteins

Many Helicobacter pylori isolates contain a 40-kb region of chromosomal DNA known as the cag pathogenicity island (PAI). The risk for development of gastric cancer or peptic ulcer disease is higher among humans infected with cag PAI-positive H. pylori strains than among those infected with cag PAI-negative strains. The cag PAI encodes a type IV secretion system that translocates CagA into gastric epithelial cells. To identify Cag proteins that are expressed by H. pylori during growth in vitro, we compared the proteomes of a wild-type H. pylori strain and an isogenic cag PAI deletion mutant using two-dimensional difference gel electrophoresis (2D-DIGE) in multiple pH ranges. Seven Cag proteins were identified by this approach. We then used a yeast two-hybrid system to detect potential protein-protein interactions among 14 Cag proteins. One heterotypic interaction (CagY/7 with CagX/8) and two homotypic interactions (involving H. pylori VirB11/ATPase and Cag5) were similar to interactions previously reported to occur among homologous components of the Agrobacterium tumefaciens type IV secretion system. Other interactions involved Cag proteins that do not have known homologues in other bacterial species. Biochemical analysis confirmed selected interactions involving five of the proteins that were identified by 2D-DIGE. Protein-protein interactions among Cag proteins are likely to have an important role in the assembly of the H. pylori type IV secretion apparatus.     

Protein-protein interaction networks: from interactions to networks

The goal of interaction proteomics that studies the protein-protein interactions of all expressed proteins is to understand biological processes that are strictly regulated by these interactions. The availability of entire genome sequences of many organisms and high-throughput analysis tools has led scientists to study the entire proteome (Pandey and Mann, 2000). There are various high-throughput methods for detecting protein interactions such as yeast two-hybrid approach and mass spectrometry to produce vast amounts of data that can be utilized to decipher protein functions in complicated biological networks. In this review, we discuss recent developments in analytical methods for large-scale protein interactions and the future direction of interaction proteomics.