Global landscape of protein complexes in the yeast Saccharomyces cerevisiae

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.     

Probing cellular protein complexes using single-molecule pull-down

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.     

The molten globule protein conformation probed by disulphide bonds

The molten globule is a compact protein conformation that has a secondary structure content like that of the native protein, but poorly defined tertiary structure. It is a stable state for a few proteins under particular conditions and could be a ubiquitous kinetic intermediate in protein folding. The extent to which native interactions, above the level of the secondary structure, are preserved in this conformation is not so far known. Here we report that alpha-lactalbumin can adopt a molten globule conformation when one of its four disulphide bonds is reduced. In this state, the three other disulphide bonds rearrange spontaneously, at the same rate as when the protein is fully unfolded, to a number of different disulphide bond isomers that tend to maintain the molten globule conformation. That the molten globule state is compatible with a variety of disulphide bond pairings suggests that it is unlikely to be stabilized by many specific tertiary interactions.     

癌症相关促凋亡蛋白Par-4的信号转导途径预测研究

利用支持向量机(SVM)技术构建Par-4关联的蛋白质相互作用网络,预测出与Par-4有相互作用的蛋白质82个;这些蛋白质按照功能划分为8大类,主要包括:蛋白激酶、泛素化蛋白酶、死亡受体相关因子、与细胞周期或DNA复制相关蛋白质、调节蛋白质、与疾病相关蛋白质、具有特定结构域结合蛋白质和其他蛋白质等。结合文献挖掘和数据库检索信息,推断出了Par-4的2条可能新的信号转导途径。首次预测到Par-4与一大类泛素化蛋白有密切的关系。研究发现,Par-4与多种蛋白质具有复杂的相互作用,并且,在多个细胞凋亡途径中扮演了重要角色。     

In vitro biological effects of magnetic nanoparticles

Magnetic nanoparticles (MNPs) have great potential for a wide use in various biomedical applications due to their unusual properties. It is critical for many applications that the biological effects of nanoparticles are studied in depth. To date, many disparate results can be found in the literature regarding nanoparticle-biological factors interactions. This review highlights recent developments in this field with particular focuses on in vitro MNPs-cell interactions. The effect of MNPs properties on cellular uptake and cytotoxicity evaluation of MNPs were discussed. Some employed methods are also included. Moreover, nanoparticle-cell interactions are mediated by the presence of proteins absorbed from biological fluids on the nanoparticle. Many questions remain on the effect of nanoparticle surface (in addition to nanoparticle size) on protein adsorption. We review papers related to this point too.     

Molecular cloning and expression of brain-derived neurotrophic factor

During the development of the vertebrate nervous system, many neurons depend for survival on interactions with their target cells. Specific proteins are thought to be released by the target cells and to play an essential role in these interactions. So far, only one such protein, nerve growth factor, has been fully characterized. This has been possible because of the extraordinarily (and unexplained) large quantities of this protein in some adult tissues that are of no relevance to the developing nervous system. Whereas the dependency of many neurons on their target cells for normal development, and the restricted neuronal specificity of nerve growth factor have long suggested the existence of other such proteins, their low abundance has rendered their characterization difficult. Here we report the full primary structure of brain-derived neurotrophic factor. This very rare protein is known to promote the survival of neuronal populations that are all located either in the central nervous system or directly connected with it. The messenger RNA for brain-derived neurotrophic factor was found predominantly in the central nervous system, and the sequence of the protein indicates that it is structurally related to nerve growth factor. These results establish that these two neurotrophic factors are related both functionally and structurally.     

Millisecond-timescale motions contribute to the function of the bacterial response regulator protein Spo0F.

Protein backbones and side chains display varying degrees of flexibility, which allows many slightly different but related conformational substates to occur. Such fluctuations are known to differ in both timescale and magnitude, from rotation of methyl groups (nanoseconds) to the flipping of buried tyrosine rings (seconds). Because many mechanisms for protein function require conformational change, it has been proposed that some of these ground-state fluctuations are related to protein function. But exactly which aspects of motion are functionally relevant remains to be determined. Only a few examples so far exist where function can be correlated to structural fluctuations with known magnitude and timescale. As part of an investigation of the mechanism of action of the Bacillus subtilis response regulator SpoOF, we have explored the relationship between the motional characteristics and protein-protein interactions. Here we use a set of nuclear magnetic resonance 15N relaxation measurements to determine the relative timescales of SpoOF backbone fluctuations on the picosecond-to-millisecond timescale. We show that regions having motion on the millisecond timescale correlate with residues and surfaces that are known to be critical for protein-protein interactions.     

平衡透析法用于有机小分子和蛋白质相互作用研究进展

生物大分子是近年来生命科学研究的热点和难点之一,有关蛋白质的各类研究也是人们比较感兴趣的课题.平衡透析法是定量研究蛋白质与有机小分子相互作用的经典方法.通过平衡透析法的研究我们可以讨论蛋白质与有机小分子的结合数目、结合平衡常数及作用力情况等.近年来,国内外学者在此方面做了大量的工作,提出了各种各样的结合模型.本文就此方面的研究进行综述.     

Interaction network containing conserved and essential protein complexes in Escherichia coli

Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism, no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames (approximately 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged successfully, whereas 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry. An interaction network of protein complexes involved in diverse biological processes was uncovered and validated by sequential rounds of tagging and purification. This network includes many new interactions as well as interactions predicted based solely on genomic inference or limited phenotypic data. This study provides insight into the function of previously uncharacterized bacterial proteins and the overall topology of a microbial interaction network, the core components of which are broadly conserved across Prokaryota.     

The protein-protein interaction map of Helicobacter pylori

With the availability of complete DNA sequences for many prokaryotic and eukaryotic genomes, and soon for the human genome itself, it is important to develop reliable proteome-wide approaches for a better understanding of protein function. As elementary constituents of cellular protein complexes and pathways, protein-protein interactions are key determinants of protein function. Here we have built a large-scale protein-protein interaction map of the human gastric pathogen Helicobacter pylori. We have used a high-throughput strategy of the yeast two-hybrid assay to screen 261 H. pylori proteins against a highly complex library of genome-encoded polypeptides. Over 1,200 interactions were identified between H. pylori proteins, connecting 46.6% of the proteome. The determination of a reliability score for every single protein-protein interaction and the identification of the actual interacting domains permitted the assignment of unannotated proteins to biological pathways.     

Superhydrophobicity: drying transition of confined water

Long-range hydrophobic interactions operating underwater are important in the mediation of many natural and synthetic phenomena, such as protein folding, adhesion and colloid stability. Here we show that rough hydrophobic surfaces can experience attractive forces over distances more than 30 times greater than any reported previously, owing to the spontaneous evaporation of the intervening, confined water. Our finding highlights the importance of surface roughness in the interaction of extended structures in water, which has so far been largely overlooked.     

Detection of molecular interactions at membrane surfaces through colloid phase transitions

The molecular architecture of-and biochemical processes within--cell membranes play important roles in all living organisms, with many drugs and infectious disease agents targeting membranes. Experimental studies of biochemical reactions on membrane surfaces are challenging, as they require a membrane environment that is fluid (like cell membranes) but nevertheless allows for the efficient detection and characterization of molecular interactions. One approach uses lipid membranes supported on solid substrates such as silica or polymers: although the membrane is trapped near the solid interface, it retains natural fluidity and biological functionality and can be implanted with membrane proteins for functional studies. But the detection of molecular interactions involving membrane-bound species generally requires elaborate techniques, such as surface plasmon resonance or total internal reflection fluorescence microscopy. Here we demonstrate that colloidal phase transitions of membrane-coated silica beads provide a simple and label-free method for monitoring molecular interactions on lipid membrane surfaces. By adjusting the lipid membrane composition and hence the pair interaction potential between the membrane-supporting silica beads, we poise our system near a phase transition so that small perturbations on the membrane surface induce dramatic changes in the macroscopic organization of the colloid. We expect that this approach, used here to probe with high sensitivity protein binding events at membrane surfaces, can be applied to study a broad range of cell membrane processes.     

A quantitative protein interaction network for the ErbB receptors using protein microarrays

Although epidermal growth factor receptor (EGFR; also called ErbB1) and its relatives initiate one of the most well-studied signalling networks, there is not yet a genome-wide view of even the earliest step in this pathway: recruitment of proteins to the activated receptors. Here we use protein microarrays comprising virtually every Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain encoded in the human genome to measure the equilibrium dissociation constant of each domain for 61 peptides representing physiological sites of tyrosine phosphorylation on the four ErbB receptors. This involved 77,592 independent biochemical measurements and provided a quantitative protein interaction network that reveals many new interactions, including ones that fall outside of our current view of domain selectivity. By slicing through the network at different affinity thresholds, we found surprising differences between the receptors. Most notably, EGFR and ErbB2 become markedly more promiscuous as the threshold is lowered, whereas ErbB3 does not. Because EGFR and ErbB2 are overexpressed in many human cancers, our results suggest that the extent to which promiscuity changes with protein concentration may contribute to the oncogenic potential of receptor tyrosine kinases, and perhaps other signalling proteins as well.     

Alternative packing arrangements in the hydrophobic core of lambda repressor

The random alteration of hydrophobic core positions in the N-terminal domain of lambda-repressor, both individually and in combination, shows that there are many ways of repacking the core of the protein. Although the number of functional sequences is limited by constraints on composition, volume and steric interactions, the simple requirement that these positions remain hydrophobic is the main determinant of whether a core sequence is compatible with the wild-type fold.     

The Caenorhabditis elegans lin-12 gene encodes a transmembrane protein with overall similarity to Drosophila Notch

The lin-12 gene seems to control certain binary decisions during Caenorhabditis elegans development, from genetic and anatomical studies of lin-12 mutants that have either elevated or reduced levels of lin-12 activity. We report here the complete DNA sequence of lin-12: 13.5 kilobases (kb) derived from genomic clones and 4.5 kb from complementary DNA clones. It is of interest that the predicted product is a putative transmembrane protein, given that many of the decisions controlled by lin-12 activity require cell-cell interactions for the correct choice of cell fate. In addition, the predicted lin-12 product may be classified into several regions, based on amino acid sequence similarities to other proteins. These include extensive overall sequence similarity to the Drosophila Notch protein, which also is involved in cell-cell interactions that specify cell fate; a repeated motif found in proteins encoded by the yeast cell-cycle control genes cdc10 (Schizosaccharomyces pombe) and SWI6 (Saccharomyces cerevisiae); and a repeated motif exemplified by epidermal growth factor, found in many mammalian proteins.     

Framework to Identify Protein Complexes Based on Similarity Preclustering

Proteins interact with each other to form protein complexes, and cell functionality depends on both protein interactions and these complexes. Based on the assumption that protein complexes are highly connected and correspond to the dense regions in Protein-protein Interaction Networks(PINs), many methods have been proposed to identify the dense regions in PINs. Because protein complexes may be formed by proteins with similar properties,such as topological and functional properties, in this paper, we propose a protein complex identification framework(KCluster). In KCluster, a PIN is divided into K subnetworks using a K-means algorithm, and each subnetwork comprises proteins of similar degrees. We adopt a strategy based on the expected number of common neighbors to detect the protein complexes in each subnetwork. Moreover, we identify the protein complexes spanning two subnetworks by combining closely linked protein complexes from different subnetworks. Finally, we refine the predicted protein complexes using protein subcellular localization information. We apply KCluster and nine existing methods to identify protein complexes from a highly reliable yeast PIN. The results show that KCluster achieves higher Sn and Sp values and f-measures than other nine methods. Furthermore, the number of perfect matches predicted by KCluster is significantly higher than that of other nine methods.     

酵母双杂交系统的研究进展

酵母双杂交系统自创建以来,已成为研究蛋白质相互作用的重要手段,揭示了大量未知蛋白质之间的相互作用。随着该系统的广泛应用,近年来又发展了三杂交系统、单杂交系统、逆向双杂交系统、SOS富集系统等。酵母双杂交及其衍生系统已经成功地运用于蛋白质之间,蛋白质与DNA、RNA、配体之间相互作用的研究。     

Aggregation and vesiculation of membrane proteins by curvature-mediated interactions

Membrane remodelling plays an important role in cellular tasks such as endocytosis, vesiculation and protein sorting, and in the biogenesis of organelles such as the endoplasmic reticulum or the Golgi apparatus. It is well established that the remodelling process is aided by specialized proteins that can sense as well as create membrane curvature, and trigger tubulation when added to synthetic liposomes. Because the energy needed for such large-scale changes in membrane geometry significantly exceeds the binding energy between individual proteins and between protein and membrane, cooperative action is essential. It has recently been suggested that curvature-mediated attractive interactions could aid cooperation and complement the effects of specific binding events on membrane remodelling. But it is difficult to experimentally isolate curvature-mediated interactions from direct attractions between proteins. Moreover, approximate theories predict repulsion between isotropically curving proteins. Here we use coarse-grained membrane simulations to show that curvature-inducing model proteins adsorbed on lipid bilayer membranes can experience attractive interactions that arise purely as a result of membrane curvature. We find that once a minimal local bending is realized, the effect robustly drives protein cluster formation and subsequent transformation into vesicles with radii that correlate with the local curvature imprint. Owing to its universal nature, curvature-mediated attraction can operate even between proteins lacking any specific interactions, such as newly synthesized and still immature membrane proteins in the endoplasmic reticulum.     

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.