Lalonde, S. et al. Molecular and cellular approaches for the detection of protein-protein interactions: latest techniques and current limitations. Plant J. 53, 610-635

Carnegie Institution, 260 Panama Street, Stanford, CA 94305, USA.
The Plant Journal (Impact Factor: 5.97). 03/2008; 53(4):610-35. DOI: 10.1111/j.1365-313X.2007.03332.x
Source: PubMed


Homotypic and heterotypic protein interactions are crucial for all levels of cellular function, including architecture, regulation, metabolism, and signaling. Therefore, protein interaction maps represent essential components of post-genomic toolkits needed for understanding biological processes at a systems level. Over the past decade, a wide variety of methods have been developed to detect, analyze, and quantify protein interactions, including surface plasmon resonance spectroscopy, NMR, yeast two-hybrid screens, peptide tagging combined with mass spectrometry and fluorescence-based technologies. Fluorescence techniques range from co-localization of tags, which may be limited by the optical resolution of the microscope, to fluorescence resonance energy transfer-based methods that have molecular resolution and can also report on the dynamics and localization of the interactions within a cell. Proteins interact via highly evolved complementary surfaces with affinities that can vary over many orders of magnitude. Some of the techniques described in this review, such as surface plasmon resonance, provide detailed information on physical properties of these interactions, while others, such as two-hybrid techniques and mass spectrometry, are amenable to high-throughput analysis using robotics. In addition to providing an overview of these methods, this review emphasizes techniques that can be applied to determine interactions involving membrane proteins, including the split ubiquitin system and fluorescence-based technologies for characterizing hits obtained with high-throughput approaches. Mass spectrometry-based methods are covered by a review by Miernyk and Thelen (2008; this issue, pp. 597-609). In addition, we discuss the use of interaction data to construct interaction networks and as the basis for the exciting possibility of using to predict interaction surfaces.

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Available from: Dominique Loqué
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    • "Membrane protein complexes are notoriously difficult to study using traditional highthroughput techniques (Lalonde et al., 2008). Intact membrane complexes are difficult to " pull down " using conventional affinity purification/mass spectrometry-based systems. "
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    DESCRIPTION: Challenges and open problems in computational prediction of protein complexes: the case of membrane complexes
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    • "Microscopy-based bimolecular fluorescence complementation (BiFC) has the tremendous advantage of allowing simultaneous assessment of protein–protein interactions and determination of their subcellular localization. The most commonly used BiFC systems, however, are prone to false positives due to both reassembly of the fluoroprotein fragments even in the absence of interaction of the test proteins and the inherent tendency of the fluoroprotein to dimerize (Lalonde et al., 2008; Kodama and Hu, 2012). Aequorea derived GFP derivatives such as Venus are approximately 239 residues long and are characterized by 11 b-sheets which fold into a b-barrel structure with a central chromophore . "
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    ABSTRACT: Protein networks and signaling cascades are key mechanisms for intra- and intercellular signal transduction. Identifying the interacting partners of a protein can provide vital clues regarding its physiological role. The Bimolecular Fluorescence Complementation (BiFC) assay has become a routine tool for in vivo analysis of protein-protein interactions and their subcellular location. Although the BiFC system has improved since its inception, the available options for in planta analysis are still subject to very low signal to noise ratios, and a systematic comparison of BiFC confounding background signals has been lacking. Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives. To overcome these problems, we performed an extensive in planta analysis of published BiFC fragments used in metazoa and plants, and then developed an optimized single vector BiFC system which utilizes monomeric Venus (mVenus) split at residue 210, and contains an integrated mTurquoise2 marker to precisely identify transformed cells in order to distinguish true negatives. Here we provide our streamlined Double ORF Expression (pDOE) BiFC system, and show that our advance in BiFC methodology functions even with an internally fused mVenus210 fragment. We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin-like (CML) protein, and by showing that heterotrimeric G-protein subunits Gα (GPA1) and Gβ (AGB1) interact in plant cells. We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1 in planta, and that mutation of the so-called PLDα1 "DRY" motif abolishes both of these interactions. This article is protected by copyright. All rights reserved.
    Full-text · Article · Sep 2014 · The Plant Journal
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    • "Investigations of protein-protein interactions (PPIs) are crucial in modern biological science research [26], and there is growing interest in the development of high throughput technologies [18], [27]. In the method developed here, proteins with different affinity of LZs localized to IBs were quantitatively analyzed in living cells using flow cytometry (Fig. 5), while the E, K coil proteins in IB fractions was detected by electrophoretic methods after cell disruption in previous study [14]. "
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    ABSTRACT: Inclusion bodies (IBs) are typically non-functional particles of aggregated proteins. However, some proteins in fusion with amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs) generate IB particles retaining the original functions in cells. Here, we attempted to generate CBD IBs displaying functional leucine zipper proteins (LZs) as bait for localizing cytosolic proteins in E. coli. When a red fluorescent protein was tested as a target protein, microscopic observations showed that the IBs red-fluoresced strongly. When different LZ pairs with KDs of 8-1,000 µM were tested as the bait and prey, the localization of the red fluorescence appeared to change following the affinities between the LZs, as observed by fluorescence imaging and flow cytometry. This result proposed that LZ-tagged CBD IBs can be applied as an in vivo matrix to entrap cytosolic proteins in E. coli while maintaining their original activities. In addition, easy detection of localization to IBs provides a unique platform for the engineering and analyses of protein-protein interactions in E. coli.
    Full-text · Article · Jun 2014 · PLoS ONE
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