Department of Biomedical Science, Faculty of Science and Technology, University of Westminster
Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta
Collection OverviewThe study of protein-protein interactions is key to understanding the network of molecular interactions underlying biological effects, and defining how individual proteins perform their functional role within a cell. Detailed understanding of these interactions often provides insight into aberrant signaling in disease. This collection brings together the wide range of techniques that have been developed to study interactions using in vitro, cell-based and in vivo approaches. Ea… Show Morech technique has its own strengths, for example enabling systematic interrogation of the complete ‘interactome’, providing detailed atomic level information about a single interaction or insight into the sub-cellular localization of interacting proteins. Some techniques are easily adaptable to high-throughput screening of libraries or pulling out novel interacting partners while others have a more limited capacity. Readouts such as microscopy and mass-spectrometry are employed in protein-protein interaction techniques and some provide quantitative information on the affinity of an interaction.
Examples of methods to be included in this collection are: co-immunoprecipitation and antibody interference; pull-down assays using fusion proteins or modular protein domains; proximity-dependent labelling techniques (e.g. BioID); far western and receptor affinity probes; affinity purification coupled with mass-spectrometry; two-hybrid technologies (yeast and mammalian); fluorescence microscopy (e.g. immunofluoresence, FRET, super-resolution microscopy); protein complementation assays (e.g. BiFC); biophysical approaches (X-ray crystallography, NMR, AUC, SPR) and microarray based techniques (i.e. tissue and protein arrays). Show Less
Calibration-Free in-vitro Quantification of Homo-Oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software
We present a calibration free approach to detect protein homo-dimerization and oligomerization that employs fluorescence fluctuation spectroscopy (FFS) using a conventional confocal microscope equipped with digital detectors. Using a purified mVenus-labelled FKBP12 before and after the application of a dimerization drug (AP20187, referred commercially as BB, Clonetech); we show that number and brightness accurately provide information on the oligomeric state of FKBP12 when using very low concent… Show Morerations of protein (100nM). We discuss the importance of using the correct microscope acquisition parameters. We also describe how to correct artifacts of bleaching. This inexpensive method can be employed to study protein-protein interactions in many biological contexts. Show Less
Structural Studies of Macromolecular Interactions in Solution
Proteins containing multiple structural domains that interact with their binding partners (e.g. proteins and nucleic acids) present technical challenges for determining how such macromolecular assemblies are formed. An emerging tool that can be used to elucidate which specific domains mediate interactions in multicomponent systems is described. A method for calculating hybrid solution structures of such assemblies is provided, and integrates data from small angle X-ray scattering (SAXS… Show More), chromatography and atomic resolution structures. The example given is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins, and forms an extended, curved nanostructure. One of its globular domains attaches to laminin in order to structure the basement membrane. This provides a basis for the technique to determine accurate structures of (flexible) multidomain macromolecular complexes, and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows large numbers of samples to be analyzed in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high resolution structures of the component proteins is also given. Show Less
Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions.
Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated α subunit (HIF-1α) and constitutively expressed β subunit (HIF-1β) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis.
The identification… Show More of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1α stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1α and ARNT. Notably, most of the current methods used to study protein-protein interactions (PPIs) are based on systems where protein overexpression is used. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1α and ARNT. This protocol can also be applied to demonstration of any interactions between transcription factors and transcriptional coregulators under hypoxic conditions. Show Less
Measuring interactions of globular and filamentous proteins by nuclear magnetic resonance spectroscopy and microscale thermophoresis
Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. This protocol describes a method for detecting and measuring such interactions using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The glob… Show Moreular protein of interest is labelled with nitrogen-15 and titrated with vimentin protein in solution. A two dimensional NMR spectrum is acquired to detect interactions by observing changes in chemical shifts and peak shape, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds vimentin, the binding interaction is quantified by microscale thermophoresis using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction. Show Less
Detection of Heterodimerization of Protein Isoforms using an in situ Proximity Ligation Assay.
Dysregulation or loss of MST1/Hippo signaling is linked to developmental disorders and carcinogenesis with poor prognosis1. In mammals, the kinases MST1 and MST2 activate (phosphorylate) MOB1 and LATS1/2, which then phosphorylates and inactivates the transcriptional co-activator Yes-associated protein (YAP)2. In its active (unphosphorylated) form, YAP acts as an oncogene and enhances transcription of genes involved in cell proliferation, and inactivation of YAP by the Hippo pathway suppresses ce… Show Morell proliferation and promotes apoptosis3. In tissues, MST1 and MST2 exist mainly as active homodimers, but oncogenic stimuli can increase levels of MST1/MST2 heterodimers, and such heterodimers are inactive4. However, how MST1/MST2 heterodimerization is regulated remains poorly understood. Both homo- and heterodimers are mediated via interactions between C-terminal coiled-coil regions of MST1 and MST2 known as SARAH domains5. Using in situ PLA demonstrated in this video, we show the presence of MST1/MST2 heterodimers in Human Schwann Cells (HSC) and Human Embryonic Kidney cells (HEK 293). PLA has an advantage over other protein/protein interaction detection methods because it allows the detection of endogenous protein-protein interactions, which can be identified and quantified without the need of transgene expression or the use of epitope tags 6. Show Less
2 in 1: One-step affinity purification for the parallel analysis of protein–protein and protein–metabolite complexes
Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein–protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein–metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our pr… Show Moreotocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass-spectrometry-(MS)-based protein and metabolite detection. 35 In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography–mass spectrometry (LC–MS or LC–MS/MS). Empty-vector control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in single experiment in near-physiological conditions (cellular lysate). The presented method is
straightforward and fast, and can be easily adapted to other systems than plant cell cultures. Show Less