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
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
1Lee Kong Chian School of Medicine, Nanyang Technological University, 2Singapore Eye Research Institute (SERI), Singapore General Hospital, 3The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, 4Cancer and Stem Cell Biology Program, Duke-NUS Medical School, 5Department of Cell and Molecular Biology, Karolinska Institutet
Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software
1Cellular Imaging Group, Wellcome Centre Human Genetics, University of Oxford, 2Division of Structural Biology, Wellcome Centre Human Genetics, University of Oxford, 3Dynamic Structural Virology Group, Biocruces Health Research Centre, 4IKERBASQUE, Basque Foundation for Science
2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes
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
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