Protein-protein interaction

A protein-protein interaction is an interaction between two or more proteins . It is based predominantly on non- covalent interactions, such as van der Waals forces and hydrogen bonds , electrostatic interactions and hydrophobic effects of the amino acid residues near the surface of the protein domains between the proteins involved.

properties

Protein-protein interactions play a key role in practically all biological processes in which proteins are involved. These include in particular signal transduction processes , transport functions and the cytoskeleton . Hence, protein-protein interactions are the subject of research in many areas of life sciences . The totality of protein-protein interactions, which form a network of around 650,000 interactions in the human organism, is generally also referred to as an interactome and is registered in databases such as KEGG and Reactome . Interactions defined in the same way are e.g. B. protein-lipid interactions , protein-RNA interactions, and protein-DNA interactions .

List of methods for elucidating protein-protein interactions

Protein-protein interactions can be investigated experimentally in the course of protein characterization with the help of biochemical and biophysical methods. These include:

• the yeast two-hybrid system and the bacterial two-hybrid system , two molecular biological processes using fusion proteins which, after interaction, form a functional transcription factor in cells
• the molecular display , e.g. B. the phage display or the yeast display , is a screening method in which proteins are expressed on the surface of bacteriophages and whose coding cDNA can be identified after interaction
• the Förster resonance energy transfer and the bioluminescence resonance energy transfer are based on a physical phenomenon in which dye-conjugated proteins show an energy transfer when interacting
• the bimolecular fluorescence complementation , a biochemical and biophysical methods, the two previously separated on a union of fragments of the green fluorescent protein for interaction with the conjugated proteins is based, the enzyme fragment complementation analog
• the fluorescence correlation spectroscopy and fluorescence lifetime correlation spectroscopy
• the scintillation proximity assay , in which scintillation is amplified after binding of a radioactively labeled protein.
• the surface plasmon resonance , a quantitative physical method, which relies on a detection of the change of the layer thickness of the bound proteins by an interaction
• the bio-layer interferometry measures the interference modified with the addition of a protein to a layer of another protein
• the NMR spectroscopy , a method for determining the spatial structure of a protein
• Ligand binding test such as B. the radioligand binding in the filter binding test , a quantitative biochemical method based on a radiation measurement of a mobile radioactively labeled protein ligand after interaction with an immobilized protein
• the alanine scan , several targeted mutageneses to identify the amino acids required for binding
• the cross-linking , a chemical method for fixation of interacting proteins for later analysis, can also be in vivo, using photo-reactive amino acid analogs take place which are installed during expression in the proteins, usually combined with a Western blot or MALDI-TOF .
• the isopeptag enzymatically links neighboring proteins.
• the label transfer transmits a signal from one molecule to another-binding.
• the affinity chromatography , a chromatographic separation method using a protein-conjugated stationary phase for the isolation of interacting proteins in the mobile phase
• the affinity electrophoresis , one of the affinity chromatography method similar electrophoretic
• Pulldown assays such as B. Co-immunoprecipitation or SPINE (Strep-protein interaction experiment), methods based on a precipitation and subsequent analysis of protein complexes
• Quantitative immunoprecipitation combined with knock-down (QUICK)
• Native gel electrophoresis shows that proteins interact with each other and their behavior in gel electrophoresis changes.
• the Far Western blot uses, after incubation of a Western blot with the interaction partner, the latter as the target of an immunostaining
• Proximity Ligation Assay
• Protein-fragment complementation assays like Split-TEV , analogous to bimolecular fluorescence complementation , but with two enzyme halves instead of two fluorophore halves.
• Thermal Shift Assay , measurement of the changed denaturation temperature with bound ligands
• Microscale Thermophoresis
• Isothermal titration calorimetry
• Surface plasmon resonance (engl. Surface plasmon resonance spectroscopy , SPR) spectroscopy is a spectroscopic analyzing method that allows binding affinities to determine proteins. For example, monoclonal antibodies can be characterized
• BioID ( engl. Proximity-dependent biotin identification ) relies on the expression of a fusion protein with a nonspecific biotin protein ligase followed by detection of biotinylated proteins
• By a density gradient centrifugation or a gel permeation chromatography of the modified can hydrodynamic radius of a protein complex can be determined.

By comparing the amino acid sequence with databases such as BLASTp and Pfam , sequence-related proteins with known functions can give an indication of the possible functions of the protein to be examined. In addition, with the help of theoretical, computer-aided methods in the course of a protein structure prediction, the prediction of protein-protein interactions is partially possible.

literature

• Adams, Peter; Golemis, Erica: Protein-protein interactions: a molecular cloning manual . Cold Spring Harbor Laboratory Press, Plainview, NY 2005, ISBN 0-87969-723-7 .
• Friedrich Lottspeich , Joachim W. Engels , Solodkoff Zettlmeier Lay: Bioanalytik . 3rd edition, Spektrum, Heidelberg, 2012, ISBN 978-3827400413 .
• Hubert Rehm , Thomas Letzel: The Experimenter: Protein Biochemistry / Proteomics . 6th edition, Spektrum, Heidelberg 2009, ISBN 978-3827423122 .
• Werther, Meike; Seitz, Harald: Protein-protein interaction. Springer 2008, ISBN 978-3-540-68820-4

Individual evidence

1. ↑ Stump M, Thorne T, et al. : Estimating the size of the human interactome . In: PNAS . 105, No. 19, 2008, p. 6959.
2. ^ S. Schmollinger, D. Strenkert, V. Offeddu, A. Nordhues, F. Sommer, M. Schroda: A protocol for the identification of protein-protein interactions based on 15N metabolic labeling, immunoprecipitation, quantitative mass spectrometry and affinity modulation. In: Journal of visualized experiments: JoVE. Number 67, 2012, S., doi : 10.3791 / 4083 , PMID 23051728 , PMC 3490270 (free full text).
3. ↑ KJ Roux, DI Kim, M. Raida, B. Burke: A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. In: Journal of Cell Biology . Volume 196, number 6, March 2012, pp. 801-810, doi : 10.1083 / jcb.201112098 , PMID 22412018 , PMC 3308701 (free full text).