AF was supported by a starting grant from the European Research Council under the European Community&#39;s Seventh Framework Programme (FP7/2007-2013) / ERC Grant agreement n&#176; 203413 and by the Minerva Center for Bio-hybrid complex systems.HA and AI are supported by the Dalia and Dan Maydan Fellowship for advanced degree students at the Hebrew University of Jerusalem.

1. Designing a Peptide Array
<ol>
	<li>Divide the sequence of the target protein into partly overlapping 10-20 residues peptides. Vary the amount of overlap on the specific experiment and the resources of the performing lab, but in principle the longer the overlap the better. When designing the peptides take into account known secondary structure elements in the protein that can be responsible for the interaction.</li>
	<li>Order the designed peptide array through commercial vendors. Here, use peptides covalently bound...

<ol>
	<li>Benyamini, H., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+peptides+to+study+protein&#8211;protein+interactions.">Using peptides to study protein&#8211;protein interactions.</a> Future Medicinal Chemistry. 2 (6), 989-1003 (2010).</li><li>Geysen, H. M., Wagner, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotope+or+mass+encoding+of+combinatorial+libraries.">Isotope or mass encoding of combinatorial libraries.</a> Chemistry &amp; Biology. 3 (8), 679-688 (1996).</li><li>Frank, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spot-synthesis:+an+easy+technique+for+the+positionally+addressable,+parallel+chemical+synthesis+on+a+membrane+support.">Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support.</a> Tetrahedron. 48 (42), 9217-9232 (1992).</li><li>Katz, C., Levy-Beladev, L., Rotem-Bamberger, S., Rito, T., Rudiger, S. G., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein-protein+interactions+using+peptide+arrays.">Studying protein-protein interactions using peptide arrays.</a> Chem Soc Rev. 40 (5), 2131-2145 (2011).</li><li>Hansen, L. B., Buus, S., Schafer-Nielsen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Mapping+of+Linear+Antibody+Epitopes+in+Human+Serum+Albumin+Using+High-Density+Peptide+Arrays.">Identification and Mapping of Linear Antibody Epitopes in Human Serum Albumin Using High-Density Peptide Arrays.</a> PLoS ONE. 8 (7), (2013).</li><li>Uecker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+substrate+peptide+for+the+FLT3+receptor+tyrosine+kinase.">A substrate peptide for the FLT3 receptor tyrosine kinase.</a> British Journal of Haematology. 144 (1), 127-130 (2009).</li><li>Gabizon, R., Faust, O., Benyamini, H., Nir, S., Loyter, A., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+relationship+studies+using+peptide+arrays:+the+example+of+HIV-1+Rev-integrase+interaction.">Structure-activity relationship studies using peptide arrays: the example of HIV-1 Rev-integrase interaction.</a> Med. Chem. Commun. 4 (1), 252-259 (2013).</li><li>Frank, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SPOT-synthesis+technique:+Synthetic+peptide+arrays+on+membrane+supports&#8212;principles+and+applications.">The SPOT-synthesis technique: Synthetic peptide arrays on membrane supports&#8212;principles and applications.</a> Journal of Immunological Methods. 267 (1), 13-26 (2002).</li><li>Falsey, J. R., Renil, M., Park, S., Li, S., Lam, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+and+Small+Molecule+Microarray+for+High+Throughput+Cell+Adhesion+and+Functional+Assays.">Peptide and Small Molecule Microarray for High Throughput Cell Adhesion and Functional Assays.</a> Bioconjugate Chemistry. 12 (3), 346-353 (2001).</li><li>Castiel, A., Danieli, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stil+protein+regulates+centrosome+integrity+and+mitosis+through+suppression+of+Chfr.">The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr.</a> Journal of Cell Science. 124 (4), 532-539 (2011).</li><li>Amartely, H., David, A., Lebendiker, M., Benyamini, H., Izraeli, S., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STIL+protein+contains+intrinsically+disordered+regions+that+mediate+its+protein-protein+interactions.">The STIL protein contains intrinsically disordered regions that mediate its protein-protein interactions.</a> Chemical Communications. 50, 5245-5247 (2013).</li><li>Izraeli, S., Lowe, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SIL+gene+is+required+for+mouse+embryonic+axial+development+and+left-right+specification.">The SIL gene is required for mouse embryonic axial development and left-right specification.</a> Nature. 399 (6737), 691-694 (1999).</li><li>Izraeli, S., Colaizzo-Anas, T., Bertness, V. L., Mani, K., Aplan, P. D., Kirsch, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+SIL+gene+is+correlated+with+growth+induction+and+cellular+proliferation.">Expression of the SIL gene is correlated with growth induction and cellular proliferation.</a> Cell Growth Differ. 8 (11), 1171-1179 (1997).</li><li>Arquint, C., Sonnen, K. F., Stierhof, Y. -. D., Nigg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Cell-cycle-regulated+expression+of+STIL+controls+centriole+number+in+human+cells.">a Cell-cycle-regulated expression of STIL controls centriole number in human cells.</a> Journal of Cell Science. 125 (5), 1342-1352 (2012).</li><li>Tang, C. J., Lin, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+microcephaly+protein+STIL+interacts+with+CPAP+and+is+required+for+procentriole+formation.">The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.</a> Embo J. 30 (23), 4790-4804 (2011).</li><li>Vulprecht, J., David, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIL+is+required+for+centriole+duplication+in+human+cells.">STIL is required for centriole duplication in human cells.</a> Journal of Cell Science. 125 (5), 1353-1362 (2012).</li><li>Campaner, S., Kaldis, P., Izraeli, S., Kirsch, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sil+phosphorylation+in+a+Pin1+binding+domain+affects+the+duration+of+the+spindle+checkpoint.">Sil phosphorylation in a Pin1 binding domain affects the duration of the spindle checkpoint.</a> Mol Cell Biol. 25 (15), 6660-6672 (2005).</li><li>Kasai, K., Inaguma, S., Yoneyama, A., Yoshikawa, K., Ikeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCL/TAL1+interrupting+locus+derepresses+GLI1+from+the+negative+control+of+suppressor-of-fused+in+pancreatic+cancer+cell.">SCL/TAL1 interrupting locus derepresses GLI1 from the negative control of suppressor-of-fused in pancreatic cancer cell.</a> Cancer Res. 68 (19), 7723-7729 (2008).</li><li>Chaturvedi, P., Sudakin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chfr+regulates+a+mitotic+stress+pathway+through+its+RING-finger+domain+with+ubiquitin+ligase+activity.">Chfr regulates a mitotic stress pathway through its RING-finger domain with ubiquitin ligase activity.</a> Cancer Res. 62 (6), 1797-1801 (2002).</li><li>Olaussen, K. a., Commo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+proapoptotic+effects+of+the+two+tyrosine+kinase+inhibitors+pazopanib+and+lapatinib+on+multiple+carcinoma+cell+lines.">Synergistic proapoptotic effects of the two tyrosine kinase inhibitors pazopanib and lapatinib on multiple carcinoma cell lines.</a> Oncogene. 28 (48), 4249-4260 (2009).</li><li>Reingewertz, T. H., Britan-Rosich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Vif&#8211;A3G+interaction+using+peptide+arrays:+A+basis+for+anti-HIV+lead+peptides.">Mapping the Vif&#8211;A3G interaction using peptide arrays: A basis for anti-HIV lead peptides.</a> Bioorganic &amp; Medicinal Chemistry. 21 (12), 3523-3532 (2013).</li><li>Katz, C., Benyamini, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+interaction+between+the+antiapoptotic+Bcl-2+family+proteins+and+the+proapoptotic+protein+ASPP2.">Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2.</a> Proc Natl Acad Sci U S A. 105 (34), 12277-12282 (2008).</li><li>Katz, C., Zaltsman-Amir, Y., Mostizky, Y., Kollet, N., Gross, A., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Basis+of+the+Interaction+between+Proapoptotic+Truncated+BID+(tBID)+Protein+and+Mitochondrial+Carrier+Homologue+2+(MTCH2)+Protein:+KEY+PLAYERS+IN+MITOCHONDRIAL+DEATH+PATHWAY.">Molecular Basis of the Interaction between Proapoptotic Truncated BID (tBID) Protein and Mitochondrial Carrier Homologue 2 (MTCH2) Protein: KEY PLAYERS IN MITOCHONDRIAL DEATH PATHWAY.</a> Journal of Biological Chemistry. 287 (18), 15016-15023 (2012).</li><li>Rosenberg, M. M., Ronen, D., Lahav, N., Nazirov, E., Ravid, S., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+characterization+of+myosin+IIC+tailpiece+and+its+effect+on+filament+assembly.">High resolution characterization of myosin IIC tailpiece and its effect on filament assembly.</a> Journal of Biological Chemistry. 288 (14), 9779-9789 (2013).</li></ol>

Peptide array screening is an excellent tool for identifying the binding sites of a target protein in its partners. This assay is fast and easy to perform and the results can be obtained in one or two working days. Peptide array screening is versatile and can be used for many purposes. It can be used for characterizing post translation modifications such as phosphorylation and identifying substrates of kinases. For example: the FLT3 kinase, which is related to acute myeloid leukemia, phosphorylates Tyr-containing substra...

Protein-protein interactions mediate most of the processes in the living cell. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions. This is because peptides can be easily synthesized and allow the focusing on a specific interaction site on one hand and on multiple protein targets in a high throughput manner on the other hand1,2. Peptide array screening is a fast, easy to perform method for obtaining a large amount of data about the interactions of a target protein with numerous partners in a short time3. Unlike other biochemical or biophysical methods for detecting and analyzing protein-protein interactions, peptide array screening requires a very low concentration of protein and can detect very weak binding. Peptide arrays can be used for many applications in peptide-protein interactions such as mapping of protein-protein or receptor-ligand interaction sites4, homo- or hetero-oligomerization interfaces, characterizing antibodies epitopes5, studying enzyme activities6 and high throughput structure-activity relationship (SAR) studies7. For an in-depth review about peptide array screening see Katz et al. 4
Several types of peptide arrays currently exist. There are two major synthetic strategies for making peptide arrays: synthesis of the peptides before attaching them to the solid support, or synthesis of peptides directly on the solid support, mainly using the SPOT technique4,8. The peptides are synthesized on the solid support usually by 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry8. Among the common synthetic schemes are peptide attachment through the N terminus (e.g., JPT pepstar arrays9) and peptide attachment through the C terminus (e.g., PEPSCAN pepchip arrays10, JPT pepspot arrays9 and INTAVIS celluspot arrays11)4. The solid support can vary and so does the chemistry of the peptide coupling to it. Cys-terminated peptides can be attached to glass slides via the thiol group12. The N terminus of a peptide can be covalently bound to a hydroxyl group on the cellulose membrane through esterification of the amino acid attached8.
Here we present a detailed protocol for screening peptide arrays as a method for studying protein-protein interactions. The array we used is the Celluspots array, which is a micro-array containing large amount of spots (duplicates of up to 384 spots) on a small cellulose membrane supported by a glass slide. This enables working with low volumes of protein and antibodies and obtaining significant amount of data per single experiment. This array also contains high peptide density that allows detection of low affinity binding. The array was used for mapping the STIL-CHFR interaction, which is highly important for controlling normal cell proliferation13. Uncontrolled interaction between the two proteins can lead to the development of cancer. By mapping this interaction we found the specific binding site and binding residues14. This paves the way for designing rational inhibitors that inhibit this protein-protein interaction.

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			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
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			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptide array</td>
			<td>INTAVIS Bioanalytical Instruments</td>
			<td></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">His-probe Antibody (H-3) HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8036 HRP</td>
			<td></td>
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			<td height="21" style="height:21px;">EZ-ECL Kit</td>
			<td>Biological industries</td>
			<td>20-500-120</td>
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			<td height="21" style="height:21px;">Skim Milk</td>
			<td>Becton, Dickinson and Company</td>
			<td style="width:119px;">232100</td>
			<td></td>
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			<td height="21" style="height:21px;">LAS-3000 camera&#160;</td>
			<td>FUJI film</td>
			<td></td>
			<td></td>
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identifying protein-protein interaction sites using peptide arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

STIL is a highly important centrosomal protein. It controls normal cell division and cell proliferation13,15&#8211;19. STIL interacts with several proteins 18,20,21, and most of the interactions occur through its central part, which is an intrinsically disordered region (IDR)14. We designed an array composed of peptides derived from the STIL binding protein CHFR. CHFR is a tumor suppressor that is induced in response to mitotic stress22. Since the structur...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction Sites Using Peptide Arrays at JoVE.com

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

identifying-protein-protein-interaction-sites-using-peptide-arrays

Research

JoVE Journal

Biology

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...