Identifying Protein-protein Interaction Sites Using Peptide Arrays

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 hand^1,2. 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 technique^4,8. 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.

Protokół

1. Designing a Peptide Array

1. 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.
2. Order the designed peptide array through commercial vendors. Here, use peptides covalently bound to the array through the C-terminus.

2. Blocking Non-specific Binding

1. Make a 50 mM Tris or Phosphate buffer solution containing 0.05% Tween 20 (TBST/PBST) and adjust to the desired pH with a measured amount of HCl or NaOH (in order to know the precise ionic strength) and the desired ionic strength with NaCl. Here, use a pH of 7.5 and an ionic strength of 150 mM.
2. Make a blocking solution of 2.5% (w/v) skimmed milk powder in TBST/PBST.
3. To prevent non-specific binding, immerse the array in 5 ml of blocking solution. Incubate the array for 2-4 hr at room temperature or overnight at 4 ºC on a shaker.

3. Incubating with the Protein

1. Wash the array first with 5 ml of blocking solution for 30 sec and then twice with 5 ml TBST/PBST for 5 min on a shaker at room temperature.
2. Incubate the washed array with 5 ml of His-tagged protein solution containing 2.5% (w/v) skimmed milk powder to prevent non-specific binding. Here, use 4.5 µM of protein solution (STIL 500-650) dissolved in the described blocking solution.
   NOTE: Usually 5-10 µM protein are used for the screening, but the protein concentration can be even as low as 2-3 µM, depending on the binding affinity and the local concentrations of the peptides that bound on the array (efficiency of the synthesis). The buffer in which the protein is dissolved can vary but is common to dilute the protein using the same blocking solution described above.
3. Incubate the array in the protein solution for 3-8 hr at room temperature or overnight at 4 ºC.

4. Incubating with the Antibody

1. Wash the array three times with 5 ml TBST/PBST. The first wash is for 30 sec followed by two 5 min washes on shaker at room temperature.
2. Incubate the washed array with 5 ml of diluted HRP-conjugated antibody (1:1,500) in an incubation buffer that contains the same ingredients at the same concentrations as the blocking solution, for 1 hr on a shaker at room temperature. The antibody can bind either the target protein or the tag.
3. Perform control experiments testing the interaction of the antibody with peptides on the array, especially if the array contains peptides from the target protein (e.g., for oligomerization studies) by repeating the same protocol without step 3, incubating with the protein.
4. Wash the array three times with 5 ml TBST/PBST. The first wash is for 30 sec followed by two 5 min washes on shaker at room temperature.

5. Reading the Array

1. Carry out chemiluminescence development with an ECL western blotting substrate kit.
2. Perform the detection with a luminescent image analyzer.
3. Analyze the results. Make sure that both duplicates on the array show the same signals, so the results are reliable. If the structure of the protein partner is known, search on the structure for the binding peptides and look for the binding sites. Even peptides that are far in the sequence can be close together in the tertiary structure and create a binding site.

Wyniki

STIL is a highly important centrosomal protein. It controls normal cell division and cell proliferation^13,15–19. 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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Peptide array	INTAVIS Bioanalytical Instruments
His-probe Antibody (H-3) HRP	Santa Cruz Biotechnology	sc-8036 HRP
EZ-ECL Kit	Biological industries	20-500-120
Skim Milk	Becton, Dickinson and Company	232100
LAS-3000 camera	FUJI film