Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases

Rune Matthiesen and L. Maria Lois (eds.), Plant Proteostasis: Methods and Protocols, Methods in Molecular Biology, vol. 1450,DOI 10.1007/978-1-4939-3759-2_7, © Springer Science+Business Media New York 2016

Chapter 7

Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases

Maria Klecker and Nico Dissmeyer

Abstract

The automated SPOT (synthetic peptide arrays on membrane support technique) synthesis technology has entrenched as a rapid and robust method to generate peptide libraries on cellulose membrane supports. The synthesis method is based on conventional Fmoc chemistry building up peptides with free N-terminal amino acids starting at their cellulose-coupled C-termini. Several hundreds of peptide sequences can be assembled with this technique on one membrane comprising a strong binding potential due to high local peptide concentrations (Wenschuh et al. Biopolymers 55(3):188–206, 2000). Peptide orientation on SPOT membranes qualifies this array type for assaying substrate specificities of N-recognins, the recogni-tion elements of the N-end rule pathway of targeted protein degradation (NERD). Pioneer studies described binding capability of mammalian and yeast enzymes depending on a peptide’s N-terminus (Erbse et al. Nature 439(7077):753–756, 2006; Choi et al. Nat Struct Mol Biol 17(10):1175–1181, 2010; Hwang et al. Science 327(5968):973–977, 2010; Thiele et al. Mol Biotechnol 49(3):283–305, 2011; Kim et al. Cell 156(1–2):158–169, 2014). SPOT arrays have been successfully used to describe substrate specificity of N-recognins (Erbse et al. Nature 439(7077):753–756, 2006; Choi et al. Nat Struct Mol Biol 17(10):1175–1181, 2010; Hwang et al. Science 327(5968):973–977, 2010; Kim et al. Cell 156(1–2):158–169, 2014) which are the recognition elements of the N-end rule pathway of targeted pro-tein degradation (NERD). Here, we describe the implementation of SPOT binding assays with focus on the identification of N-recognin substrates, applicable also for plant NERD enzymes.

Key words SPOT assay, N-end rule, Ubiquitin ligase, Protein-protein interaction, ResPep SL, Substrate screen, Peptide library

1 Introduction

Screening of protein activity and elucidating binding parameters are at the basis of enzyme characterization and functional assi-gnment. However, the identification of substrates, their enzyme- binding sites or of potent inhibitors may imply extensive work of cloning, mutant generation, protein expression, and purification when performed on full-length recombinant proteins. Another drawback of many rather classical protein–protein interaction assays such as co-immunoprecipitation or pull-downs is the often intrinsically low binding affinity between enzyme and substrate.

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1.1 Peptide Arrays for Protein Interaction Studies

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For this reason, the use of synthetic peptide libraries on membrane supports with high local peptide concentrations at each “spot” has become increasingly popular for protein interaction studies since the full automation of SPOT synthesis with robots was launched.

Publications involving SPOT assays for mapping of epitopes, kinase, and phosphatase interaction sites or for protease substrate identification run into the hundreds [7, 8]. In the last decade, the method was further seized for the study of enzymes which are functionally comprised by the pathway of N-end rule degradation (NERD) [2–4, 6]. This brings about a new type of SPOT design which contains peptides derived only from the very N-termini of substrate candidates and opens up the technique for the applica-tion on enzymes such as E3 ubiquitin ligases.

The N-end rule of protein degradation relates the in vivo half-life of a protein to the nature of its N-terminal amino acid [9, 10]. In eukaryotes, the machinery behind involves a variety of enzyme classes including Met-aminopeptidases, N-terminal acetyltransfer-ases, amidases, cysteine oxidases, Arg-tRNA transferases, and E3 ubiquitin ligases, all of which appear to select their substrates chiefly based on their very N-terminal residues.

On a SPOT membrane, the lengths of the arrayed peptides usually limit their ability to adopt the correct conformation. Furthermore, in vivo peptide binding may be dependent on the cellular environment (membranes, scaffold proteins) which differs from the situation on the SPOT membrane. However, according to the current understanding, the ability of a given N-terminus to act as a NERD degradation signal, i.e. as an N-degron, does not require the adoption of a higher order structure over the primary sequence, but is in contrast promoted when a disordered region allows for flexibility and protrusion of the N-terminal degron. This condition of substrate recognition by NERD components predes-tines these enzymes for the study by the SPOT method.

So far, SPOT assays have been applied to characterize the substrate specificity of the ClpAP-specific adapter protein ClpS from E. coli [2] and of the unique NERD ubiquitin ligase Ubr1p from Saccharomyces cerevisiae [3, 4]. Furthermore, the importance of the penultimate amino acid for recognition by NERD ubiquitin ligases in S. cerevisiae and mouse was revealed by SPOT assay appli-cation [6].

The peptide synthesis using the ResPep SL method, side chain de-protection, and general protein-protein interaction screening was recently protocolled [11]. Here, it will be described how to perform binding assays on SPOT membranes with NERD enzymes, particularly E3 ubiquitin ligases. The presented protocol applies to peptide arrays synthesized by the ResPep SL method on acid- hardened cellulose membranes, derivatized with polyethylene gly-col (PEG) spacers.

1.2 SPOT Assays on N-End Rule Enzymes


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2 Materials

If not indicated otherwise, all solutions should be prepared freshly before every experiment. The studied protein should be available in a microgram scale as a purified, active enzyme solution of known concentration. If specific antibodies are unavailable, an epitope tag for detection should be strongly considered (see Notes 1 and 7).

1. Synthesized SPOT array on PEG-functionalized cellulose (see Notes 2, 16, and 17).

2. Rocking platform for incubation at room temperature and in a 4 °C environment.

3. Rolling device or a tube rotator for 3D shaking of tubes (e.g. SB3 (Stuart) equipped with appropriate tube holders).

4. Vapor-proof polypropylene (PP) box (e.g. a sealable lunch box). 5. Polyethylene boxes of the area of the SPOT membrane and

about 2 cm in height (e.g. Hartenstein, #AD01) (see Note 5). 6. Flat tweezers for membrane transfer. 7. Pipettes accurately delivering 2.5 or up to 200 μL, depending

on the concentration of the stored enzyme solution. 8. Flasks with screw caps comprising at least 250 mL.

1. Methanol or ethanol. 2. Washing/binding buffer: any buffer system in which the pro-

tein of interest (POI) is known to be active; alternatively: TBST (20 mM Tris–HCl, pH 7.4, 135 mM NaCl, 0.1 % (v/v) Tween 20) can be tried as the basis for a binding buffer and stored as a 2× stock solution. According to protein requi-rements, it should be supplemented before use with redu-cing agents (such as dithiothreitol) and compatible solutes (see Notes 10, 11, and 12).

3. Blocking buffer: Binding buffer (see item 2; Note 9) contain-ing a blocking agent (e.g. 3 % (w/v) PVP40, 3 % (w/v) BSA or up to 10 % (w/v) milk powder).

1. Binding buffer (see item 2 in Subheading 2.2) (see Notes 3, 10, and 11).

2. Optional: protease inhibitors. If the purified protein samples used for binding assays might contain protease contaminations or the ambient air bears high titers of bacterial or fungal contam-inants, use of protease inhibitors is indicated as follows: 1 mg/mL Pepstatin A (Fluka, store at −80 °C), 1 mg/mL 4-(2-ami-noethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, Santa Cruz, sc-202041B; store at −80 °C).

2.1 General Equipment and Infrastructure

2.2 Membrane Activation and Blocking

2.3 Enzyme Binding on SPOT Membranes

SPOT Arrays for Characterization of E3 Ubiquitin Ligases

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1. Cathode buffer: 25 mM Tris-base, 40 mM 6-aminohexanoic acid (6-aminocaproic acid/ε-aminocaproic acid, Santa Cruz, sc-202146), 0.01 % (w/v) SDS, 20 % (v/v) methanol (pH of the solution should be 9.4).

2. Anode buffer I: 30 mM Tris-base, 20 % (v/v) methanol, (pH should be 10.4).

3. Anode buffer II: 300 mM Tris-base, 20 % (v/v) methanol. 4. Semi-dry blot apparatus (such as Bio-Rad Trans-Blot SD Semi-

Dry Transfer Cell) with power supply. 5. Filter paper (Whatman). 6. PVPF membrane (Amersham/GE Healthcare).

3 Methods

The experiment takes in total about 2 days.

Before performing each binding assay, the membrane must be acti-vated in order to fully hydrate the coupled peptides. This can be done directly after side chain de-protection (for method refer to [18]) before an assay.

1. If the membrane was stored de-protected at refrigerated con-ditions, let it reach room temperature before exposing it to the procedure.

2. Place the membrane into a vapor-tight box and cover it com-pletely with methanol or ethanol and incubate until the visible spots disappear (at least 1 min, up to half an hour) (see Note 4).

3. Wash and equilibrate the membrane at least three times for 10 min with binding buffer. Be quick upon transfer from alcohol to the buffer since methanol and ethanol are volatile and the membrane must be prevented from drying at this step (see Note 6).

4. Cover the membrane completely with appropriate blocking solution and incubate on a rocker for at least 1 h at room tem-perature or overnight at 4 °C (see Notes 8 and 9).

Ubiquitin E3 ligases are characterized by relatively high dissocia-tion constants [19] and one may face problems when trying to approach their substrate binding capacities by pull-down experi-ments. This issue is circumvented in a SPOT binding assay through extremely high local substrate concentrations (see Fig. 1). In epitope mapping experiments, peptide–antibody interactions with dissocia-tion constants as high as 1–0.1 mM remained detectable [7].

1. Prepare at least 100 mL of the binding buffer. 2. Add your recombinant enzyme of interest at a starting concen-

tration of 50 nM (see Note 13) to a volume of binding buffer

2.4 Western Blotting of SPOT-Array- Bound Proteins

3.1 Membrane Activation and Blocking

3.2 Enzyme Binding on SPOT Membranes

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that is sufficient to completely cover the membrane. Incubate the solution for 5 min under rotation.

3. Add the binding buffer containing enzyme to the blocked membrane and incubate for 1–3 h on a rocking platform at room temperature (see Notes 14 and 18).

4. Wash the membrane 3–6 times for 10 min with washing buffer.

In order to immobilize the relatively weak binding enzyme for antibody incubations, the NERD SPOT assay usually involves elec-trotransfer of the bound protein to a PVDF membrane. Here, the three buffer blot system according to [21] is the method of choice for semi-dry electrotransfer (Fig. 2). Unlike when blotting an SDS- PAGE gel, the blotted protein from a SPOT membrane is only very briefly incubated with the transfer buffer, including only mild

3.3 Western Blotting of SPOT-Array- Bound Proteins

Fig. 1 SPOT array seen at different stages of synthesis and assay. (a) Shown is the standard grid of 600 posi-tions available for peptide spots to be synthesized on one cellulose membrane predefined by the ResPepSL software. (b) Example of one membrane divided into four identical subsets of each 126 spots seen under UV light directly after SPOT synthesis. The peptides appear as light or gray spots according to the presence of UV absorbing groups of the amino acid residues and side chain protection groups. (c) Immunodetection of a recombinant enzyme from the plant NERD after binding assay with one of the four membranes shown in (b) and electrotransfer. (d) UV light view of the SPOT membrane used in (c) after side chain de-protection


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charging by SDS, and can be considered to be in a partly native form. For this reason, the migration properties of one enzyme might differ depending on its stability and isoelectric point.

1. Pre-wet up to three filter papers in each cathode, anode I, and anode II buffer.

2. Activate a PVDF membrane by incubation in methanol for at least 1 min.

3. Equilibrate the PVDF membrane in anode I buffer. 4. Equilibrate the POI-charged SPOT membrane briefly (max.

1 min) in cathode buffer. 5. Assemble the blot sandwich as follows and strictly avoid air

bubbles between all layers: ● Filter papers with anode II buffer facing the anode. ● Up to three layers of filter papers soaked with anode I

buffer. ● PVDF membrane. ● SPOT membrane with the protein-bound site facing the

PVDF membrane. ● Up to three layers of filter papers soaked in cathode buffer

facing the cathode. 6. Perform the electrotransfer for 30 min at 0.8 mA/cm2 of

SPOT membrane.

Fig. 2 Assembly of the semi-dry western blot using the three buffer blot system. Up to three layers of filter papers soaked in each Anode II buffer (1) and Anode I buffer (2) are placed on top of the anode (only one layer each is shown). The activated PVDF membrane (3) is equilibrated in Anode I buffer and faces the SPOT side of the SPOT membrane (4) bearing the POI. This is covered by again up to three filter papers soaked in cathode buffer (5) and mounted by the cath-ode. The system was described by Kyhse-Anderson, 1984


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7. Transfer the PVDF membrane to your favored western blot blocking solution (see Note 15).

8. Transfer the SPOT membrane to distilled water for subsequent stripping and reuse (for protocol, see [22]).The PVDF membrane can be treated with antibodies specific

to the target protein and detected like a normal western blot.

4 Notes

1. When using affinity tags, be aware of the fact that those may have binding capacity to parts of your membrane. A negative control with the affinity-tag protein itself is recommended. Particular care must be taken when using maltose binding pro-tein which has strong affinity for cellulose units resulting in a massive background signal also on the PEG-ylated membranes. This can be blocked by addition of maltose (10 mM) to the binding buffer.

2. The Synthesis of a SPOT membrane using the ResPep SL method from INTAVIS (Cologne, Germany) was recently described [11]. A customized SPOT membrane bearing the requested peptide sequences can be purchased from JPT Peptide Technologies (Berlin, Germany).

3. It can be advisable to supplement the binding buffer (see item 2 in Subheading 2.2) with low amounts of the blocking agent (see item 3 in Subheading 2.2). This is especially the case if the protein tends to bind to plastic surfaces.

4. Incompletely hydrated peptides remain apparent on the mem-brane as white spots. Activation needs to be prolonged until the visible spots disappear. Depending on the overall hydro-phobicity of each peptide sequence, the time required can differ between the spots.

5. The size of the box should not exceed the membrane diame-ters, otherwise higher volumes of the protein solution will have to be incubated with the membrane.

6. From this step on, polyethylene boxes can be used. 7. In any case, especially if there is no positive control present on

the SPOT membrane, the activity of the used protein should be proven by additional methods like enzymatic assays.

8. The PEG-derivatized cellulose membrane is considered to give very little background binding. However, blocking of the membrane is recommended in order to inactivate unspecific binding sites.

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9. When blocking the membrane overnight, avoid the use of sugars in the blocking solution, since this might promote bac-terial growth.

10. The optimal binding condition for each tested enzyme will have to be defined and fine-tuning of the buffer conditions has the potential to alter binding properties. For instance, hydro-phobic interactions by E3 ligases may tolerate salt concentra-tions up to 1 M [20], whereas this will obviously interfere with ionic binding situations. The opposite effect may be achieved by varying detergent strength of the binding buffer.

11. So far, binding buffer systems based on Tris [2, 4], MES [3], or HEPES buffers [6] have been reported to work successfully for SPOT assays investigating NERD components. If the enzyme of interest is known to be active in a certain buffer system, e.g. the storage buffer, this will be the first condition to use in the SPOT assay. For enzymes sensitive to pH shifts it should be considered that the assay will be performed at room temperature which will cause changes in the pH of buffers such as Tris when the enzyme is usually handled at low tem-perature conditions.

12. Frequently in the literature, a compatible solute like 5 % (w/v) sucrose or 10 % (v/v) glycerol is also present in the buffer systems.

13. Due to the high binding capacity of SPOT membranes, the pro-tein concentration in the binding buffer can be as low as 20 nM depending on the enzyme binding properties. However, if the peptide set comprises lots of strong binders, enough protein has to be supplied to prevent depletion of the solution and overall loss of signal strength. On the other hand, it will be easier to discriminate between the interaction strength of different spots when using lower amounts of enzyme to be studied.

14. Usually, the binding assay is robust enough to tolerate also residuals of the blocking solution and the membrane does not necessarily need to be washed with the washing buffer before incubation with the enzyme mixture.

15. If high amounts of protein are bound to the SPOT membrane, it can be subjected to another turn of blotting giving rise to western blots with more stringent protein signals. Often, only this sequential blotting gives clear results due to unclear load-ing or binding status of the spots and unclear binding affinity of the POI to the highly concentrated peptides.

16. Concerning array size, it should be taken into account that the cost-effectiveness of the SPOT method will improve with increased number of different peptides on a membrane.


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This makes SPOT assays excellently suited for broad affinity screens, rather than the testing of a small set of predefined substrate candidates. Subsets of 120 peptides were described in the NERD field [3]. This array size also appears reasonable for its workable dimensions (about 6 × 4 cm2; spot diameter of ~0.4 cm). The standard grid provided by the INTAVIS ResPep SL software is defined by 600 possible spot positions for one customary membrane (see Fig. 1). This implies that a set of 120 peptides can be synthesized four times in parallel on one mem-brane, giving rise to an appropriate set of membrane replicates with sufficient interspace left for cutting the membrane in four pieces.

17. The optimal range of peptide length with respect to synthesis success is stated between 6 and 15 amino acids, with higher amounts of amino acid couplings increasing the probability of possible side reactions [12]. For NERD E3 ubiquitin ligase screens, peptides of 5–13 residues in length were applied. In theory, substrate recognition by NERD components should depend entirely on the very N-terminal amino acids and pep-tides of five amino acids are theoretically sufficient. However, one might miss the potential influence of more distal residues which might affect the total peptide properties by charge and hydrophobicity.

18. The duration of the binding depends mostly on the protein binding strength, the concentration, and the stability of the tested protein.

Acknowledgements

We thank Christian Behn from INTAVIS for personal advices on the ResPep SL SPOT peptide synthesis and we are grateful to Petra Majovsky and Wolfgang Hoehenwarter for constant support in mass spectrometry and proteome analytics. This work was sup-ported by a grant for setting up the junior research group of the ScienceCampus Halle—Plant-based Bioeconomy to N.D., a Ph.D. fellowship of the ScienceCampus Halle to M.K. Financial support came from the Leibniz Association, the state of Saxony-Anhalt, the Deutsche Forschungsgemeinschaft (DFG) Graduate Training Center GRK1026 “Conformational Transitions in Macromolecular Interactions” at Halle, and the Leibniz Institute of Plant Bio-chemistry (IPB) at Halle, Germany.


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References[AU4]

1. Wenschuh H, Volkmer-Engert R, Schmidt M, Schulz M, Schneider-Mergener J, Reineke U (2000) Coherent membrane supports for par-allel microsynthesis and screening of bioactive peptides. Biopolymers 55(3):188–206

2. Erbse A, Schmidt R, Bornemann T, Schneider- Mergener J, Mogk A, Zahn R, Dougan DA, Bukau B (2006) ClpS is an essential compo-nent of the N-end rule pathway in Escherichia coli. Nature 439(7077):753–756

3. Choi WS, Jeong BC, Joo YJ, Lee MR, Kim J, Eck MJ, Song HK (2010) Structural basis for the recognition of N-end rule substrates by the UBR box of ubiquitin ligases. Nat Struct Mol Biol 17(10):1175–1181

4. Hwang CS, Shemorry A, Varshavsky A (2010) N-terminal acetylation of cellular proteins cre-ates specific degradation signals. Science 327 (5968):973–977

5. Thiele A, Stangl GI, Schutkowski M (2011) Deciphering enzyme function using peptide arrays. Mol Biotechnol 49(3):283–305

6. Kim HK, Kim RR, Oh JH, Cho H, Varshavsky A, Hwang CS (2014) The N-terminal methio-nine of cellular proteins as a degradation signal. Cell 156(1-2):158–169

7. Hilpert K, Winkler DF, Hancock RE (2007) Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a par-allel and addressable fashion. Nat Protoc 2(6):1333–1349

8. Thiele A, Zerweck J, Schutkowski M (2009) Peptide arrays for enzyme profiling. Methods Mol Biol 570:19–65

9. Bachmair A, Finley D, Varshavsky A (1986) In vivo half-life of a protein is a function of its amino-terminal residue. Science 234(4773): 179–186

10. Varshavsky A (1996) The N-end rule: func-tions, mysteries, uses. Proc Natl Acad Sci U S A 93(22):12142–12149

11. Yim YY, Betke K, Hamm H (2015) Using pep-tide arrays created by the SPOT method for defining protein-protein interactions. Methods Mol Biol 1278:307–320

12. Winkler DF, Hilpert K, Brandt O, Hancock RE (2009) Synthesis of peptide arrays using

SPOT-technology and the CelluSpots-method. Methods Mol Biol 570:157–174

13. Bachmair A, Varshavsky A (1989) The degra-dation signal in a short-lived protein. Cell 56(6):1019–1032

14. Suzuki T, Varshavsky A (1999) Degradation signals in the lysine-asparagine sequence space. EMBO J 18(21):6017–6026

15. Gibbs DJ, Lee SC, Isa NM, Gramuglia S, Fukao T, Bassel GW, Correia CS, Corbineau F, Theodoulou FL, Bailey-Serres J, Holdsworth MJ (2011) Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants. Nature 479(7373):415–418

16. Licausi F, Kosmacz M, Weits DA, Giuntoli B, Giorgi FM, Voesenek LA, Perata P, van Dongen JT (2011) Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature 479(7373):419–422

17. Mendiondo GM, Gibbs DJ, Szurman- Zubrzycka M, Korn A, Marquez J, Szarejko I, Maluszynski M, King J, Axcell B, Smart K, Corbineau F, Holdsworth MJ (2016) Enhanced waterlogging tolerance in barley by manipulation of expres-sion of the N-end rule pathway E3 ligase PROTEOLYSIS6. Plant Biotechnol J 14:40

18. Gausepohl H, Behn C (2002) Automated syn-thesis of solid-phase bound peptides. In: Koch J, Mahler M (eds) Peptide arrays on membrane supports, vol 4, Springer lab manuals. Springer, New York, NY, pp 55–68

19. Ayad NG, Rankin S, Ooi D, Rape M, Kirschner MW (2005) Identification of ubiquitin ligase substrates by in vitro expression cloning. Methods Enzymol 399:404–414

20. Tasaki T, Zakrzewska A, Dudgeon DD, Jiang Y, Lazo JS, Kwon YT (2009) The substrate recognition domains of the N-end rule path-way. J Biol Chem 284(3):1884–1895

21. Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10(3-4):203–209

22. Cretich M, Longhi R, Corti A, Damin F, Di Carlo G, Sedini V, Chiari M (2009) Epitope mapping of human chromogranin A by peptide microarrays. Methods Mol Biol 570:221–232


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Documents

Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases