An Improved Method for the Synthesis of Cellulose Membrane-Bound Peptides with Free C Termini Is Useful for PDZ Domain Binding Studies

Abstract

SPOT synthesis permits parallel synthesis and screening of thousands of cellulose membrane-bound peptides to study protein-protein interactions in a proteomic context. Recognition of C-terminal residues is one of the most common binding features of PDZ domains. Unfortunately, most solid support-bound peptide libraries lack a free C terminus due to C-terminal fixation on the solid support. To overcome this restriction, we developed a robust methodology based on our previous strategy for generating peptides with authentic C termini. To validate this improved method, we screened a human peptide library of 6223 C termini with the syntrophin PDZ domain. Furthermore, using the same library, new peptide ligands derived from membrane proteins and receptors were found for the ERBIN PDZ domain. Finally, we identified the protein kinase breakpoint cluster region, which is known as a negative regulator of cell proliferation and oncogenic transformation, as an ERBIN ligand.

Crystal Structure of NblA from Anabaena sp. PCC 7120, a Small Protein Playing a Key Role in Phycobilisome Degradation

“…For quantification, the spot-signal intensities were measured in Boehringer light units. We used the intensity of background signal plus three times its standard deviation as the threshold for sufficient binding affinity on the cellulose membrane as described (26,27). To exclude false-positive results in the NblA incubation experiment, the membrane was pre-examined with GST/anti-GST and anti-mouse monoclonal antibodies.…”

Bcr (breakpoint cluster region) protein binds to PDZ-domains of scaffold protein PDZK1 and vesicle coat protein Mint3

“…In addition to these GST-Bcr pulldown experiments, coimmunoprecipitation of Bcr and Mint3 could also be detected. at least four [AF-6 (Radziwill et al, 2003), erbin (Boisguerin et al, 2004), PDZK1 and Mint3], there does seem to be considerable selectivity of the Bcr C-terminal ligand among PDZ-domain proteins, since EBP-50, GOPC and more than 20 other proteins represented on the array are not recognized. Selectivity among the different Mint proteins is particularly striking since both of their PDZ domains are quite highly conserved (Okamoto and Sudhof, 1998) and some protein ligands are known to bind to all three isoforms (Hill et al, 2003).…”

Synthetic peptide arrays for investigating protein interaction domains

“…Unfortunately, standard SPOT-synthesized peptides lack free C-termini due to their coupling to the cellulose support. The first reliable and robust SPOT synthesis concept for synthesizing inverted peptide arrays with free C-termini was published in 2004 by the Volkmer lab, and the approach was recently improved [52,75]. Such an inverted peptide array requires a special, ether-type cellulose membrane called N-CAPE membrane [76] with amino-loading capacities of 200-800 nmol/cm 2 .…”

“…These peptides are defined by some shared characteristics, e.g. the targets of a consensus in a sequence data bank such as an array of 12-mer human PPxY sequences [50,51] or an array of human C-terminal protein sequences (''Humlib'') [52]. Further systematic sets of peptides for simple functional assays applied for mapping protein binding are scans of overlapping peptides [53,54], amino acid substitution scans such as the alanine scanning approach [55,56], or (complete) substitution analyses.…”