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Peptide Array (peptide + array)
Selected AbstractsPalladin is a novel binding partner for Ena/VASP family membersCYTOSKELETON, Issue 1 2004Malika Boukhelifa Abstract Palladin is an actin-associated protein that contains proline-rich motifs within its amino-terminal sequence that are similar to motifs found in zyxin, vinculin, and the Listeria protein ActA. These motifs are known to be potential binding sites for the Vasodilator-Stimulated Phosphoprotein (VASP). Here, we demonstrate that palladin is an additional direct binding partner for VASP, by using co-immunoprecipitation and blot overlay techniques with both endogenous palladin and recombinant myc-tagged palladin. These results show that VASP binds to full-length palladin and also to the amino-terminal half of palladin, where the polyproline motifs are located. Using a synthetic peptide array, two discrete binding sites for VASP were identified within palladin's proline-rich amino-terminal domain. Using double-label immunofluorescence staining of fully-spread and actively-spreading fibroblasts, the extent of co-localization of palladin and VASP was explored. These proteins were found to strongly co-localize along stress fibers, and partially co-localize in focal adhesions, lamellipodia, and focal complexes. These results suggest that the recently described actin-associated protein palladin may play an important role in recruiting VASP to sites of actin filament growth, anchorage, and crosslinking. Cell Motil. Cytoskeleton 58:17,29, 2004. © 2004 Wiley-Liss, Inc. [source] Inhibition of human ether à go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-terminiFEBS JOURNAL, Issue 5 2006Ulrike Ziechner Human ether à go-go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca2+/calmodulin (CaM), presumably binding to the C-terminal domain of the channel subunits. Deletion of the cytosolic N-terminal domain resulted in complete abolition of Ca2+/CaM sensitivity suggesting the existence of further CaM binding sites. A peptide array-based screen of the entire cytosolic protein of hEAG1 identified three putative CaM-binding domains, two in the C-terminus (BD-C1: 674,683, BD-C2: 711,721) and one in the N-terminus (BD-N: 151,165). Binding of GST-fusion proteins to Ca2+/CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and precipitation assays. In the presence of Ca2+, BD-N and BD-C2 provided dissociation constants in the nanomolar range, BD-C1 bound with lower affinity. Mutations in the binding domains reduced inhibition of the functional channels by Ca2+/CaM. Employment of CaM-EF-hand mutants showed that CaM binding to the N- and C-terminus are primarily dependent on EF-hand motifs 3 and 4. Hence, closure of EAG channels presumably requires the binding of multiple CaM molecules in a manner more complex than previously assumed. [source] Investigations into the development of catalytic activity in anti-acetylcholinesterase idiotypic and anti-idiotypic antibodiesJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2009Glynis Johnson Abstract We have previously described anti-acetylcholinesterase antibodies that display acetylcholinesterase-like catalytic activity. No evidence of contaminating enzymes was found, and the antibodies are kinetically and apparently structurally distinct from both acetylcholinesterase (AChE) and butyrylcholinesterase. We have also mimicked the antibody catalytic sites in anti-anti-idiotypic (Ab3) antibodies. Independently from us, similar acetylcholinesterase-like antibodies have been raised as anti-idiotypic (Ab2) antibodies against a non-catalytic anti-acetylcholinesterase antibody, AE-2. In this paper, we describe an epitope analysis, using synthetic peptides in ELISA and competition ELISA, and a peptide array, of five catalytic anti-acetylcholinesterase antibodies (Ab1s), three catalytic Ab3s, as well as antibody AE-2 and a non-catalytic Ab2. The catalytic Ab1s and Ab3s recognized three Pro- and Gly-containing sequences (40PPMGPRRFL, 78PGFEGTE, and 258PPGGTGGNDTELVAC) on the AChE surface. As these sequences do not adjoin in the AChE structure, recognition would appear to be due to cross-reaction. This was confirmed by the observation that the sequences superimpose structurally. The non-catalytic antibodies, AE-2 and the Ab2, recognized AChE's peripheral anionic site (PAS), in particular, the sequence 70YQYVD, which contains two of the site's residues. The crystal structure of the AChE tetramer (Bourne et al., 1999) shows direct interaction and high complementarity between the 257CPPGGTGGNDTELVAC sequence and the PAS. Antibodies recognizing the sequence and the PAS may, in turn, be complementary; this may account for the apparent paradox of catalytic development in both Ab1s and Ab2s. The PAS binds, but does not hydrolyze, substrate. The catalytic Ab1s, therefore, recognize a site that may function as a substrate analog, and this, together with the presence of an Arg-Glu salt bridge in the epitope, suggests mechanisms whereby catalytic activity may have developed. In conclusion, the development of AChE-like catalytic activity in anti-AChE Ab1s and Ab2s appears to be the result of a combination of structural complementarity to a substrate-binding site, charge complementarity to a salt bridge, and specific structural peculiarities of the AChE molecule. Copyright © 2008 John Wiley & Sons, Ltd. [source] A study to assess the cross-reactivity of cellulose membrane-bound peptides with detection systems: an analysis at the amino acid levelJOURNAL OF PEPTIDE SCIENCE, Issue 6 2010Carsten C. Mahrenholz Abstract The growing demand for binding assays to study protein,protein interaction can be addressed by peptide array-based methods. The SPOT technique is a widespread peptide-array technology, which is able to distinguish semi-quantitatively the binding affinities of peptides to defined protein targets within one array. The quality of an assay system used for probing peptide arrays depends on the well-balanced combination of screening and read-out methods. The former address the steady-state of analyte capture, whereas the latter provide the means to detect captured analyte. In all cases, however, false-positive results can occur when challenging a peptide array with analyte or detecting captured analyte with label conjugates. Little is known about the cross-reactivity of peptides with the detection agents. Here, we describe at the amino acid level the potential of (i) 5-(and 6)-carboxytetramethylrhodamine (5(6)-TAMRA), (ii) fluoresceinisothiocyanate in form of the peptide-bound fluorescein-substituted thiourea derivative (FITC), and (iii) biotin/streptavidin-POD to cross-react with individual amino acids in a peptide sequence. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. [source] | ||||||||||