A microarray-based approach to evaluate the functional significance of protein-binding motifs.

Intracellular proteins comprise numerous peptide motifs that interact with protein-binding domains. However, using sequence information alone, the identification of functionally relevant interaction motifs remains a challenge. Here, we present a microarray-based approach for the evaluation of peptides as protein-binding motifs.

A peptide-functionalized polymer as a minimal scaffold protein to enhance cluster formation in early T cell signal transduction.

In cellular signal transduction, scaffold proteins provide binding sites to organize signaling proteins into supramolecular complexes and act as nodes in the signaling network. Furthermore, multivalent interactions between the scaffold and other signaling proteins contribute to the formation of protein microclusters. Such microclusters are prominent in early T cell signaling.

Multivalent presentation of the cell-penetrating peptide nona-arginine on a linear scaffold strongly increases its membrane-perturbing capacity.

Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation.

Peptide microarrays to probe for competition for binding sites in a protein interaction network.

Unlabelled: Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins will greatly overestimate connectivity within the network. Here, we addressed the impact of intracellular complexity on signalling networks using microarrays that carried a collection of peptides binding to the GRB2 SH2 and SH3 domains.

Analyzing the homeostasis of signaling proteins by a combination of Western blot and fluorescence correlation spectroscopy.

The determination of intracellular protein concentrations is a prerequisite for understanding protein interaction networks in systems biology. Today, protein quantification is based either on mass spectrometry, which requires large cell numbers and sophisticated measurement protocols, or on quantitative Western blotting, which requires the expression and purification of a recombinant protein as a reference. Here, we present a method that uses a transiently expressed fluorescent fusion protein of the protein-of-interest as an easily accessible reference in small volumes of crude cell lysates.