A fluorescence polarization assay for the experimental validation of an in silico model of the chemokine CXCL8 binding to receptor-derived peptides.

Peptide based inhibitors of protein-protein interactions are of great interest in proteomics, structural biology and medicinal chemistry. Optimized inhibitors can be designed by experimental approaches or by computational prediction. Ideally, computational models are adjusted to the peptide-protein complex of interest according to experimental data obtained in specific binding experiments. The chemokine CXCL8 (interleukin-8) is an interesting target for drug discovery due to its role in inflammatory diseases. Given the available structural data and information on its receptor interactions it constitutes a basis for the rational design of inhibitor peptides. Starting from the reported structure of CXCL8 in complex with a peptide derived from its receptor CXCR1 we developed a computational docking procedure to estimate the changes in binding energy as a function of individual amino acid exchanges. This indicates whether the respective amino acid residue must be preserved or can be substituted to maintain or improve affinity, respectively. To validate and improve the assumptions made in this docking simulation we established a fluorescence polarization assay for receptor-derived peptides binding to CXCL8. A peptide library was tested comprising selected mutants characterized by docking simulations. A number of predictions regarding electrostatic interactions were confirmed by these experiments and it was revealed that the model needed to be corrected for backbone flexibility. Therefore, the assay presented here is a promising tool to systematically improve the computational model by iterative cycles of modeling, experimental validation and refinement of the algorithm, leading to a more reliable model and peptides with improved affinity.

Interdigitated multicolored bioink micropatterns by multiplexed polymer pen lithography.

Multiplexing, i.e., the application and integration of more than one ink in an interdigitated microscale pattern, is still a challenge for microcontact printing (µCP) and similar techniques. On the other hand there is a strong demand for interdigitated patterns of more than one protein on subcellular to cellular length scales in the lower micrometer range in biological experiments. Here, a new integrative approach is presented for the fabrication of bioactive microarrays and complex multi-ink patterns by polymer pen lithography (PPL). By taking advantage of the strength of microcontact printing (µCP) combined with the spatial control and capability of precise repetition of PPL in an innovative way, a new inking and writing strategy is introduced for PPL that enables true multiplexing within each repetitive subpattern. Furthermore, a specific ink/substrate platform is demonstrated that can be used to immobilize functional proteins and other bioactive compounds over a biotin-streptavidin approach. This patterning strategy aims specifically at application by cell biologists and biochemists addressing a wide range of relevant pattern sizes, easy pattern generation and adjustment, the use of only biofriendly, nontoxic chemicals, and mild processing conditions during the patterning steps. The retained bioactivity of the fabricated cm(2) area filling multiprotein patterns is demonstrated by showing the interaction of fibroblasts and neurons with multiplexed structures of fibronectin and laminin or laminin and ephrin, respectively.

Maskless projection lithography for the fast and flexible generation of grayscale protein patterns.

Protein patterns of different shapes and densities are useful tools for studies of cell behavior and to create biomaterials that induce specific cellular responses. Up to now the dominant techniques for creating protein patterns are mostly based on serial writing processes or require templates such as photomasks or elastomer stamps. Only a few of these techniques permit the creation of grayscale patterns. Herein, the development of a lithography system using a digital mirror device which allows fast patterning of proteins by immobilizing fluorescently labeled molecules via photobleaching is reported. Grayscale patterns of biotin with pixel sizes in the range of 2.5 µm are generated within 10 s of exposure on an area of about 5 mm(2) . This maskless projection lithography method permits the rapid and inexpensive generation of protein patterns definable by any user-defined grayscale digital image on substrate areas in the mm(2) to cm(2) range.