Integrin-Targeting Fluorescent Proteins: Exploration of RGD Insertion Sites.

The potential of the fluorescent protein scaffold to control peptide sequence functionality is illustrated by an exploration of fluorescent proteins as novel probes for targeting integrins. A library of fluorescent mCitrine proteins with RGD motifs incorporated at several positions in loops within the protein main chain was generated and characterized. Amino acid mutations to RGD as well as RGD insertions were evaluated: both led to constructs with typical mCitrine fluorescent properties.

Single Particle Tracking Reveals that EGFR Signaling Activity Is Amplified in Clathrin-Coated Pits.

Signaling from the epidermal growth factor receptor (EGFR) via phosphorylation on its C-terminal tyrosine residues requires self-association, which depends on the diffusional properties of the receptor and its density in the plasma membrane. Dimerization is a key event for EGFR activation, but the role of higher order clustering is unknown. We employed single particle tracking to relate the mobility and aggregation of EGFR to its signaling activity.

Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells.

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest.

Site-specific protection and dual labeling of human epidermal growth factor (hEGF) for targeting, imaging, and cargo delivery.

Well-defined human epidermal growth factor (hEGF) constructs featuring selectively addressable labels are urgently needed to address outstanding questions regarding hEGF biology. A protein-engineering approach was developed to provide access to hEGF constructs that carry two cysteine-based site-specific orthogonal labeling sites in multi-milligram quantities. Also, a site-selective (de)protection and labeling approach was devised, which allows selective modification of these hEGF constructs.

Multivalent protein assembly using monovalent self-assembling building blocks.

Discotic molecules, which self-assemble in water into columnar supramolecular polymers, emerged as an alternative platform for the organization of proteins. Here, a monovalent discotic decorated with one single biotin was synthesized to study the self-assembling multivalency of this system in regard to streptavidin. Next to tetravalent streptavidin, monovalent streptavidin was used to study the protein assembly along the supramolecular polymer in detail without the interference of cross-linking.

Modular columnar supramolecular polymers as scaffolds for biomedical applications.

Self-assembly of discotic molecules into supramolecular polymers offers a flexible approach for the generation of multicomponent one-dimensional columnar architectures with tuneable biomedical properties. Decoration with ligands induces specific binding of the self-assembled scaffold to biological targets. The modular design allows the easy co-assembly of different discotics for the generation of probes for targeted imaging and cellular targeting with adjustable ligand density and composition.

Supramolecular polymers as dynamic multicomponent cellular uptake carriers.

Supramolecular synthesis represents a flexible approach to the generation of dynamic multicomponent materials with tunable properties. Here, cellular uptake systems based on dynamic supramolecular copolymers have been developed using a combination of differently functionalized discotic molecules. Discotics featuring peripheral amine functionalities that endow the supramolecular polymer with cellular uptake capabilities were readily synthesized.