Cells are steadily influenced by their surrounding microenvironment. In vivo, ligand presentation is largely affected by the extracellular matrix. To dynamically control the microenvironment of cells, the design and development of model systems that recapitulate these spatiotemporal dynamics is important. This project thus aims to photo-pattern and assemble (bio-)chemical cues in biocompatible frameworks utilizing light-responsive interaction pairs.
The PhD project involves the design and chemical synthesis of new wavelength-tuned, photo-activatable amino acids and their introduction in novel light-sensitive nanotools. As the uniform presentation of ligands is critical for guiding specific interactions with cell surface receptors, the second aim of the project is the implementation and application of the developed light-addressable nanotools at functionalized interfaces and cell-compatible hydrogels. By exploring optochemical nanotools as anchoring units for biochemical cues, the dynamic structuring of matrices in a spatial precise and temporal controlled manner will be established. In vitro proof-of-concept studies will be performed on various functionalized interfaces (2D PEG surfaces, 3D hydrogels, etc.) to demonstrate the multiplexed in-situ protein organization. Despite this, the expansion the optochemical nanotools towards two-photon activatable interaction pairs (in collaboration with A2.2) allows the activation of the anchoring units with higher spatial precision as well as higher accuracy. Thus another challenge is the establishment of two-photon addressable interaction pairs.
The in situ patterning of proteins is broadly useful to study spatial gradients of biochemical cues in 2D/3D cell culture in a non-invasive way. The light-responsive interfaces and hydrogels will enable to study cell behavior and even direct cell signaling in vivo and real-time.
Despite this, the PhD student will closely work together with the PhD theses B4.1 as well as PhD theses B4.5 and contribute to the development of novel photo-activatable lock-and-key elements and synthetic viral immune evasins to probe and manipulate cellular processes by light.
B7.2: Optical control of peptide-membrane interaction for spatiotemporally precise membrane perturbation
The controlled localization of proteins to specific regions of the cellular membrane is critical to regulate processes involved in signaling, transport, membrane shaping and curvature. Natural mechanisms to recruit proteins to membranes are typically complex and require multiple steps as well as accessory components. Current approaches to specifically target proteins to membranes lack the ability to control the interactions in a spatiotemporal manner.
This project aims to design and develop light-sensitive, lipid-sensing peptides for the on demand recruitment of membrane-modulating moieties/ proteins. The project will consist in the organic synthesis of “caged” peptides, their photophysical characterization and the in vitro analysis of the light-induced binding to the surface of different model membrane systems.
The PhD student will design and synthesize various wavelength-addressable caged amino acids followed by their installation in diverse membrane-interacting peptide motifs. To optically control membrane interaction, the peptide sequences will be altered in a systematic fashion, such that gradual gain of a new function (here, the in-situ membrane targeting activity after photoactivation) will be achieved. A library of tailor-designed caged peptides bearing light-sensitive modifications at single or multiple sites as well as orthogonal addressable caged amino acids will be generated. In addition, the synthetic access will also allow the implementation of native chemical ligation to chemoselectively ligate photo-activatable peptides with recombinantly expressed proteins. To study how the developed photoactivatable peptides perturb the membrane organization and induce curvature, membrane binding will be followed in vitro by CLSM using various model membrane systems (e.g. giant unilamellar vesicles) with defined lipid composition.
The developed photo-activatable, membrane-binding peptides offer the ability to deliberately localize and recruit probes to desired cellular membranes and to study membrane reorganization and bending with high time and spatial precision. Ultimately, this project aims to optically regulate membrane perturbation in response to light and will thus provide new interventions and information of dynamic regulation processes in membrane shaping and reorganization.
The PhD student will closely work together with PhD theses A2.7 for the photophysical investigation of various uncaging scenarios and with PhD theses B2.2 to obtain global as well as local structural information of peptide flexibility and conformational folding in hydrophobic environment. In addition, the PhD student will contribute to the incorporation of caged lysines into proteins via amber suppression systems (in collaboration with PhD theses B4.4).