B4.1: Small lock-and-key elements to track and manipulate receptors in time and space

The PhD student will explore wavelength-addressable optochemical tools for the spatiotemporal labeling and assembly of proteins. Novel small lock-and-key elements will be developed in order to site-specifically label, track and manipulate receptor proteins in time and space. The photophysical properties (e.g. uncaging quantum yield, two-photon cross-section) of the light-sensitive moieties will be analyzed and optimized in close collaboration with A2.1. Further performance tests of the newly developed PA-trisNTAs will be undertaken at functionalized interfaces (surface plasmon resonance, TIRFM, etc.). By strategic positioning of black-hole quenchers, photo-cleavage will cause fluorescence dequenching and allow high affinity labeling upon illumination for super-resolution microscopy (in collaboration with B5.1).

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B4.2: In-situ assembly of receptors triggered by light

This project aims at introducing the newly designed PA-trisNTAs into cell surface receptors by bioorthogonal labeling. Based on the self-inactivation, the PA-trisNTA and His-tagged receptors will interact only upon light activation, which can be performed in dual or triple color mode by LSM or TIRF microscopy. By the generation of protein binding regions upon illumination, the process of iterative protein binding and sequential clustering of membrane receptors will be examined. The project will take advantage of a range of receptors fused to a fluorescent reporter protein and advanced microscopy in collaboration with B5.1.

The PhD student will be able to control receptor clustering in vitro and later in vivo. The lateral organization of membrane receptors will be realized in giant unilamellar vesicles (GUV), solid supported membranes, as well as the plasma membrane of living cells. The receptor mobility before and after photo-activation will be analyzed with fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and single-particle tracking in combination with photo-switchable fluorophores (in collaboration with B5.1).

Light induced receptor clustering in time and space. A) Chemical structure of PA-trisNTA and examples of wavelength selective caging groups. B) PA-trisNTA is covalently attached to receptors on living cells. This provides interaction of orthogonal receptor pairs triggered by light.

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B4.3: 3D organization and manipulation of proteins by light

This project will focus on the in-situ assembly of biomolecules in 3D scaffolds. Laser scanning lithography combined with light-triggered protein assembly offers a powerful tool to organize protein gradients and clustering for a broad range of applications. The PhD student will employ the site-specific and spatiotemporal organization of proteins as well as the sequential protein clustering in 3D scaffolds. For elaborated applications, the photophysical properties, like wavelength selective-activation and two-photon accessibility will be systematically optimized (with A2.2 and A3.2). The process of iterative binding and sequential protein clustering of proteins, including the fabrication of lateral protein gradients will then be examined. Mask-patterned illumination and laser lithography will be employed to write in-situ regions and protein gradients.

Figure: In-situ protein assembly by optochemical tools. A) Comparison of one and two photon activation in hydrogels by light (a) and three dimensional protein assembly by two photon laser-scanning microscopy (b).

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B4.4 and B4.5

In the first period of CLiC, Alina Klein (née Kollmannsperger) successfully delivered small lock-and-key elements to the cytosol of living cells for the spatiotemporal tracking of proteins. Cargo delivery was efficiently facilitated by microfluidic cell squeezing and enabled the transfer of a plethora of lock-and-key elements as well as light-addressable modules. This allowed to precisely control the site-specific labeling, tracking and manipulation of receptor proteins at single-cell level by light and culminated in the live-cell super resolution of the nuclear lamina by high affinity multivalent chelator heads. In close collaboration with the Heilemann lab, the live-cell fluorescence microscopy was performed and the photophysical properties as well as the fluorogenic behavior of the developed light-sensitive moieties were analyzed in contact with A2.1.

In parallel, Karl Gatterdam aimed at developing new photo-conditional, ultrasmall lock-and-key pairs for the organization of receptor proteins by light. He engineered chemical inducible dimerizers based on peptide as well as DNA platforms and was further able to generate multivalent chelator heads, which exhibit subnanomolar to picomolar affinities (super binder). Chemical induced dimerization was analyzed in contact with Tobias Lieblein (B6).

On the basis of these achievements, we will explore the newly established lock-and-key elements for the rapid fluorescence labeling of receptor proteins in living cells. The PhD student will work on the light-mediated, traceless modification of receptors as well as the site-directed conjugation of single-stranded DNA/PNA (finally caged strands) to cell surface receptors. The analysis of the traceless receptor modification will be performed

by live-cell and high-end fluorescence microscopy. To promote the light driven dimerization/oligomerization of receptors, real-time confocal laser scanning microscopy will be performed and the start of protein-protein interaction will be tested at different time points by illumination in live, intact cells. In addition, the extension of the UV-controlled assembly of proteins at functionalized interfaces (PEGylated SiO2, PLL-PEG surfaces) as well as three-dimensional scaffolds (e.g. DNA origami, hydrogels) will be explored to control protein assembly and ultimately receptor clustering at the molecular level. To gain even higher spatiotemporal resolution for site-specific protein trapping, optochemical tools will be designed, which can be photo-activated even by two-photon processes (in contact with A2.6).

 

 

B4.6

In the first period of CLiC, Andres Arrigada established the site directed installation of photoactivatable lock-and-key elements in various model systems (GFP, the ribosome recycling factor ABCE1 and ABC transporter TmrAB). By incorporation of unnatural amino acids and subsequent bioorthogonal click chemistry, he facilitated their equipment with multivalent chelator heads and light-sensitive moieties. The semi-synthetic proteins were biochemically investigated in detail (size exclusion chromatography, in gel fluorescence, etc.) and led to a better understanding of the design and incorporation of photoactivatable tags in proteins. He was able to induce protein dimerization in vitro as well as facilitated the reconstitution of membrane proteins into giant unilamellar vesicles for the control of their lateral organization (together with Karl Gatterdam). In teamwork with Alina Klein, first in-cell manipulation studies were performed. Efficient transfer of cell-impermeable multivalent chelator heads as well as light-sensitive moieties was established by cell squeezing, allowing us now to employ and incorporate a multitude of optochemical tools for intracellular applications such as super-resolution microscopy.

In the second term, we will extend the incorporation of unnatural amino acids in proteins. On the one hand, we will install photoactivatable amino acids into elected protein scaffolds to control the protein-protein interaction face upon illumination. On the other hand, light-activatable amino acids and tags will be developed. Their in vitro and in vivo incorporation into protein modifiers will be studied by state-of-the art biochemical techniques (size exclusion chromatography, in gel fluorescence, thermophoresis, high-resolution mass spectrometry, etc.). The PhD student will take part in the design and evaluation of protein scaffolds as well as the strategic introduction of amber stop mutants thereof. Performance tests of the developed photoactivatable protein modifiers will be undertaken first in vitro (surface plasmon resonance, thermophoresis, etc.) and subsequent in vivo (live-cell microscopy, real-time CLSM). Ultimately, the control of protein-protein interaction, localization and/or oligomerization in living cells will be analyzed with fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and single-particle tracking and, whenever possible, by super-resolution microscopy.