Annett Reichel

In situ assembly of macromolecular complexes triggered by light

Chemical biology aims for a perfect control of protein complexes in time and space by their site-specific labeling, manipulation, and structured organization. Here we developed a self-inactivated, lock-and-key recognition element whose binding to His-tagged proteins can be triggered by light from zero to nanomolar affinity. Activation is achieved by photocleavage of a tethered intramolecular ligand arming a multivalent chelator head for high-affinity protein interaction. We demonstrate site-specific, stable, and reversible binding in solution as well as at interfaces controlled by light with high temporal and spatial resolution. Multiplexed organization of protein complexes is realized by an iterative in situ writing and binding process via laser scanning microscopy. This light-triggered molecular recognition should allow for a spatiotemporal control of protein-protein interactions and cellular processes by light-triggered protein clustering.

Functional Immobilization and Patterning of Proteins by an Enzymatic Transfer Reaction

Functional immobilization and lateral organization of proteins into micro- and nanopatterns is an important prerequisite for miniaturizing bioanalytical and biotechnological devices. Here, we report an approach for efficient site-specific protein immobilization based on enzymatic phosphopantetheinyl transfer (PPT) from coenzyme A (CoA)-functionalized glass-type surfaces to specific peptide tags. We devised a bottom-up surface modification approach for coupling CoA densely to a molecular poly(ethylene glycol) polymer brush. Site-specific enzymatic immobilization of proteins fused to different target peptides for the PPTase Sfp was confirmed by real-time label-free detection. Quantitative protein-protein interaction experiments confirmed that significantly more than 50% of the immobilized protein was fully active. The method was successfully applied with different proteins. However, different immobilization efficiencies of PPT-based immobilization were observed for different peptide tags being fused to the N- and C-termini of proteins. On the basis of this immobilization method, we established photolithographic patterning of proteins into functional binary microstructures.

Self-Assembled Monolayers Containing Terminal Mono-, Bis-, and Tris-nitrilotriacetic Acid Groups: Characterization and Application

We have undertaken a structural and functional study of self-assembled monolayers (SAMs) formed on gold from a series of alkylthiol compounds containing terminal multivalent chelators (MCHs) composed of mono-, bis-, and tris-nitrilotriacetic acid (NTA) moieties. SAMs were formed from single-component solutions of the mono-, bis-, and tris-NTA compounds, as well as from mixtures with a tri(ethylene glycol)-terminated alkylthiol (EG(3)). Contact angle goniometry, null ellipsometry, and infrared spectroscopy were used to explore the structural characteristics of the MCH SAMs. Ellipsometric measurements show that the amount of the MCH groups on surfaces increases with increasing mol % of the MCH thiols in the loading solution up to about 80 mol %. We also conclude that mixed SAMs, prepared in the solution composition regime 0-30 mol % of the MCH thiols, consist of a densely packed alkyl layer, an amorphous ethylene glycol layer, and an outermost layer of MCH groups exposed toward the ambient. Above 30 mol %, a significant degree of disorder is observed in the SAMs. Finally, functional evaluation of the three MCH SAMs prepared at 0-30 mol% reveals a consistent increase in binding strength with increasing multivalency. The tris-NTA SAM, in particular, is enabled for stable and functional immobilization of a His6-tagged extracellular receptor subunit, even at low chelator surface concentrations, which makes it suitable for applications when a low surface density of capturing sites is desirable, e.g., in kinetic analyses.

Protein-Protein Interactions in Reversibly Assembled Nanopatterns

We describe herein a platform to study protein-protein interactions and to form functional protein complexes in nanoscopic surface domains. For this purpose, we employed multivalent chelator (MCh) templates, which were fabricated in a stepwise procedure combining dip-pen nanolithography (DPN) and molecular recognition-directed assembly. First, we demonstrated that an atomic force microscope (AFM) tip inked with an oligo(ethylene glycol) (OEG) disulfide compound bearing terminal biotin groups can be used to generate biotin patterns on gold achieving line widths below 100 nm, a generic platform for fabrication of functional nanostructures via the highly specific biotin-streptavidin recognition. Subsequently, we converted such biotin/streptavidin patterns into functional MCh patterns for reversible assembly of histidine-tagged (His-tagged) proteins via the attachment of a tris-nitriloacetic acid (trisNTA) biotin derivative. Fluorescence microscopy confirmed reversible immobilization of the receptor subunit ifnar2-His10 and its interaction with interferon-alpha2 labeled with fluorescent quantum dots in a 7 x 7 dot array consisting of trisNTA spots with a diameter of approximately 230 nm. Moreover, we carried out characterization of the specificity, stability, and reversibility as well as quantitative real-time analysis of protein-protein interactions at the fabricated nanopatterns by imaging surface plasmon resonance. Our work offers a route for construction and analysis of functional protein-based nanoarchitectures.

Organization of Motor Proteins into Functional Micropatterns Fabricated by a Photoinduced Fenton Reaction

The functional organization of proteins on solid supports is a key prerequisite for the integration of the powerful capabilities of biomolecules into miniaturized biomedical and biotechnological devices. Motor proteins are particularly attractive building blocks for the construction of such devices. Numerous approaches have been reported for the organization of motor proteins into functional microand nanostructures, which have inspired the development of novel bioanalytical devices. For these purposes, techniques for the functional organization of proteins on surfaces into micrometerand submicrometer-sized assemblies are required. Despite substantial developments in this field, simple and generic techniques for functional protein patterning are scarce. A critical prerequisite is that the functionality of proteins immobilized on the surface must be fully maintained. As many proteins denature upon interaction with solid supports, surface modifications, for example, in the form of thin protein-repellent polymer layers, are required to render the surface biocompatible. Moreover, suitable, spatially resolved functionalization of these layers is required for the site-specific capturing of target molecules onto the surface. We previously developed multivalent head groups containing nitrilotriacetic acid (NTA) moieties (such as tris-NTA, Figure 1a) as generic, high-affinity adapters for oligohistidine-tagged proteins. These multivalent chelators have proven powerful for stable, yet reversible protein binding in solution and for immobilization onto various supports. Through the use of such chelators in combination with a dense poly(ethylene glycol) (PEG) polymer brush, we demonstrated the oriented capturing of highly active kinesin on glass surfaces. Herein, we present a generic method for the functional micropatterning of such surface architectures. This method is based on selective photodestruction by a lightinduced Fenton reaction (Figure 1b), whereby suitable transition-metal ions are complexed by the immobilized NTA moieties. For the implementation of this approach, we used both UV illumination through a mask and the UV laser of a standard confocal microscope. The principle of the first technique is depicted schematically in Figure 1c: After loading of the NTA moieties with Co ions, the surface is illuminated with UV light through a mask. All metal ions are then removed by washing with HCl or ethylenediaminetetraacetic acid (EDTA), and the remaining NTA groups are loaded with Ni ions prior to protein binding. The protein-binding efficiencies of tris-NTA-functionalized surfaces after UV illumination with the NTA moieties loaded with different transition-metal ions are compared in Figure 1b. Illumination in the presence of Ni ions did not affect the binding capacity of the surface; in contrast, no protein binding was observed after illumination of surfaces loaded with Co, Cu, or Fe ions. These three transitionmetal ions mediate photoinduced Fenton reactions, which are probably responsible for the destruction of the NTA moieties on the surface. The same effect was observed for surfaces functionalized with mono-NTA. A decrease in the efficiency of photodestruction on surfaces was observed when the length of the PEG chain was decreased (see the Supporting Information). Thus, the surface architecture has some influence on the destruction process. To further characterize the photodestruction process, we quantitatively assessed protein binding to surfaces after illumination for shorter periods of time than required for full destruction of the binding capacity. The unstable protein binding observed under these conditions (see the Supporting Information) indicated that the tris-NTA groups were partially destroyed and thus lost binding affinity. Moreover, leaching of Ni ions was observed, which indicated that the NTA moieties themselves were decomposed by the Fenton reaction. This result is in line with studies carried out on the hydroxyl-radical-mediated oxidation of chelating agents such as NTA and EDTA. The oxidation of NTA yielded species with a weaker metal-ion-coordination ability, such as imidodiacetic acid, glycolic acid, oxalic acid, and glycine. Since the active oxidant in the Fenton reaction is also a hydroxyl radical, a similar oxidation pathway can be assumed. Thus, the metal-ion-mediated photodestruction appears to selectively eliminate the transition-metal-ion-binding moieties, but not the protein-repelling PEG polymer brush. This conclusion is [*] M. Bhagawati, A. Reichel, Prof. Dr. J. Piehler Institut f r Biophysik, Universit t Osnabr ck Barbarastrasse 11, 49076 Osnabr ck (Germany) Fax: (+49)541-9692262 E-mail: piehler@uos.de Homepage: http://www.biologie.uni-osnabrueck.de/Biophysik/ Piehler/ Dr. S. Ghosh, Dr. T. Surrey Cell Biology and Biophysics Unit, EMBL Heidelberg (Germany)

Accelerating phage-display library selection by reversible and site-specific biotinylation

Immobilization of a target molecule to a solid support is an indispensable step in phage display library sorting. Here we describe an immobilization method that addresses shortcomings of existing strategies. Our method is based on the use of a polyhistidine-tagged (His-tagged) target molecule and (BT)tris-NTA, a high-affinity capture reagent for His-tags that also contains a biotin moiety. (BT)tris-NTA provides a stable and reversible linkage between a His-tag and a streptavidin-coated solid support. Because His-tags are the de facto standard for recombinant protein purification, this method dramatically simplifies target preparation for phage display library sorting. Here, we demonstrate the utility of this method by selecting high-affinity binding proteins based on the fibronectin type III (FN3) scaffold to two His-tagged protein targets, yeast small ubiquitin-like modifier and maltose-binding protein. Notably, a significant number of FN3 clones binding either targets selected using the new immobilization method exhibited only very weak binding when the same target was immobilized by coating on a polystyrene surface. This suggests that the His-tag-mediated immobilization exposes epitopes that are masked by commonly used passive adsorption methods. Together, these results establish a method with the potential to streamline and enhance many binding-protein engineering experiments.

Conformation of receptor adopted upon interaction with virus revealed by site-specific fluorescence quenchers and FRET analysis.

Human rhinovirus serotype 2 (HRV2) specifically binds to very-low-density lipoprotein receptor (VLDLR). Among the eight extracellular repeats of VLDLR, the third module (V3) has the highest affinity for the virus, and 12 copies of the genetically engineered concatamer V33333-His(6) were found to bind per virus particle. In the present study, ring formation of V33333-His(6) about each of the 12 5-fold symmetry axes on HRV2 was demonstrated by fluorescence resonance energy transfer (FRET) between donor and acceptor on N- and C-terminus, respectively. In particular, the N-terminus of V33333-His(6) was labeled with fluorescein, and the C-terminus with a new quencher which was bound to the His(6) tag with nanomolar affinity (K(d) approximately 10(-8) M) in the presence of 2 microM NiCl(2).

Probing Protein Conformations by in Situ Non-Covalent Fluorescence Labeling

The conformational dynamics of proteins plays a key role in their complex physiological functions. Fluorescence resonance energy transfer (FRET) is a particular powerful tool for studying protein conformational dynamics, but requires efficient site-specific labeling with fluorescent reporter probes. We have employed different tris-NTA/fluorophore conjugates, which bind histidine-tagged proteins with high affinity, for site-specific incorporation of FRET acceptors into proteins, which were covalently labeled with a donor fluorophore. We demonstrate versatile application of this approach for exploring the conformation of the type I interferon receptor ectodomains ifnar1-EC and ifnar2-EC. Substantial ligand-induced conformational changes of ifnar1-EC, but not ifnar2-EC, were observed by monitoring the fluorescence intensity and the fluorescence lifetime of the FRET donor. Time-resolved fluorescence correlation spectroscopy revealed a substantial conformational flexibility of ifnar1-EC and a ligand-induced tightening. Our results demonstrate that protein labeling with tris-NTA/fluorophores enables for efficient quantitative intramolecular FRET analysis.

Selectivity of competitive multivalent interactions at interfaces.

The development of synthetic, low‐molecular‐weight ligand receptor systems for the selective control of biomolecular interactions remains a major challenge. Binding of oligohistidine peptides to chelators containing Ni2+‐loaded nitrilotriacetic acid (NTA) moieties is one of the most widely used and best‐characterised recognition systems. Recognition units containing multiple NTA moieties (multivalent chelator headgroups, MCHs) recognise oligohistidines with substantially increased binding affinities. Different multivalencies both at the level of the MCH and at that of the oligohistidine ligand provide a powerful means to vary the affinity of the interaction systematically. Here we have explored the selectivity for the binding of different oligohistidines to immobilised MCH. Using microarrays of mono‐, bis‐, tris‐ and tetrakis‐NTA chelators spotted at different surface densities, we explored the ability of these binders to discriminate fluorescently labelled hexa‐ and decahistidine peptides. When hexa‐ and decahistidine were tested alone, the discrimination of ligands showed little dependence either on the nature or on the density of the chelator. In contrast, coincubation of both peptides decreased the affinity of hexahistidine, increased the affinity of decahistidine, and made the binding of decahistidine highly dependent on MCH density. Kinetic binding assays by dual‐colour total internal reflection fluorescence spectroscopy revealed active exchange of His6 by His10 and confirmed the high selectivity towards His10. Our results establish the key role of surface multivalency for the selectivity of multivalent interactions at interfaces.