Maniraj Bhagawati

Affinity capturing for targeting proteins into micro and nanostructures

Protein immobilization into micro and nanoscaled patterns opens exciting possibilities in fundamental and applied research. Developing efficient capturing techniques while preserving the structural and functional integrity of the proteins on surfaces is a key challenge for surface scientists. In this paper, current techniques for site-specific protein immobilization into engineered surface architectures are reviewed. Fundamental principles for functional protein immobilization on solid supports are discussed and popular affinity-based recognition pairs and their application for capturing proteins into nano and microstructures are presented.

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.

Stimuli-sensitive intrinsically disordered protein brushes

Grafting polymers onto surfaces at high density to yield polymer brush coatings is a widely employed strategy to reduce biofouling and interfacial friction. These brushes almost universally feature synthetic polymers, which are often heterogeneous and do not readily allow incorporation of chemical functionalities at precise sites along the constituent chains. To complement these synthetic systems, we introduce a biomimetic, recombinant intrinsically disordered protein that can assemble into an environment-sensitive brush. This macromolecule adopts an extended conformation and can be grafted to solid supports to form oriented protein brushes that swell and collapse dramatically with changes in solution pH and ionic strength. We illustrate the value of sequence specificity by using proteases with mutually orthogonal recognition sites to modulate brush height in situ to predictable values. This study demonstrates that stimuli-responsive brushes can be fabricated from proteins and introduces them as a new class of smart biomaterial building blocks.

Native Laser Lithography of His-Tagged Proteins by Uncaging of Multivalent Chelators

We report a generic approach for targeting proteins into micropatterns by in situ laser lithography. To this end, we have designed a photocleavable oligohistidine peptide for caging tris(nitrilo triacetic acid) (tris-NTA) groups on surfaces by multivalent interactions. Local photofragmentation of the peptide by UV illumination through a photomask or by a confocal laser beam uncages tris-NTA, thus generating free binding sites for rapid, site-specific capturing of His-tagged proteins into micropatterns. Iterative writing of proteins by laser lithography enabled for assembly of multiplexed functional protein microstructures on surfaces. Thus, versatile, user-defined protein micropatterns can be assembled under physiological conditions with a standard confocal laser-scanning microscope.

Modulating Surface Density of Proteins via Caged Surfaces and Controlled Light Exposure

We demonstrate the possibility of tuning the degree of functionalization of a surface using photoactivatable chemistries and controlled light exposure. A photosensitive organosilane with a protected amine terminal group and a tetraethyleneglycol spacer was synthesized. A o-nitrobenzyl cage was used as the photoremovable group to cage the amine functionality. Surfaces with phototunable amine densities were generated by controlled irradiation of silica substrates modified with the photosensitive anchor. Protein layers with different densities could be obtained by successive coupling and assembly steps. Protein surface concentrations were quantified by reflectance interference. Our results demonstrate that the protein density correlates with the photogenerated ligand density. The density control was proved over four coupling steps (biotin, SAv, (BT)tris-NTA, MBP, or GFP), indicating that the interactions between underlying layer and soluble targets are highly specific and the immobilized targets at the four levels maintain their full functionality. Protein micropatterns with a gradient of protein density were also obtained.

Maleimide Photolithography for Single-Molecule Protein-Protein Interaction Analysis in Micropatterns

Spatial organization of proteins into microscopic structures has important applications in fundamental and applied research. Preserving the function of proteins in such microstructures requires generic methods for site-specific capturing through affinity handles. Here, we present a versatile bottom-up surface micropatterning approach based on surface functionalization with maleimides, which selectively react with organic thiols. Upon UV irradiation through a photomask, the functionality of illuminated maleimide groups was efficiently destroyed. Remaining maleimides in nonilluminated regions were further reacted with different thiol-functionalized groups for site-specific protein immobilization under physiological conditions. Highly selective immobilization of His-tagged proteins into tris(nitrilotriacetic acid) functionalized microstructures with very high contrast was possible even by direct capturing of proteins from crude cell lysates. Moreover, we employed phosphopantetheinyl transfer from surface-immobilized coenzyme A to ybbR-tagged proteins in order to implement site-specific, covalent protein immobilization into microstructures. The functional integrity of the immobilized protein was confirmed by monitoring protein-protein interactions in real time. Moreover, we demonstrate quantitative single-molecule analysis of protein-protein interactions with proteins selectively captured into these high-contrast micropatterns.

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)

Single Cell GFP-Trap Reveals Stoichiometry and Dynamics of Cytosolic Protein Complexes

We developed in situ single cell pull-down (SiCPull) of GFP-tagged protein complexes based on micropatterned functionalized surface architectures. Cells cultured on these supports are lysed by mild detergents and protein complexes captured to the surface are probed in situ by total internal reflection fluorescence microscopy. Using SiCPull, we quantitatively mapped the lifetimes of various signal transducer and activator of transcription complexes by monitoring dissociation from the surface and defined their stoichiometry on the single molecule level.

Quantitative real-time imaging of protein-protein interactions by LSPR detection with micropatterned gold nanoparticles.

Localized surface plasmon resonance (LSPR) offers powerful means for sensitive label-free detection of protein-protein interactions in a highly multiplexed format. We have here established self-assembly and surface modification of plasmonic nanostructures on solid support suitable for quantitative protein-protein interaction analysis by spectroscopic and microscopic LSPR detection. These architectures were obtained by layer-by-layer assembly via electrostatic attraction. Gold nanoparticles (AuNP) were adsorbed on a biocompatible amine-terminated poly(ethylene glycol) (PEG) polymer brush and further functionalized by poly-l-lysine graft PEG (PLL-PEG) copolymers. Stable yet reversible protein immobilization was achieved via tris(nitrilotriacetic acid) groups incorporated into the PLL-PEG coating. Thus, site-specific immobilization of His-tagged proteins via complexed Ni(II) ions was achieved. Functional protein immobilization on the surface was confirmed by real-time detection of LSPR scattering by reflectance spectroscopy. Association and dissociation rate constants obtained for a reversible protein-protein interaction were in good agreement with the data obtained by other surface-sensitive detection techniques. For spatially resolved detection, AuNP were assembled into micropatterns by means of photolithographic uncaging of surface amines. LSPR imaging of reversible protein-protein interactions was possible in a conventional wide field microscope, yielding detection limits of ∼30 protein molecules within a diffraction-limited surface area.

A Versatile Toolbox for Multiplexed Protein Micropatterning by Laser Lithography

Photocleavable oligohistidine peptides (POHP) allow in situ spatial organization of multiple His-tagged proteins onto surfaces functionalized with tris(nitrilotriacetic acid) (tris-NTA). Here, a second generation of POHPs is presented with improved photoresponse and site-specific covalent coupling is introduced for generating stable protein assemblies. POHPs with different numbers of histidine residues and a photocleavable linker based on the 4,5-dimethoxy-o-nitrophenyl ethyl chromophore are prepared. These peptides show better photosensitivity than the previously used o-nitrophenyl ethyl derivative. Efficient and stable caging of tris-NTA-functionalized surfaces by POHPs comprising 12 histidine residues is demonstrated by multiparameter solid-phase detection techniques. Laser lithographic uncaging by photofragmentation of the POHPs is possible with substantially reduced photodamage as compared to previous approaches. Thus, in situ micropatterning of His-tagged proteins under physiological conditions is demonstrated for the first time. In combination with a short peptide tag for a site-specific enzymatic coupling reaction, covalent immobilization of multiple proteins into target micropatterns is possible under physiological conditions.