[Show abstract][Hide abstract] ABSTRACT: Single molecule applications, saturated pattern excitation microscopy, or
stimulated emission depletion (STED) microscopy demand for bright and highly
stable fluorescent dyes1,2. Despite of intensive research the choice of
fluorphores is still very limited. Typically a stable fluorescent dyes is
covalently attached to the target. This methodology brings forward a number of
limitations, in particular, in case of protein labeling. First of all the
fluorescent probes need to be attached selectively and site-specifically to
prevent unspecific background. This often requires single cysteine mutations
for covalent protein modification. Employing quantum dots allows overcoming
problems of photo-bleaching3-6. However, the downsides are their large size,
rendering the probe inaccessible to spatially confined architectures, issues in
biocompatibility due to proper particle coating, and cellular toxicity6-8. Here
we propose a new method to overcome the above outlined problems.
[Show abstract][Hide abstract] ABSTRACT: Single-molecule applications, saturated pattern excitation microscopy, and stimulated emission depletion (STED) microscopy demand bright as well as highly stable fluorescent dyes. Here we describe the synthesis of quantum-yield-optimized fluorophores for reversible, site-specific labeling of proteins or macromolecular complexes. We used polyproline-II (PPII) helices as sufficiently rigid spacers with various lengths to improve the fluorescence signals of a set of different trisNTA-fluorophores. The improved quantum yields were demonstrated by steady-state and fluorescence lifetime analyses. As a proof of principle, we characterized the trisNTA-PPII-fluorophores with respect to in vivo protein labeling and super-resolution imaging at synapses of living neurons. The distribution of His-tagged AMPA receptors (GluA1) in spatially restricted synaptic clefts was imaged by confocal and STED microscopy. The comparison of fluorescence intensity profiles revealed the superior resolution of STED microscopy. These results highlight the advantages of biocompatible and, in particular, small and photostable trisNTA-PPII-fluorophores in super-resolution microscopy.
Journal of the American Chemical Society 06/2011; 133(21):8090-3. · 10.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In medical technologies concerning the surface immobilization of proteins in a defined orientation, maintaining their activity is a critical aspect. Therefore, in this study, the authors have investigated the activity of an elongated protein attached to a self-assembled monolayer supported streptavidin layer for different relative orientations of the protein with regard to the surface. Several mutants of this protein, human guanylate-binding protein 1 (hGBP1) showing GTPase catalytic activity, have been furnished with either one or two biotin anchors. Various independent methods that are based on different biophysical properties such as surface plasmon resonance, atomic force microscopy, and quartz crystal microbalance have been used to determine the orientation of the hGBP1 variants after anchoring them via a streptavidin-linker to a biotinylated surface. The activity of guanosine-triphosphate hydrolysis of hGBP1 monomers bound on the surface is found to depend on their orientation relative to the substrate, relating to their ability to form dimers with other neighboring anchored mutants; the maximum activity is lower than that observed in solutions, as might be expected from diffusion limitations at the solid/liquid interface on the one hand and prevention from homodimer formation due to immobilization on the other hand.
[Show abstract][Hide abstract] ABSTRACT: We introduce a nanofabricated silicon chip for massively multiplexed analysis of membrane channels and transporters in suspended lipid membranes that does not require any surface modification or organic solvent. Transport processes through single membrane complexes are monitored by fluorescence. The chip consists of an array of well-defined nanopores, addressing an individual pyramidal back-reflecting 30-fL compartment. The setup allows simultaneous analyses of ∼1000 single transmembrane events in one field of view, observing translocation kinetics of transmembrane complexes.
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 03/2010; 107(14):6146-51. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Crystalline bacterial cell surface layers (S-layers) show the ability to recrystallize into highly regular pattern on solid supports. In this study, the genetically modified S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177, carrying a hexa-histidine tag (His(6)-tag) at the C-terminus, was used to generate functionalized two-dimensional nanoarrays on a silicon surface. Atomic force microscopy (AFM) was applied to explore the topography and the functionality of the fused His(6)-tags. The accessibility of the His(6)-tags was demonstrated by in-situ anti-His-tag antibody binding to the functional S-layer array. The metal binding properties of the His(6)-tag was investigated by single molecule force microscopy. For this purpose, newly developed tris-NTA was tethered to the AFM tips via a flexible polyethylene glycol (PEG) linker. The functionalized tips showed specific interactions with S-layer containing His(6)-tags in the presence of nickel ions. Thus the His(6)-tag is located at the outer surface of the S-layer and can be used for stable but reversible attachment of functional tris-NTA derivatives.
Journal of Structural Biology 03/2009; 168(1):217-22. · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this paper we present a modular approach for the fabrication of surfaces to characterize protein-protein interactions. The approach is based on azido peptides with an optimized sequence which are then thiol-functionalized using an alkynyl thiol and "click" chemistry. From these peptide thiols we fabricated SAMs on gold to evaluate the protein resistance, using surface plasmon resonance spectroscopy, toward streptavidin, bovin serum albumin (BSA), and fibronectin.
Journal of the American Chemical Society 11/2008; 130(45):14952-3. · 10.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The analytical performance of surface plasmon resonance imaging with charge coupled device detection can be improved significantly by splitting a macroscopic sensing surface into multiple microscopic neighboring sensing and referencing subareas. It is shown that such a multiple referencing reduces intensity fluctuations across the total sensing area and, therefore, improves the signal/noise (S/N) ratio proportional to the splitting factor. The approach is demonstrated by detection of biotin binding to a monolayer of streptavidin. An effective variation of the reflected intensity of about 10(-4), which corresponds to the refraction index variation of 3x10(-6), was detected with a S/N ratio about 10 without any temperature stabilization of the sensing area.
[Show abstract][Hide abstract] ABSTRACT: The adsorption of multiple protein layers on biotinylated organic surfaces has been characterized using surface plasmon resonance (SPR) and atomic force microscopy (AFM). Diffusion-limited loading of the biotinylated self-assembled monolayers (SAMs) ensures a precise control of the streptavidin surface density. For the subsequent interaction with biotinylated peroxidase, SPR data hint at a streptavidin density dependent orientation during peroxidase adsorption. Microcontact printed well-defined two-dimensional patterned surfaces of biotinylated organothiols and protein-resistant OEG-thiols allow an in-situ differentiation of specific and nonspecific adsorption (e.g., mono- vs multilayer adsorption). Additionally, the very important issue of biological activity of surface-bound enzymes is addressed by comparing the enzyme activities in solution with that for surface-bound species.
The Journal of Physical Chemistry A 01/2008; 111(49):12295-303. · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new imaging technique for high-throughput surface plasmon resonance (SPR) measurements is described. It is the application of a CCD camera for simultaneous processing of two images at two different wavelengths provided by two laser diodes. The two lasers are brought to resonance by tuning of the angle of incidence so that the detection power and the dynamic range are optimized for the wavelength pair selected. Applying a special differential processing of the two images, SPR measurements can be performed near the shot noise limit taking into account the number of CCD pixels involved. It is shown that the detection limit of imaging methods can be improved significantly if the working point is set near to the reflection minimum instead of choosing the angle with the steepest slope of the reflection curve. The technique is demonstrated by simultaneous measurement of hybridization reactions of three different types of thiolated oligonucleotides in 30 small areas set by a commercial spotter. A noise level of 1.5 x 10(-6) refractive index units (RIU) was obtained for single, 500 x 500 microm2 reaction areas. The noise level was about 6 x 10(-7) RIU when five areas were taken into account. The present arrangement and the particular spotter applied would allow simultaneous measurements of up to 400 binding reactions with a noise level of about 1.5 x 10(-6) RIU.
[Show abstract][Hide abstract] ABSTRACT: To establish a semiartificial device for (bio-)hydrogen production utilizing photosynthetic water oxidation, we report on the immobilization of a Photosystem 2 on electrode surfaces. For this purpose, an isolated Photosystem 2 with a genetically introduced His tag from the cyanobacterium Thermosynechococcus elongatus was attached onto gold electrodes modified with thiolates bearing terminal Ni(II)-nitrilotriacetic acid groups. Surface enhanced infrared absorption spectroscopy showed the binding kinetics of Photosystem 2, whereas surface plasmon resonance measurements allowed the amount of protein adsorbed to be quantified. On the basis of these data, the surface coverage was calculated to be 0.29 pmol protein cm(-2), which is in agreement with the formation of a monomolecular film on the electrode surface. Upon illumination, the generation of a photocurrent was observed with current densities of up to 14 microA cm(-2) . This photocurrent is clearly dependent on light quality showing an action spectrum similar to an isolated Photosystem 2. The achieved current densities are equivalent to the highest reported oxygen evolution activities in solution under comparable conditions.
Photochemistry and Photobiology 01/2006; 82(5):1385-90. · 2.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The control of unspecific adsorption of proteins to natural and technical surfaces plays an important role in biology and also for many applications. Organic model surfaces, e.g., self-assembled monolayers, are often used to identify fundamental surface and/or protein properties that rule protein adsorption. Some techniques involved in biointerface research require the use of heavy water, e.g. neutron scattering techniques. Also in NMR studies D(2)O is the solvent of choice when focusing on biomolecular and hydration dynamics. So far several studies have been concerned with the characterization of the unspecific adsorption of proteins from normal water buffers. In the present work, we report a comparison of the unspecific protein adsorption from normal and heavy water buffers. So far it has been assumed that the surface kinetic of the unspecific adsorption is unaffected by the substitution of water by D(2)O. However, for the four proteins investigated here, this assumption does not hold. The ratio k(H)/k(D) of the adsorption rate constants of the different buffer conditions describe the strength of the isotope effect. We have measured ratios between 1.0 and 2.6, indicating that the adsorption kinetics are strongly affected by a H(2)O-D(2)O substitution.
[Show abstract][Hide abstract] ABSTRACT: A new technique for on-line monitoring of analyte binding to sensor surfaces by surface plasmon resonance (SPR) detection is described. It is based on differential measurements using two wavelengths provided by two diode lasers. The technique is as simple and robust as the conventional SPR detection measuring the reflected radiation at fixed incidence angle, but it has the advantage of being nonsensitive to variations of the resonance width and providing essentially higher signal/noise ratios. The paper presents the first four channel prototype system for parallel 2D-monitoring at four different spots. One channel is always used as a reference to compensate temperature fluctuations and nonspecific adsorptions. Calibration with sucrose solutions revealed an absolute sensitivity of Deltan approximately 5 x 10(-6). The new technique is tested with a biotin-streptavidin binding and with hybridization/denaturation of DNA. Biotin binding to a streptavidin monolayer is detected with a signal/noise ratio of about 5, which demonstrates the high potential of the new technique for applications in drug discovery. Applications to gene analysis are tested with short oligonucleotides of the sequences used for genotyping human hepatitis C viruses. A selective response to complementary oligonucleotides is observed. The high reproducibility in subsequent cycles of hybridization/denaturation (by formamide or by heating) points out potential applications of the technique in medical diagnostics, food industry, genomics, and proteomics too.