Christian Grunwald

Goethe-Universität Frankfurt am Main, Frankfurt am Main, Hesse, Germany

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Publications (20)130.5 Total impact

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    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. DOI:10.1021/ja200967z · 11.44 Impact Factor
  • Ruoshan Wei, Christian Grunwald, Robert Tampé, Ulrich Rant
    Biophysical Journal 02/2011; 100(3). DOI:10.1016/j.bpj.2010.12.1145 · 3.83 Impact Factor
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    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.
    Biointerphases 12/2010; 5(4):131-8. DOI:10.1116/1.3516461 · 1.91 Impact Factor
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    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.
    Nano Letters 10/2010; 10(12). DOI:10.1021/nl1033528 · 13.03 Impact Factor
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    ABSTRACT: Nanopatterning of biomolecules on functionalized surfaces offers an excellent route for ultrasensitive protein immobilization, for interaction measurements, and for the fabrication of devices such as protein nanoarrays. An improved understanding of the physics and chemistry underlying the device properties and the recognition process is necessary for performance optimization. This is especially important for the recognition and immobilization of intrinsically disordered proteins (IDPs), like the prion protein (PrP), a partial IDP, whose folding and stability may be influenced by local environment and confinement. Atomic force microscopy allows for both highly controllable nanolithography and for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of protein interactions in a liquid environment, thus different environmental parameters affecting the biorecognition phenomenon can be investigated in situ. Using nanografting, a tip-induced lithographic technique, and an affinity immobilization strategy based on two different histidine tagged antibodies, with high nM affinity for two different regions of PrP, we successfully demonstrated the immobilization of recombinant mouse PrP onto nanostructured surfaces, in two different orientations. Clear discrimination of the two molecular orientations was shown by differential height (i.e., topographic) measurements, allowing for the estimation of binding parameters and the full characterization of the nanoscale biorecognition process. Our work opens the way to several high sensitivity diagnostic applications and, by controlling PrP orientation, allows for the investigation of unconventional interactions with partially folded proteins, and may serve as a platform for protein misfolding and refolding studies on PrP and other thermodynamically unstable, fibril forming, proteins.
    ACS Nano 10/2010; 4(11):6607-16. DOI:10.1021/nn101872w · 12.03 Impact Factor
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    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. DOI:10.1073/pnas.0912617107 · 9.81 Impact Factor
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    ABSTRACT: We demonstrate that scattering scanning near-field infrared microscopy (s-SNIM) is a label free analytical method allowing hybridization detection of nanografted DNA patches on a sub-mu M scale. Oil the basis of their distinct dielectric properties in the IR. we can distinguish between single stranded and double stranded DNA. The sensitivity of s-SNIM is found to be increased by 7 orders of magnitude compared to conventional FTIR spectroscopy due to the tip enhanced interaction with the Substrate.
    The Journal of Physical Chemistry C 01/2010; 114(2-2):1306-1311. DOI:10.1021/jp906813f · 4.84 Impact Factor
  • Biophysical Journal 01/2010; 98(3). DOI:10.1016/j.bpj.2009.12.3585 · 3.83 Impact Factor
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    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. DOI:10.1016/j.jsb.2009.02.003 · 3.37 Impact Factor
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    ABSTRACT: To understand better enzyme/DNA interactions and to design innovative detectors based on DNA nanoarrays, we need to study the effect of nanometric confinement on the biochemical activity of the DNA molecules. We focus on the study of the restriction enzyme reactions (DpnII) within DNA nanostructures on flat gold films by atomic force microscopy (AFM). Typically we work with a few patches of DNA self assembled monolayers (SAMs) that are hundred nm in size and are lithographically fabricated within alkylthiol SAMs by AFM nanografting. We start by nanografting a few patches of a single-stranded DNA (ssDNA) molecule of 44 base pairs (bps) with a 4 bps recognition sequence (specific for DpnII) in the middle. Afterwards, reaction-ready DNA nanopatches are obtained by hybridization with a complementary 44bps ssDNA sequence. The enzymatic reactions were carried out over nanopatches with different density. By carrying out AFM height measurements, we are able to show that the capability of the DpnII enzyme to reach and react at the recognition site is easily varied by controlling the DNA packing in the nanostructures. We have found strong evidence that inside our ordered DNA nanostructures the enzyme (that works as a dimer) can operate down to the limit in which the space between adjacent DNA molecules is equal to the size of the DNA/enzyme complex. Similar experiments were carried out with a DNA sequence without the recognition site, clearly finding that in that case the enzymatic reaction did not lead to digestion of the molecules. These findings suggest that it is possible to tune the efficiency of an enzymatic reaction on a surface by controlling the steric hindrance inside the DNA nanopatches without vary any further physical or chemical variable. These findings are opening the door to novel applications in both the fields of biosensing and fundamental biophysics.
    Nano Letters 01/2009; 8(12):4140-5. DOI:10.1021/nl802370g · 12.94 Impact Factor
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    ABSTRACT: We demonstrate that, contrary to current understanding, the density of probe molecules is not responsible for the lack of hybridization in high density single-stranded DNA (ss-DNA) self-assembled monolayers (SAMs). To this end, we use nanografting to fabricate well packed ss-DNA nanopatches within a "carpet matrix" SAM of inert thiols on gold surfaces. The DNA surface density is varied by changing the "writing" parameters, for example, tip speed, and number of scan lines. Since ss-DNA is 50 times more flexible than ds-DNA, hybridization leads to a transition to a "standing up" phase. Therefore, accurate height and compressibility measurements of the nanopatches before and after hybridization allow reliable, sensitive, and label-free detection of hybridization. Side-by-side comparison of self-assembled and nanografted DNA-monolayers shows that the latter, while denser than the former, display higher hybridization efficiencies.
    Nano Letters 01/2009; 8(12):4134-9. DOI:10.1021/nl802722k · 12.94 Impact Factor
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    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. DOI:10.1021/ja8065754 · 11.44 Impact Factor
  • Christian Grunwald
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    ABSTRACT: This review presents the molecular basis of the high affinity between (strept)avidin and biotin as it was discovered from different protein crystal structures using wild type and mutant streptavidin. Optimization strategies for further improving the applicability of the (strept)avidin-biotin system and prospects for modulating the affinity arc discussed. The characterization and the application of the streptavidin-biotin system in surface-based biosensing assays are demonstrated with selected examples focussing on Surface plasmon resonance (SPR) and atomic force microscopy (AFM). Recent trends to further enhance the utility of convential SPR e.g. parallel detection of biological molecules and sensitivity enhancement towards small molecules are covered as well.
    Zeitschrift für Physikalische Chemie 05/2008; 222(5-6):789-821. DOI:10.1524/zpch.2008.6009 · 1.18 Impact Factor
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    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.
    Review of Scientific Instruments 03/2008; 79(2 Pt 1):023110. DOI:10.1063/1.2888527 · 1.58 Impact Factor
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    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. DOI:10.1021/jp074847u · 2.78 Impact Factor
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    ABSTRACT: We report on the infrared spectroscopic characterization of microstructured self-assembled monolayers by scanning near-field infrared microscopy. Using a CO laser as the radiation source in the characteristic amide band (at 1711 cm(-1) (lambda = 5.85 mu m)), we were able to image self-assembled monolayers of thiols. The measurements were carried out on well-defined microcontact printed line patterns of monomolecular films of 1-octadecanethiolate and biotinylated alkylthiolate (BAT). The lateral resolution was similar to 90 x 90 nm(2), which is well below the Abbe limit. The detection limit was 5 x 10(-20) mol corresponding to 27 attogram or 30 000 molecules of BAT. This demonstrates the high sensitivity of our setup, which allows recording IR spectra of a single monolayer.
    The Journal of Physical Chemistry C 06/2007; 111(23-23):8166-8171. DOI:10.1021/jp070201q · 4.84 Impact Factor
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    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.
    Analytical Chemistry 02/2007; 79(2):702-9. DOI:10.1021/ac061623j · 5.83 Impact Factor
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    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. DOI:10.1562/2006-07-14-RC-969 · 2.68 Impact Factor
  • Christian Grunwald, Jürgen Kuhlmann, Christof Wöll
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    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.
    Langmuir 10/2005; 21(20):9017-9. DOI:10.1021/la0515930 · 4.38 Impact Factor
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    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.
    Analytical Chemistry 05/2005; 77(8):2393-9. DOI:10.1021/ac048156v · 5.83 Impact Factor

Publication Stats

357 Citations
130.50 Total Impact Points

Institutions

  • 2009–2011
    • Goethe-Universität Frankfurt am Main
      • Institut für Biochemie
      Frankfurt am Main, Hesse, Germany
  • 2008
    • Max Planck Institute of Molecular Physiology
      Dortmund, North Rhine-Westphalia, Germany
  • 2006–2008
    • Ruhr-Universität Bochum
      • • Institut für Physiologische Chemie
      • • Fachbereich Physikalische Chemie
      Bochum, North Rhine-Westphalia, Germany
  • 2007
    • Technische Universität Dortmund
      • Leibniz Institute for Analytical Sciences (ISAS)
      Dortmund, North Rhine-Westphalia, Germany