Bernhard H Geierstanger

Genomics Institute of the Novartis Research Foundation, San Diego, California, United States

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Publications (61)444 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: To expand the utility of proteinaceous FRET biosensors, we have developed a dual-labeling approach based on two small bio-orthogonal tags: pyrroline-carboxy-lysine (Pcl) and the S6 peptide. The lack of cross-reactivity between those tags enables site-specific two-color protein conjugation in a one-pot reaction. Moreover, Pcl/S6 dual-tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE-Fc, respectively. Both proteins could be efficiently dual-labeled with FRET-compatible fluorescent dyes at neutral pH. In the case of IgE-Fc, the resulting conjugate enabled the monitoring of IgE binding to its high-affinity receptor FcεRI, which is a key event in allergic disease.
    ChemBioChem 07/2014; 12(12). DOI:10.1002/cbic.201402204 · 3.09 Impact Factor
  • Hsien-Po Chiu · Jan Grünewald · Xueshi Hao · Ansgar Brock · Linda Okach · Tetsuo Uno · Bernhard H Geierstanger ·
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    ABSTRACT: Sticky residue: Pyrroline-carboxy-lysine (Pcl) can be readily incorporated into proteins expressed in E. coli and mammalian cells by using the pyrrolysyl tRNA/tRNA synthetase pair. Pcl can be used as a single amino acid purification tag and can be site-specifically modified with functional probes during the elution process.
    ChemBioChem 02/2012; 13(3):364-6. DOI:10.1002/cbic.201100684 · 3.09 Impact Factor
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    ABSTRACT: Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F(420) deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F(420) and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.
    Structure 01/2012; 20(1):101-12. DOI:10.1016/j.str.2011.11.001 · 5.62 Impact Factor
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    ABSTRACT: The site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO(2)Phe) into autologous proteins overcomes self-tolerance and induces a long-lasting polyclonal IgG antibody response. To determine the molecular mechanism by which such simple modifications to amino acids are able to induce autoantibodies, we incorporated pNO(2)Phe, sulfotyrosine (SO(3)Tyr), and 3-nitrotyrosine (3NO(2)Tyr) at specific sites in murine TNF-α and EGF. A subset of TNF-α and EGF mutants with these nitrated or sulfated residues is highly immunogenic and induces antibodies against the unaltered native protein. Analysis of the immune response to the TNF-α mutants in different strains of mice that are congenic for the H-2 locus indicates that CD4 T-cell recognition is necessary for autoantibody production. IFN-γ ELISPOT analysis of CD4 T cells isolated from vaccinated mice demonstrates that peptides with mutated residues, but not the wild-type residues, are recognized. Immunization of these peptides revealed that a CD4 repertoire exists for the mutated peptides but is lacking for the wild-type peptides and that the mutated residues are processed, loaded, and presented on the I-A(b) molecule. Overall, our results illustrate that, although autoantibodies are generated against the endogenous protein, CD4 cells are activated through a neo-epitope recognition mechanism. Therefore, tolerance is maintained at a CD4 level but is broken at the level of antibody production. Finally, these results suggest that naturally occurring posttranslational modifications such as nitration may play a role in antibody-mediated autoimmune disorders.
    Proceedings of the National Academy of Sciences 08/2011; 108(31):12821-6. DOI:10.1073/pnas.1110042108 · 9.67 Impact Factor
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    ABSTRACT: Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pcl is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. Here, we describe a broadly applicable conjugation chemistry that is specific for Pcl and orthogonal to all other reactive groups on proteins. The reaction of Pcl with 2-amino-benzaldehyde or 2-amino-acetophenone reagents proceeds to near completion at neutral pH with high efficiency. We illustrate the versatility of the chemistry by conjugating Pcl proteins with poly(ethylene glycol)s, peptides, oligosaccharides, oligonucleotides, fluorescence, and biotin labels and other small molecules. Because Pcl is genetically encoded by TAG codons, this conjugation chemistry enables enhancements of the pharmacology and functionality of proteins through site-specific conjugation.
    Proceedings of the National Academy of Sciences 06/2011; 108(26):10437-42. DOI:10.1073/pnas.1105197108 · 9.67 Impact Factor
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    ABSTRACT: D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and biochemical studies point to specific roles of the poorly understood Pyl biosynthetic enzymes PylC and PylD in converting L-lysine and D-ornithine to Pcl and confirm intermediates in the biosynthesis of Pyl.
    Nature Chemical Biology 04/2011; 7(8):528-30. DOI:10.1038/nchembio.586 · 13.00 Impact Factor
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    ABSTRACT: The solution structures of two different DNA duplexes (one containing a G-T mismatched base pair and the other a non-hydrogen-bonding G-F pair, where F is difluorotoluene) in complex with the peptide antibiotic actinomycin D (ActD) are presented. Using (1)H, (19)F NMR, and molecular dynamics simulations, we show that there are three major differences between the complexes: (1) ActD binds to the GF duplex in an orientation that is flipped 180° relative to its position in the GT duplex; (2) whereas the difluorotoluene moiety takes the typical anti glycosidic conformation in the "free" (uncomplexed) GF duplex, it takes the syn conformation in the GF:ActD complex; and (3) in GF:ActD, the difluorotoluene moiety is completely unstacked in the helix; however, the guanine of the G-F pair is stacked quite well with the ActD intercalator and the flanking base on the 5' side. In GT:ActD, the G-T base pair (although pushed into the major groove from the non-Watson-Crick hydrogen-bonding pattern) stacks favorably with the ActD intercalator and the flanking base pair on the 5' side. The results described here indicate that a sequence-specific DNA binding ligand such as actinomycin D will, indeed, recognize and bind with high affinity to a DNA incorporating a non-natural, non-hydrogen-bonding nucleoside mimic despite the presentation of modified functionality in the binding site.
    Journal of the American Chemical Society 11/2010; 132(49). DOI:10.1021/ja107575f · 12.11 Impact Factor
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    Chang C Liu · Susan E Cellitti · Bernhard H Geierstanger · Peter G Schultz ·
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    ABSTRACT: Tyrosine sulfation is an important post-translational modification that occurs in higher eukaryotes and is involved in cell-cell communication, viral entry and adhesion. We describe a protocol for the heterologous expression of selectively tyrosine-sulfated proteins in Escherichia coli through the use of an expanded genetic code that co-translationally inserts sulfotyrosine in response to the amber nonsense codon, TAG. The components required for this process, an orthogonal aminoacyl-tRNA synthetase specific for sulfotyrosine and its cognate orthogonal tRNA that recognizes the amber codon, are encoded on the plasmid pSUPAR6-L3-3SY, and their use, along with a simple chemical synthesis of sulfotyrosine, are outlined in this protocol. Specifically, the gene for a protein of interest is mutated such that the codon corresponding to the desired location of tyrosine sulfate is TAG. Co-transformation of an expression vector containing this gene and pSUPAR6-L3-3SY into an appropriate E. coli strain allows the overexpression of the site-specifically sulfated protein with high efficiency and fidelity. The resulting protein contains tyrosine sulfate at any location specified by a TAG codon, making this method significantly simpler and more versatile than competing methods such as in vitro enzymatic sulfation, chemical sulfation and peptide synthesis. Once the proper expression vectors are cloned, our protocol should allow the production of the desired sulfated proteins in <1 week.
    Nature Protocol 11/2009; 4(12):1784-9. DOI:10.1038/nprot.2009.188 · 9.67 Impact Factor
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    ABSTRACT: A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.
    Journal of Biomolecular NMR 09/2009; 46(1):89-100. DOI:10.1007/s10858-009-9365-4 · 3.14 Impact Factor
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    Peng R Chen · Dan Groff · Jiantao Guo · Weijia Ou · Susan Cellitti · Bernhard H Geierstanger · Peter G Schultz ·
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    ABSTRACT: A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl-tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl-tRNA synthetase (PylRS) mutant was evolved in E. coli that selectively aminoacylates a cognate nonsense suppressor tRNA with a photocaged lysine derivative. Transfer of this orthogonal tRNA-aaRS pair into mammalian cells made possible the selective incorporation of this unnatural amino acid into proteins.
    Angewandte Chemie International Edition 05/2009; 48(22):4052-5. DOI:10.1002/anie.200900683 · 11.26 Impact Factor
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    ABSTRACT: The crystal structures of two homologous endopeptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme were determined at 1.05 and 1.60 A resolution, respectively, and contain a bacterial SH3-like domain (SH3b) and a ubiquitous cell-wall-associated NlpC/P60 (or CHAP) cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that these two proteins act as gamma-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.
    Structure 03/2009; 17(2):303-13. DOI:10.1016/j.str.2008.12.008 · 5.62 Impact Factor
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    ABSTRACT: In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.
    Journal of the American Chemical Society 08/2008; 130(29):9268-81. DOI:10.1021/ja801602q · 12.11 Impact Factor
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    ABSTRACT: The incorporation of synthetic nucleoside analogues into DNA duplexes provides a unique opportunity to probe both structure and function of nucleic acids. We used 1H and 19F NMR and molecular dynamics calculations to determine the solution structures of two similar DNA decamer duplexes, one containing a central G-T mismatched or "wobble" base pair, and one in which the thymine in this base pair is replaced by difluorotoluene (a thymine isostere) creating a G-F pair. Here, we show that the non-hydrogen-bonding G-F pair stacks relatively well into the helix and that the distortions caused by each non-Watson-Crick G-T or G-F base pair are quite localized to a three base pair site around the mismatch. A detailed structural analysis reveals that the absence of hydrogen bonding introduces more dynamic motion into the G-F pair relative to G-T and permits the G-F pair to exhibit stacking and conformational features characteristic of both a Watson-Crick base pair (on the guanine containing strand) and a wobble base pair (on the strand containing the difluorotoluene). We used these results to posit a rationale for recognition and repair of mismatch sites in DNA.
    Journal of the American Chemical Society 05/2008; 130(14):4869-78. DOI:10.1021/ja7103608 · 12.11 Impact Factor
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    ABSTRACT: A small molecule inhibitor of alpha4 integrin-dependent cell migration was identified through a cell-based screen of small molecule libraries. Biochemical and cellular experiments suggest that this molecule functions by interacting with gamma-parvin. This molecule should serve as a useful tool to study alpha4 integrin signaling and may lead to new therapeutics for the treatment of autoimmune diseases.
    Bioorganic & medicinal chemistry 03/2008; 17(3):977-80. DOI:10.1016/j.bmc.2008.02.067 · 2.79 Impact Factor
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    James J Graziano · Wenshe Liu · Roshan Perera · Bernhard H Geierstanger · Scott A Lesley · Peter G Schultz ·
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    ABSTRACT: A protein evolution strategy is described by which double-stranded DNA fragments encoding defined Escherichia coli protein secondary structural elements (alpha-helices, beta-strands, and loops) are assembled semirandomly into sequences comprised of as many as 800 amino acid residues. A library of novel polypeptides generated from this system was inserted into an enhanced green fluorescent protein (EGFP) fusion vector. Library members were screened by fluorescence activated cell sorting (FACS) to identify those polypeptides that fold into soluble, stable structures in vivo that comprised a subset of shorter sequences ( approximately 60 to 100 residues) from the semirandom sequence library. Approximately 108 clones were screened by FACS, a set of 1149 high fluorescence colonies were characterized by dPCR, and four soluble clones with varying amounts of secondary structure were identified. One of these is highly homologous to a domain of aspartate racemase from a marine bacterium (Polaromonas sp.) but is not homologous to any E. coli protein sequence. Several other selected polypeptides have no global sequence homology to any known protein but show significant alpha-helical content, limited dispersion in 1D nuclear magnetic resonance spectra, pH sensitive ANS binding and reversible folding into soluble structures. These results demonstrate that this strategy can generate novel polypeptide sequences containing secondary structure.
    Journal of the American Chemical Society 02/2008; 130(1):176-85. DOI:10.1021/ja074405w · 12.11 Impact Factor
  • Edward A Lemke · Daniel Summerer · Bernhard H Geierstanger · Scott M Brittain · Peter G Schultz ·
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    ABSTRACT: We genetically encoded the photocaged amino acid 4,5-dimethoxy-2-nitrobenzylserine (DMNB-Ser) in Saccharomyces cerevisiae in response to the amber nonsense codon TAG. This amino acid was converted to serine in living cells by irradiation with relatively low-energy blue light and was used to noninvasively photoactivate phosphorylation of the transcription factor Pho4, which controls the cellular response to inorganic phosphate. When substituted at phosphoserine sites that control nuclear export of Pho4, blocks phosphorylation and subsequent export by the receptor Msn5 (ref. 2). We triggered phosphorylation of individual serine residues with a visible laser pulse and monitored nuclear export of Pho4-GFP fusion constructs in real time. We observed distinct export kinetics for differentially phosphorylated Pho4 mutants, which demonstrates dynamic regulation of Pho4 function. This methodology should also facilitate the analysis of other cellular processes involving free serine residues, including catalysis, biomolecular recognition and ion transport.
    Nature Chemical Biology 01/2008; 3(12):769-72. DOI:10.1038/nchembio.2007.44 · 13.00 Impact Factor
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    ABSTRACT: 2D 1H-15N HMBC NMR acquired at natural abundance and DMSO titration monitored by 1D 1H NMR verified the existence of an intramolecular hydrogen bond that was designed to mimic the pyrimidinone ring of a class of kinase inhibitors. A scalar coupling across the hydrogen bond was detected in organic and aqueous solvent, suggesting a simple and general approach for testing the propensity of intramolecular hydrogen bonds to stabilize pseudo-rings in drug scaffolds.
    Journal of Medicinal Chemistry 12/2007; 50(24):5875-7. DOI:10.1021/jm700983a · 5.45 Impact Factor
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    ABSTRACT: Expansion of the genetic alphabet has been a long-time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semisynthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogues, such as propynylisocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogues inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogues that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluorobenzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self-pair formed between two d3FB analogues is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self-pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl-substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analogue (dBEN). In all, the data suggest that the structure, electrostatics, and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.
    Journal of the American Chemical Society 09/2007; 129(34):10466-73. DOI:10.1021/ja072276d · 12.11 Impact Factor
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    ABSTRACT: Four different immunoassay and antibody microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate-anticoagulated-plasma, EDTA-anticoagulated-plasma, or heparin-anticoagulated-plasma) and (2) evaluate the dependence on concentration of MS-based protein identifications from data sets using the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of antibody-based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances. Continued developments in antibody-based methods will further advance the scientific goals of the PPP.
    Exploring the Human Plasma Proteome, 11/2006: pages 91 - 113; , ISBN: 9783527609482
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    ABSTRACT: Hunter-killer peptides (HKPs) are synthetic peptides that target specific cell types for apoptosis. These studies report functional and structural characteristics of HKP9, an hunter-killer peptide that specifically targets tumor vasculature with a new apoptotic sequence. Vesicle leakage experiments were performed as a model for membrane perturbing activity. Placement of the homing sequence reduces both cell toxicity and vesicle leakage activity. NMR studies elucidate the conformation and orientation of HKP9 in micelles. The positively charged end of the HKP9 killing sequence is solvent exposed; however, the central portion of the peptide is helical and buried in dodecylphosphorylcholine micelles. The homing sequence is less solvent exposed than in a previously reported tumor-homing peptide. The results suggest that solvent accessibility of the homing sequence should be considered in design of future peptides.
    Chemical Biology &amp Drug Design 07/2006; 67(6):417-24. DOI:10.1111/j.1747-0285.2006.00394.x · 2.49 Impact Factor

Publication Stats

3k Citations
444.00 Total Impact Points


  • 2002-2012
    • Genomics Institute of the Novartis Research Foundation
      San Diego, California, United States
  • 2008
    • The Scripps Research Institute
      • Department of Chemistry
      La Jolla, CA, United States
  • 2001
    • Stanford Medicine
      • Department of Biochemistry
      Stanford, California, United States
  • 1998-2001
    • Goethe-Universität Frankfurt am Main
      • Institut für Organische Chemie und Chemische Biologie
      Frankfurt am Main, Hesse, Germany
    • Stanford University
      • Department of Biochemistry
      Palo Alto, California, United States
  • 2000
    • Max Planck Institute for Biophysical Chemistry
      Göttingen, Lower Saxony, Germany
  • 1991-1999
    • University of California, Berkeley
      • • Department of Chemistry
      • • Biophysics Graduate Group
      Berkeley, California, United States