Richard Wombacher

Live-cell super-resolution imaging with trimethoprim conjugates

The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at ∼20 nm resolution.

Ordered and Dynamic Assembly of Single Spliceosomes

Fluorescently labeled yeast spliceosome proteins reveal the events of intron splicing as it happens. The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

The majority of structural efforts addressing RNAs catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a λ-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.

Chemical tags: Applications in live cell fluorescence imaging

Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chemical tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected experiments in which chemical tags have been successfully applied to live cell imaging will be discussed and evaluated.

Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells.

Light‐Induced Protein Dimerization by One‐ and Two‐Photon Activation of Gibberellic Acid Derivatives in Living Cells

We developed a highly efficient system for light-induced protein dimerization in live cells using photo-caged derivatives of the phytohormone gibberellic acid (GA3 ). We demonstrate the application of the photo-activatable chemical inducer of dimerization (CID) for the control of protein translocation with high spatiotemporal precision using light as an external trigger. Furthermore, we present a new two-photon (2P)-sensitive caging group, whose exceptionally high two-photon cross section allows the use of infrared light to efficiently unleash the active GA3 for inducing protein dimerization in living cells.

Two-Step Protein Labeling Utilizing Lipoic Acid Ligase and Sonogashira Cross-Coupling

Labeling proteins in their natural settings with fluorescent proteins or protein tags often leads to problems. Despite the high specificity, these methods influence the natural functions due to the rather large size of the proteins used. Here we present a two-step labeling procedure for the attachment of various fluorescent probes to a small peptide sequence (13 amino acids) using enzyme-mediated peptide labeling in combination with palladium-catalyzed Sonogashira cross-coupling. We identified p-iodophenyl derivatives from a small library that can be covalently attached to a lysine residue within a specific 13-amino-acid peptide sequence by Escherichia coli lipoic acid ligase A (LplA). The derivatization with p-iodophenyl subsequently served as a reactive handle for bioorthogonal transition metal-catalyzed Sonogashira cross-coupling with alkyne-functionalized fluorophores on both the peptide as well as on the protein level. Our two-step labeling strategy combines high selectivity of enzyme-mediated labeling with the chemoselectivity of palladium-catalyzed Sonogashira cross-coupling.

A trimethoprim-based chemical tag for live cell two-photon imaging.

Two-photon excitation results from the near simultaneous absorption of two relatively low-energy photons by a fluorophore, causing a transition to an excited state with an energy difference close to that of the combined two photons (Figure 1 A).1 For most biologically relevant two-photon fluorophores, the excitation light used is in the near infrared (NIR) region.2 Several characteristics of two-photon microscopy make it an attractive technique for biological imaging. Two-photon absorption is strongly dependent on the intensity of the incident light, and therefore, excitation only takes place in a small volume centered at the focal plane, giving inherent sectioning and greatly reducing background fluorescence from out-of-focus excitation. Because the two-photon excitation is limited to the focal plane, photodamage and bleaching to the sample are minimized.3 Additionally, the greatly reduced absorption and scattering of NIR light allows for deeper penetration into biological samples.4 Here we report the development of a trimethoprim (TMP) chemical tag suitable for two-photon imaging in live cells; this adds a robust two-photon fluorophore to the repertoire of labels available with this technology. Figure 1 A trimethoprim (TMP)-based chemical tag for two-photon imaging. A) Energy level diagram illustrating one- and two-photon excitation. Two-photon excitation occurs from the near simultaneous absorption of two photons that are approximately half of the energy ... With the chemical tags, rather than tagging the protein of interest with a fluorescent protein (FP), the protein of interest is tagged with a polypeptide that is subsequently modified with an organic fluorophore.5–8 Thus, the chemical tags combine the selectivity of genetic encoding with a modular organic fluorophore.9–11 We have previously established that proteins tagged with E. coli dihydrofolate reductase (eDHFR) can be labeled noncovalently12–14 (and recently covalently)15 with cell-permeable trimethoprim (TMP)–fluorophore conjugates in mammalian cells. The TMP-based tag is an attractive chemical tag because eDHFR is small in size (18 kD), TMP analogues are straightforward to synthesize, and labels based on the TMP antibiotic have no apparent cross reactivity or toxicity in mammalian cells.16, 17 Currently, the TMP-tag is one of the few available chemical tags capable of specifically labeling intracellular proteins in living cells.14, 18 To our knowledge, while a variety of organic fluorophores, quantum dots,19 and lanthanide chelates17 have been adapted for the chemical tag technology, currently there are no reported chemical tags optimized for two-photon imaging. Key to the design of a TMP-tag for two-photon microscopy was selection of a two-photon fluorophore for conjugation to TMP that would have the desired photophysical properties yet also be cell permeable. The fluorophore must have a high two-photon action (TPA) cross section, which is the product of the two-photon absorption cross section and the fluorescence quantum yield. Second, when linked to TMP, the two-photon fluorophore must behave well in the cell; it must be both cell-permeable and not partition into lipid vesicles.14 In this study, we employed a 2H-benzo[h]chromene-2-one derivative, which we refer to as BC575. This fluorophore was recently reported by Kim and co-workers to have a high TPA cross-section (67 Goeppert–Mayer, or GM [1 GM=10−50 cm4 s per photon]), and to be cell permeable.20, 21 The retrosynthetic analysis of the TMP-BC575 conjugate is shown in Scheme 1. As previously reported,14 the synthesis began with the selective hydrolysis of the 4′-methoxy group of TMP in 48 % HBr; this resulted in a phenol, which was alkylated with ethyl 5-bromovalerate. Saponification of the ester produced a carboxylic acid, which was coupled to mono-Boc protected 1,13-diamino-4,7,10-trioxatridecane and then deprotected to generate a free amine. The synthesis of BC575 was completed in four steps.20 The amino group of 6-aminotetralone was methylated by iodomethane to yield the corresponding dimethylaniline, which was formylated by treatment with ethyl formate. The ring was aromatized and condensed with dimethyl malonate, giving BC575. The methyl ester was then hydrolyzed, and the resulting carboxylic acid was coupled to the free amine of the TMP portion by using standard peptide-coupling conditions. Thus, the TMP-BC575 conjugate was synthesized from two components in six linear steps in 0.3 % overall yield from 6-amino-3,4-dihydronapthalen-1(2 H)-one, the longest linear route. Scheme 1 >Retrosynthetic analysis of the TMP-BC575 conjugate. To verify the performance of BC575, its two-photon excitation and emission spectra were first measured in vitro. Rectangular glass capillaries (300×50 microns) were filled with a 100 μm solution of BC575 dissolved in DMF. The capillaries were sealed and fixed on a coverslip immersed in a drop of water. The two-photon fluorescence data were acquired by using a custom-made two-photon laser scanning microscope based on the Olympus FV-300 system (FV-300 side-mounted to a BX50WI microscope with a 60×, 1.1 numerical aperture, water immersion objective) and a Ti:sapphire laser (Chameleon Ultra II, Coherent).22 Fluorescence of BC575 was evaluated following two-photon excitation by using wavelengths from 750–1050 nm, which are typically used for two-photon biological imaging applications. Additionally, we measured the fluorescence intensity of a 100 μm sample of rhodamine B in H2O by using the same microscope setting; this allowed for the direct comparison of BC575 to rhodamine B, which is known to be a bright two-photon dye.23 The total signal intensities were then determined by using the program ImageJ24 and were used to calculate the normalized fluorescence intensities for the dyes (Figure 2 A).23, 25 The results show that BC575 has appreciable two-photon excited fluorescence from 750–950 nm and confirms the utility of BC575 for two-photon microscopy. Figure 2 Characterization of the two-photon fluorescent chemical tag TMP-BC575. A) In vitro characterization of two-photon excitation of BC575. To evaluate the utility of the fluorophore for two-photon imaging, the two-photon excited fluorescence intensity (in ... Having demonstrated in vitro that BC575 is a good two-photon fluorophore, we then evaluated the utility of the TMP-BC575 conjugate for in vivo imaging. Human embryonic kidney (HEK) 293 cells were seeded on coverslips and transiently transfected with vector DNA encoding nucleus-targeted eDHFR.13 The cells were then incubated in media containing 1 μm TMP-BC575 for 10 min at 37 °C, washed and then imaged by oblique illumination and by two-photon microscopy by using excitation at 940 nm (Figure 2 B). Notably, the transfected cells showed distinct nuclear labeling without any significant staining of the cytoplasm or untransfected cells. Comparison of the fluorescence signal intensities from two-photon excitation of both transfected and untransfected cells incubated with TMP-BC575 verified that the conjugate can be used to label proteins of interest with high signal-to-noise (Figure S5 in the Supporting Information). These results establish that, significantly, the TMP-BC575 conjugate has the combination of cell permeability and two-photon brightness necessary for two-photon live cell imaging. Thus, TMP-BC575 is an immediately viable tool for imaging proteins in live cells by using two-photon microscopy. This two-photon fluorophore expands the TMP-tag tool kit, adding to the value of this modular-labeling technology. A protein of interest can be tagged with eDHFR, and different labels can then be swapped in, allowing the protein to be readily analyzed by multiple techniques. While cell permeability and lipid partitioning appear to be tag dependent, these experiments suggest BC575 might also be compatible with other chemical tags. Given its broad excitation maxima and distinct emission wavelength, TMP-BC575 offers an alternative to other FPs for multicolor two-photon imaging with enhanced green fluorescent protein (EGFP; see Figures S2 and S3 in the Supporting Information),26–28 Alternatively, an orthogonal chemical tag with a nonoverlapping two-photon fluorophore could be developed for multicolor two-photon microscopy. Furthermore, TMP-BC575 might be advantageous in applications in which the larger FP fusion interferes with biological function or where the reversibility of the fluorophore labeling can be exploited. The next step is to challenge TMP-BC575 to label a variety of intracellular proteins. We are also exploring the utility of TMP-BC575 for two-photon imaging of tissue sections and live animals. This work opens the door for two-photon imaging with chemical tags and further illustrates how the modular organic label of chemical tags can be harnessed for state-of-the-art biological imaging.

Probing the Active Site of a Diels#Alderase Ribozyme by Photoaffinity Cross-Linking

The active site of a Diels-Alderase ribozyme is located in solution by photoaffinity cross-linking using a productlike azidobenzyl probe. Two key nucleotides are identified that contact the Diels-Alder product in a conformation-dependent fashion. The design of such probes does not require knowledge of the three-dimensional structure of the ribozyme, and the technique yields both static and dynamic structural information. This work establishes photoaffinity cross-linking as an empirical approach that is applied here for the first time to an artificial ribozyme.

Two-step protein labeling by using lipoic acid ligase with norbornene substrates and subsequent inverse-electron demand Diels-Alder reaction.

Inverse‐electron‐demand Diels–Alder cycloaddition (DAinv) between strained alkenes and tetrazines is a highly bio‐orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two‐step labeling protocol for the site‐specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplAW37V), followed by the covalent attachment of tetrazine‐modified fluorophores to the norbornene moiety through the bio‐orthogonal DAinv . We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13‐residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two‐step labeling strategy to label proteins in cell lysates in a site‐specific manner and performed cell‐surface labeling on living cells.