Author: Christine Koehler

Synthesis and Evaluation of Novel Ring‐Strained Noncanonical Amino Acids for Residue‐Specific Bioorthogonal Reactions in Living Cells

Abstract Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels–Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)‐a method that allows to incorporate noncanonical amino acids (ncAAs) site‐specifically into proteins in vivo. These reactions enable residue‐specific fluorophore attachment to proteins in living mam…

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Inducible Genetic Code Expansion in Eukaryotes

Abstract Genetic code expansion (GCE) is a versatile tool to site‐specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl‐tRNA‐synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression whe…

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Bistetrazine-Cyanines as Double-Clicking Fluorogenic Two-Point Binder or Crosslinker Probes

Fluorogenic probes can be used to minimize the background fluorescence of unreacted and nonspecifically adsorbed reagents. The preceding years have brought substantial developments in the design and synthesis of bioorthogonally applicable fluorogenic systems mainly based on the quenching effects of azide and tetrazine moieties. The modulation power exerted by these bioorthogonal motifs typically becomes less efficient on more conjugated systems; that is, on probes with redshifted emission wavelength. To reach efficient quenching, that is, fluorogenicity, even in the red range of the spectrum, we present the synthesis, fluorogenic, and conjugation characterization of bistetrazine-cyanine pro…

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Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET.

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Forster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Be…

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MultiBacTAG-Genetic Code Expansion Using the Baculovirus Expression System in Sf21 Cells

The combination of genetic code expansion (GCE) and baculovirus-based protein expression in Spodoptera frugiperda cells is a powerful tool to express multiprotein complexes with site-specifically introduced noncanonical amino acids. This protocol describes the integration of synthetase and tRNA gene indispensable for GCE into the backbone of the Bacmid, the Tn7-mediated transposition of various genes of interest, as well as the final expression of protein using the MultiBacTAG system with different noncanonical amino acids.

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