Publications by Kai Johnsson | EPFL Graph Search

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Centrioles are microtubule-based organelles found in most eukaryotic cells and that are critical for the formation of cilia and flagella, as well as of centrosomes in animal cells. The number of centrioles must be strictly regulated in proliferating cells ...

We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to opt ...

We have designed molecules that permit the selective cross-linking (S-CROSS) of interacting proteins in cell lysates and the sensitive detection of the trapped complexes through in-gel fluorescence scanning. S-CROSS requires the expression of the putative interacting proteins as fusion to CLIP-tag or SNAP-tag, two protein tags that can be specifically labeled with synthetic probes. Bifunctional molecules that contain the substrates of the two tags connected via a fluorophore are used to selectively cross-li

Protein kinases control most cellular processes and aberrant kinase activity is involved in numerous diseases. Here we introduce molecular recorders of kinase activities for later analysis to investigate the link between specific kinase activities and cell ...

The development of molecular probes to visualize cellular processes is an important challenge in chemical biology. One possibility to create such cellular indicators is based on the selective labeling of proteins with synthetic probes in living cells. Over ...

We introduce a photoswitchable O6-benzylguanine derivative and demonstrate its use for super-resolution microscopy of SNAP-tagged proteins based on single fluorophore localization. Stochastic Optical Reconstruction Microscopy (STORM) reveals SNAP-tagged mi ...

Photosensitive probes are powerful tools to study cellular processes with high temporal and spatial resolution. However, most synthetic fluorophores suited for biomolecular imaging have not been converted yet to appropriate photosensitive analogues. Here we describe a generally applicable strategy for the generation of photoactivatable and photoconvertible fluorescent probes that can be selectively coupled to SNAP-tag fusion proteins in living cells. Photoactivatable versions of fluorescein and Cy3 as well

We report here the generation of mutants of the human O6-alkylguanine-DNA alkyltransferase (hAGT) for the efficient in vivo labeling of fusion proteins with synthetic reporter mols. Libraries of hAGT were displayed on phage, and mutants capable of efficiently reacting with the inhibitor O6-benzylguanine were selected based on their ability to irreversibly transfer the benzyl group to a reactive cysteine residue. Using synthetic O6-benzylguanine derivs., the selected mutant proteins allow for a highly effici

The enzymic oxidn. of isoniazid by the catalase-peroxidase of Mycobacterium tuberculosi was studied. The reaction products suggest two reaction pathways involving both electrophilic and radical species. Labeling expts. are consistent with a reaction mechanism involving the formation of an acyldiimide intermediate followed by decompn. to the corresponding aldehyde and peracid. The results suggest that isoniazid is oxidized by the catalase-peroxidase to a reactive species that may act by inactivating an essen

A general method for the specific and covalent labeling of fusion proteins in vivo is described. The approach is based on the unusual mechanism of the human DNA repair protein O6-alkylguanine-DNA alkyl-transferase, which irreversibly transfers an alkyl group from O6-alkylguanine-DNA to one of its cysteine residues. Labeling is possible in bacterial as well as eukaryotic cells and is independent of the nature of the label, thereby opening up new ways to study proteins in vivo. [on SciFinder (R)]

Triplet imaging is a novel optical technique that allows investigating oxy- gen metabolism at the single cell and the sub-cellular level. The method combines high temporal and spatial resolution, which are required for the monitoring of fast kinetics of ox ...

Combinatorial mutagenesis was used to investigate the role of three key residues in cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae, Arg48, Trp51, and Trp191, in control of the reactivity and selectivity of the heme-contg. enzyme. Libraries were prepd. by randomization of these residues and were subsequently screened for activity against the phenolic substrate guaiacol. Screening conditions were employed that favor either mutants with high activity or those with both high activity and stability

We review new and established methods for the chemical modification of proteins in living cells and highlight recent applications. The review focuses on tag-mediated protein labeling methods, such as the tetracysteine tag and SNAP-tag, and new developments in this field such as intracellular labeling with lipoic acid ligase. Recent promising advances in the incorporation of unnatural amino acids into proteins are also briefly discussed. We describe new tools using tag-mediated labeling methods including the

Investigating the interplay of cellular proteins with optical microscopy requires multitarget labeling. Spectral multiplexing using high-affinity or covalent labels is limited in the number of fluorophores that can be discriminated in a single imaging expe ...

The cellular level of nicotinamide adenine dinucleotide (NAD(+)), through its different functions, affects cellular metabolism and signalling(1-3). A decrease in the NAD(+) content has been associated with various pathologies and physiological aging(4,5), ...

Mycobacterium goes yeast: Target deconvolution of anti-tuberculosis drugs can be a very challenging task. Here we report a yeast 3-hybrid system that allows promising small molecules to be screened for protein targets of a pathogen in nontoxic yeast cells. ...

Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O-6-alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to incre ...

We introduce far-red, fluorogenic probes that combine minimal cytotoxicity with excellent brightness and photostability for fluorescence imaging of actin and tubulin in living cells. Applied in stimulated emission depletion (STED) microscopy, they reveal the ninefold symmetry of the centrosome and the spatial organization of actin in the axon of cultured rat neurons with a resolution unprecedented for imaging cytoskeletal structures in living cells.

A review. Recombinant proteins are often expressed with an addnl. polypeptide the role of which is to aid purifn., detection, or functional studies. Recently, the role of these tags has been extended to mediate the labeling of the protein of interest with ...

Human genetic studies have revealed an association between GTP cyclohydrolase 1 polymorphisms, which decrease tetrahydrobiopterin (BH4) levels, and reduced pain in patients. We now show that excessive BH4 is produced in mice by both axotom- ized sensory neurons and macrophages infiltrating damaged nerves and inflamed tissue. Constitutive BH4 overproduction in sensory neurons increases pain sensitivity, whereas blocking BH4 production only in these cells reduces nerve injury-induced hy- persensitivity withou

Kai Johnsson

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Chemical Genetic Screen Identifies Natural Products that Modulate Centriole Number

Computational design of environmental sensors for the potent opioid fentanyl

Selective cross-linking of interacting proteins using self-labeling tags

Molecular recording of cellular protein kinase activity with chemical labeling

Visualizing Biochemical Activities in Living Cells through Chemistry

Targeted Photoswitchable Probe for Nanoscopy of Biological Structures

Photoactivatable and photoconvertible fluorescent probes for protein labeling

Directed Evolution of O6-Alkylguanine-DNA Alkyltransferase for Efficient Labeling of Fusion Proteins with Small Molecules In Vivo

Mechanistic Studies of the Oxidation of Isoniazid by the Catalase Peroxidase from Mycobacterium tuberculosis

Covalent labeling of fusion proteins with chemical probes in living cells

Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5)

Examining reactivity and specificity of cytochrome c peroxidase by using combinatorial mutagenesis

How to obtain labeled proteins and what to do with them

Synergizing Exchangeable Fluorophore Labels for Multitarget STED Microscopy

A biosensor for measuring NAD(+) levels at the point of care

Yeast Three-Hybrid Screening for Identifying Anti-Tuberculosis Drug Targets

Directed evolution of the suicide protein O⁶-alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability

Fluorogenic probes for live-cell imaging of the cytoskeleton

Adding value to fusion proteins through covalent labeling

Reduction of Neuropathic and Inflammatory Pain through Inhibition of the Tetrahydrobiopterin Pathway