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Kai Johnsson

Publications and source records attributed to Kai Johnsson.

At least 19 recordsLinked to original sources
PubMed2006

Evolving the substrate specificity of O6-alkylguanine-DNA alkyltransferase through loop insertion for applications in molecular imaging.

We introduce a strategy for evolving protein substrate specificity by the insertion of random amino acid loops into the protein backbone. Application of this strategy to human O6-alkylguanine-DNA alkyltransferase (AGT) led to the isolation of mutants that react with the non-natural substrate O6-propargylguanine. Libraries generated by conventional random or targeted saturation mutagenesis, by contrast, did not yield any mutants with activity towards this new substrate. The strategy of loop insertion to alter enzyme specificity should be general and applicable to other classes of proteins. An important application of the isolated AGT mutant is in molecular imaging, where the mutant and parental AGTs are used to label two different AGT fusion proteins with different fluorophores in the same living cell or in vitro . This allowed the establishment of fluorescence-based assays to detect protein-protein interactions and measure enzymatic activities.

Amino Acid Sequence↗
PubMed2006

Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling.

The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivatives.

Binding Sites↗
PubMed2006

Covalent labeling of cell-surface proteins for in-vivo FRET studies.

Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor-acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell-surface neurokinin-1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.

Acyl Carrier Protein↗
PubMed2006

FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells.

The lateral organization of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R), was investigated in living cells by fluorescence resonance energy transfer (FRET) microscopy, taking advantage of the recently developed acyl carrier protein (ACP) labeling technique. The NK1R was expressed as fusion protein with ACP to which small fluorophores were then covalently bound. Our approach allowed the recording of FRET images of receptors on living cells with unprecedented high signal-to-noise ratios and a subsequent unequivocal quantification of the FRET data owing to (i) the free choice of optimal fluorophores, (ii) the labeling of NK1Rs exclusively on the cell surface, and (iii) the precise control of the donor-acceptor molar ratio. Our single-cell FRET measurements exclude the presence of constitutive or ligand-induced homodimers or oligomers of NK1Rs. The strong dependence of FRET on the receptor concentration further reveals that NK1Rs tend to concentrate in microdomains, which are found to constitute approximately 1% of the cell membrane and to be sensitive to cholesterol depletion.

Acyl Carrier Protein↗
PubMed2006

Protein function microarrays based on self-immobilizing and self-labeling fusion proteins.

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction.

Carrier Proteins↗
PubMed2005

Multicolor imaging of cell surface proteins.

We report on a method for the multicolor imaging of cell surface proteins which is based on the labeling of carrier protein (CP) fusion proteins with different fluorophores. In one application, different generations of a cell surface protein can be sequentially labeled to discriminate between old and newly made copies. In another application, fusions to different CPs can be selectively labeled with different fluorophores in one sample. Both applications open up new ways for studying the properties of cell surface proteins of living cells.

Carrier Proteins↗
PubMed2005

Protein chemistry on the surface of living cells.

The interplay between carbohydrates, lipids, and proteins determines the stability and flexibility as well as the adhesive and responsive features of the surfaces of all cells. The molecular understanding of the interactions among and between the different classes of these biomolecules is rudimentary at best, a lack of suitable experimental methods being the major reason. Here we discuss a new approach for the specific labeling of fusion proteins of carrier proteins with synthetic compounds on cell surfaces and describe how this approach can be used to investigate the properties of the labeled molecules.

Acyl Carrier Protein↗
PubMed2005

Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells.

Fusion proteins of human O(6)-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.

Animals↗
PubMed2005

The laboratory in a droplet.

In this issue of Chemistry and Biology, the groups of Tawfik and Griffiths present fluorescence-activated cell sorting of double emulsions as a generally applicable screen for enzyme activity. This novel methodology increases the throughput of a typical enzyme screen by two orders of magnitude.

Cell Separation↗
PubMed2005

Adding value to fusion proteins through covalent labelling.

Recombinant proteins are often expressed with an additional polypeptide the role of which is to aid purification, detection or functional studies. Recently, the role of these tags has been extended to mediate the labelling of the protein of interest with chemically diverse compounds. These approaches are of importance for protein science as they allow proteins to be equipped with properties that cannot be genetically encoded and also enable the use of a single fusion protein for several different applications. Several new approaches have been developed for the covalent labelling of fusion proteins both in living cells and in vitro.

Recombinant Fusion Proteins↗
PubMed2004

Specific labeling of cell surface proteins with chemically diverse compounds.

The specific and covalent labeling of fusion proteins with synthetic molecules opens up new ways to study protein function in the living cell. Here we present a novel method that allows for the specific and exclusive extracellular labeling of proteins on the surfaces of live cells with a large variety of synthetic molecules including fluorophores, protein ligands, or quantum dots. The approach is based on the specific labeling of fusion proteins of acyl carrier protein with synthetic molecules through post-translational modification catalyzed by phosphopantetheine transferase. The specificity and versatility of the labeling should allow it to become an important tool for studying and manipulating cell surface proteins and for complementing existing approaches in cell surface engineering.

Acyl Carrier Protein↗
PubMed2004

Labeling of fusion proteins with synthetic fluorophores in live cells.

A general approach for the sequential labeling of fusion proteins of O(6)-alkylguanine-DNA alkyltransferase (AGT) with different fluorophores in mammalian cells is presented. AGT fusion proteins with different localizations in the cell can be labeled specifically with different fluorophores, and the fluorescence labeling can be used for applications such as multicolor analysis of dynamic processes and fluorescence resonance energy transfer measurements. The facile access to a variety of different AGT substrates as well as the specificity of the labeling reaction should make the approach an important tool to study protein function in live cells.

Animals↗
PubMed2004

Synthesis and characterization of bifunctional probes for the specific labeling of fusion proteins.

Labeling proteins with synthetic probes is important for studying and characterizing protein function. We have recently introduced a general method for the specific in vivo and in vitro labeling of fusion proteins that is based on the reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine derivatives. Here we report two complementary routes for the synthesis of O6-benzylguanine derivatives, which allow for the labeling of AGT fusion proteins with bifunctional synthetic probes and demonstrate the specific labeling of AGT fusion proteins with these probes. These molecules should become useful tools for various applications in functional proteomics.

Cloning, Molecular↗
PubMed2004

Transforming a (beta/alpha)8--barrel enzyme into a split-protein sensor through directed evolution.

Split-protein sensors have become an important tool for the analysis of protein-protein interactions in living cells. We present here a combinatorial method for the generation of new split-protein sensors and demonstrate its application toward the (beta/alpha)(8)-barrel enzyme N-(5'-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae. The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan. This powerful selection complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening.

Aldose-Ketose Isomerases↗
PubMed2004

Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro.

The in vivo and in vitro labeling of fusion proteins with synthetic molecules capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics. We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivatives. Here, we report optimized protocols for the synthesis of O6-benzylguanine derivatives and the use of such derivatives for the labeling of different hAGT fusion proteins in vivo and in vitro.

Bacterial Proteins↗