Structural Studies and Protein Engineering of Human O6-Alkylguanine-DNA Alkyltransferase

The specific labeling of proteins with synthetic probes is a powerful approach to study protein function and protein tags have been widely used for this purpose. A well-established example for a self-labeling protein tag is SNAP-tag. It specifically reacts with a wide variety of O6-benzylguanine derivatives (BG-derivatives) and was derived from the human O6-alkylguanine-DNA alkyltransferase (hAGT) by protein engineering. Relative to hAGT, SNAP-tag possesses a 52-fold higher reactivity towards BG-derivatives, does not bind to DNA and expresses well in cells as on cell surfaces. It is known that alkylation of hAGT results in protein unfolding and degradation. However, an increased degradation of SNAP-tag fusion proteins after labeling has not been observed. The first part of this work focused on the structural basis underlying the differences in protein stability between SNAP-tag and hAGT. A detailed biochemical and structural analysis was performed to determine (i) the interaction of SNAP-tag with its substrate, (ii) the factors responsible for its increased reactivity and (iii) how the labeling affected the stability of the protein. Besides an increased reactivity with BG-derivatives the superior stability of SNAP-tag compared to the parent protein hAGT could be confirmed. Whereas wild-type hAGT was rapidly degraded in cells after alkyl transfer, benzylated SNAP-tag showed a higher stability against proteolytic degradation. Moreover, the combination of our crystallographic and computational data provided further insight into the structural basis for the improved properties. The data indicated that the intrinsic stability of a key alpha helix was an important factor in triggering the unfolding and degradation of wild-type hAGT and provided new insights into the structure-function relationship of this DNA repair protein. The second part was aiming for the generation of a new SNAP-tag-based inhibitor complex. It was envisaged that this complex would interact with the target protein via amino acid loops and decrease its function only upon labeling with BG-inhibitor molecules. Therefore, SNAP-tag was modified by the insertion of stretches of randomized amino acids and the generated protein libraries were screened for binding affinity. The utilization of two yeast-based systems, the yeast three-hybrid and two hybrid technologies, allowed for the differentiation of small-molecule dependent and independent binding interactions. It could be demonstrated that a specific protein-loop interaction can be generated by this approach. It could further be shown that inhibition of the catalytic activity of the target protein E.coli dihydrofolate reductase by a SNAP-loop mutant was possible. In summary this work revealed new insights into the stability of hAGT and SNAP-tag and the structure-function relationship of AGTs in general. Further, SNAP-tag engineering generated a new protein-binder whose affinity towards the target protein was leading to protein inhibition

Ruthenium Nanoparticles Intercalated in Hectorite: A Reusable Hydrogenation Catalyst for Benzene and Toluene

The cationic organometallic aqua complexes formed by hydrolysis of [(C6H6)RuCl2]2 in water, mainly [(C6H6)Ru(H2O)3]2+, intercalate into sodium hectorite by ion exchange, replacing the sodium cations between the anionic silicate layers. The yellow hectorite thus obtained reacts in ethanol with molecular hydrogen (50 bar, 100°C) with decomposition of the organometallic aqua complexes to give a black material, in which ruthenium(0) nanoparticles (9-18nm) are intercalated between the anionic silicate layers, the charges of which being balanced by hydronium cations. The black ruthenium-modified hectorite efficiently catalyses the hydrogenation of benzene and toluene in ethanol (50 bar H2, 50°C), the turnover frequencies attaining 7000 catalytic cycles per hou

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

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 increased protein activity but also to that the reactivity of the suicide enzyme can be influenced by higher stability, especially of the alkylated protein, suggesting stabilizing the product of the irreversible reaction. Whereas wild-type hAGT is rapidly degraded in cells after alkyl transfer, the high stability of benzylated SNAP-tag prevents proteolytic degradation. Our data indicate that the intrinstic stability of a key a helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure-function relationship of the DNA repair protein

Ruthenium Nanoparticles Intercalated in Hectorite: A Reusable Hydrogenation Catalyst for Benzene and Toluene

The cationic organometallic aqua complexes formed by hydrolysis of [(C6H6)RuCl2]2 in water, mainly [(C6H6)Ru(H2O)3]2+, intercalate into sodium hectorite by ion exchange, replacing the sodium cations between the anionic silicate layers. The yellow hectorite thus obtained reacts in ethanol with molecular hydrogen (50 bar, 100°C) with decomposition of the organometallic aqua complexes to give a black material, in which ruthenium(0) nanoparticles (9–18 nm) are intercalated between the anionic silicate layers, the charges of which being balanced by hydronium cations. The black ruthenium-modified hectorite efficiently catalyses the hydrogenation of benzene and toluene in ethanol (50 bar H2, 50°C), the turnover frequencies attaining 7000 catalytic cycles per hour

Visualizing Biochemical Activities in Living Cells through Chemistry

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 the last years, our laboratory has developed different labeling approaches for monitoring protein activity and for localizing synthetic probes inside living cells. In this article, we review two of these labeling approaches, the SNAP-tag and CLIP-tag technologies, and their use for studying cellular processes

Directed Evolution of the Suicide Protein <i>O</i>⁶-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability

Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein <i>O</i>⁶-alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to increased protein activity but also to higher stability, especially of the alkylated protein, suggesting that the reactivity of the suicide enzyme can be influenced by stabilizing the product of the irreversible reaction. Whereas wild-type hAGT is rapidly degraded in cells after alkyl transfer, the high stability of benzylated SNAP-tag prevents proteolytic degradation. Our data indicate that the intrinstic stability of a key α helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure–function relationship of the DNA repair protein

Size and surface effects on the MRI relaxivity of manganese ferrite nanoparticle contrast agents

Superparamagnetic MnFe2O4 nanocrystals of different sizes were synthesized in high-boiling ether solvent and transferred into water using three different approaches. First, we applied a ligand exchange in order to form a water soluble polymer shell. Second, the particles were embedded into an amphiphilic polymer shell. Third, the nanoparticles were embedded into large micelles formed by lipids. Although all approaches lead to effective negative contrast enhancement, we observed significant differences concerning the magnitude of this effect. The transverse relaxivity, in particular r(2)*, is greatly higher for the micellar system compared to the polymer-coated particles using same-sized nanoparticles. We also observed an increase in transverse relaxivities with increasing particle size for the polymer-coated nanocrystals. The results are qualitatively compared with theoretical models describing the dependence of relaxivity on the size of magnetic spheres