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From Multi-fluorinated DNA Polymerases to Insights into DNA Synthesis by NMR spectroscopy

From Multi-fluorinated DNA Polymerases to Insights into DNA Synthesis by NMR spectroscopy


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HOLZBERGER, Bastian, 2012. From Multi-fluorinated DNA Polymerases to Insights into DNA Synthesis by NMR spectroscopy [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Holzberger2012Multi-20589, title={From Multi-fluorinated DNA Polymerases to Insights into DNA Synthesis by NMR spectroscopy}, year={2012}, author={Holzberger, Bastian}, address={Konstanz}, school={Universität Konstanz} }

Holzberger, Bastian 2012-10-05T07:19:03Z terms-of-use From Multi-fluorinated DNA Polymerases to Insights into DNA Synthesis by NMR spectroscopy eng 2012-10-05T07:19:03Z 2012 Holzberger, Bastian The 20 canonical amino acids comprise a range of different properties and side chain functionalities that are used to assemble polypeptides, proteins and enzymes in a combinatorial fashion. Researchers are using this defined amino acid pool to create proteins and enzymes with new properties or modified functions and to study biological processes. The use of artificial or non-canonical amino acids offers the possibility to modify proteins beyond this restricted pool of the 20 canonical amino acids. The addition of amino acids with new functionalities to the existing repertoire allows the generation of proteins with novel compositions enhancing their chemical and biological diversity. Nowadays, different methods provide the possibility to selectively and efficiently introduce various new building blocks into target proteins. Among these methods, the most efficient ones utilize the natural protein translation machinery for the introduction of non-canonical amino acids in proteins in vivo.[2-15] Directed reprogramming of protein translation pathways based on the substrate tolerance of the natural components allows the genetic encoding of non-canonical amino acids in bacteria, yeast, and mammalian cells.[2-15] Aside from methods to site-specifically introduce non-canonical amino acids at single positions,[2-5, 22] natural amino acids can be globally replaced by non-canonical analogs by selective pressure incorporation (SPI).[9, 26, 27] This offers the possibility to modify the overall physical and chemical properties of target proteins. By using auxotrophic host cells that are unable to biosynthesize the specific natural amino acid, the appropriate sense-codons can be reassigned by adding a desired amino acid analog. This leads to the overall replacement of the specific natural amino acid in a residue-specific manner.<br /><br />However, non-canonical amino acid engineering has been only very sparsely exploited to modify and to study DNA polymerases,[121] although these molecular machines are an interesting target in many respects. Engineered DNA polymerases are used in biotechnological applications for the amplification, detection, and analysis of nucleic acids and are thereby constantly subject to new requirements.[98, 99] Additionally, DNA polymerases catalyze all DNA synthesis in nature and therefore actively contribute to the fidelity and accuracy of genome replication.[83-88] Thus, there is great interest in developing DNA polymerases with characteristics that can not be found under the natural occurring enzymes and in methods to study the enzymatic mechanisms of DNA polymerases in more detail.<br /><br /><br /><br />The first part of this work deals with the general applicability of non-canonical amino acids to create DNA polymerases with novel compositions. For this purpose, the 32 proline and separately the 13 methionine residues of the thermophilic KlenTaq DNA polymerase, which is composed of 540 amino acids in total (>60 kDa), were replaced throughout the entire enzyme by the fluorinated analogs 4 fluoroproline (4 FPro) and 6,6,6 trifluoro-methione (TFM), respectively. Trifluorinated analogs of the hydrophobic amino acids isoleucine, leucine, valine, and methionine are generally known to modify the properties of peptides and proteins due to the increased hyrophobicity of their side chains compared to their natural counterparts.[10, 53, 55] However, in particular TFM has been only introduced in a few globular proteins, so far.[55, 60-63] 4-FPro has been shown in the past to be very potent in modifying biophysical properties of peptides and proteins such as conformations, folding, and stability.[31-33, 44, 45, 55, 70-81] This has been mainly attributed to the conformational preferences of the two diastereomers (4R)- and (4S)-FPro and the formation of new interactions between fluorine atoms and adjacent amino acids. Nevertheless, the reasons for the complex effects of 4 FPro on globular proteins are still under discussion.<br /><br />By using the SPI method, the methionine and separately the proline residues could be almost quantitatively replaced by TFM and (4R)-FPro yielding the highly fluorinated DNA polymerases TFM-KlenTaq and (4R)-FPro-KlenTaq. Interestingly, efforts towards the recombinant expression of KlenTaq DNA polymerase in presence of (4S)-FPro failed as protein yields declined dramatically. By performing numerous functional studies on TFM- and (4R)-FPro-KlenTaq, it turned out that enzymatic properties like activity, sensitivity, and fidelity of the fluorinated enzymes are almost unaltered in comparison to the wild-type properties. However, both fluorinated enzymes show reduced half-lives at 95 °C compared to the wild-type KlenTaq DNA polymerase. Nevertheless, TFM- and (4R)-FPro-KlenTaq are both still highly active and thermophilic DNA polymerases that can be used for biotechnological applications. Thus, the KlenTaq DNA polymerase is apparently able to tolerate fluorinated amino acids at multiple positions without markedly losing enzymatic activity. These findings are the prerequisite for further investigations, such as the directed evolution of DNA polymerases with new characteristics utilizing an expanded amino acid repertoire.<br /><br />To elucidate the molecular reasons for the loss of thermostability of TFM-KlenTaq, mutational studies were carried out. On the one hand, six methionines were replaced by natural alanine instead of TFM to decrease the overall impact of the non-canonical amino acid TFM. Furthermore, three additional KlenTaq variants that bear instead of the 13 methionine residues 13 isoleucines, leucines, and alanines, respectively, were generated and investigated concerning their activity and stability. In doing so, it became apparent that the hydrophobic methionine side chains significantly contribute to the stabilization of KlenTaq DNA polymerase as all investigated variants show loss in thermostability. In addition to the hydrophobic potential of the side chains, also steric demands turned out to be critical for the altered stabilities upon replacing KlenTaq´s methionine residues. The non-canonical amino acid TFM was best suited to replace methionine as TFM-KlenTaq is the only variant that is still stable and active enough to maintain PCR activity.<br /><br />To gain detailed insight into the structure of the multi-fluorinated (4R)-FPro-KlenTaq DNA polymerase in comparison to the wild-type enzyme, attempts to solve the overall structures of the enzymes by X ray crystallography were performed. In close collaboration with Samra Obeid and the Welte and the Diederichs groups of the University of Konstanz, (4R)-FPro-KlenTaq and the wild-type enzyme could be crystallized in ternary complex with DNA and a bound nucleotide. The structures were solved at resolutions of 1.9 (wild type) and 2.4 Å ((4R) FPro), respectively, and revealed that the substitution of proline with (4R)-FPro did neither affect neighbored amino acid conformations nor the overall structure of the DNA polymerase. The decrease in thermostability upon (4R)-FPro introduction can be explained by considering different effects in a combined fashion: the consequences of (4R)-FPro’s specific conformational preferences, the newly established interactions of fluorine atoms in their microenvironments, the dispersion of fluorine atoms over buried and exposed positions, and the modified surface of (4R)-FPro-KlenTaq DNA polymerase. Interestingly, the introduction of (4R)-FPro appeared to enhance the crystallization competence of the KlenTaq DNA polymerase in comparison to the wild-type protein. This might be caused by decreased local conformational heterogeneities and the fluorinated surface of (4R)-FPro-KlenTaq that displays also new crystal contacts of fluorine atoms to symmetry-related KlenTaq molecules.<br /><br /><br /><br />Since directed enzyme evolution has been shown in the past to be a promising method to develop DNA polymerases with altered or new properties,[99-106] non-canonical protein engineering was combined with directed evolution to generate (4R)-FPro-KlenTaq variants with altered properties. To the best of my knowledge, directed evolution has so far not been applied to DNA polymerase engineering in combination with the use of non-canonical amino acids. A small DNA polymerase library was generated by diversifying the KlenTaq gene by random mutagenesis[154] as a proof of concept. KlenTaq variants were then expressed under SPI of (4R)-FPro in multi-well plates and screened by real-time PCR in 384-well format using E. coli lysates. Doing so, active variants could be identified in high-throughput. In subsequent screening steps, (4R)-FPro-KlenTaq variants were selected that showed primer extension activity in absence of dATP and thereby an increased substrate tolerance as mismatch incorporation has to be performed. Finally, two promising variants were expressed in large-scale and purified to verify the altered properties. In functional studies it turned out that these (4R)-FPro-KlenTaq variants show in fact an increased substrate spectrum. Both variants were more efficient in bypassing DNA lesions and more active in reverse transcription in comparison to (4R)-FPro-KlenTaq.<br /><br />Thus, I could show that directed DNA polymerase evolution applying non-canonical amino acids can be used to create DNA polymerase variants with altered characteristics and a non-canonical composition. This may open up new possibilities for non-canonical enzyme engineering.<br /><br /><br /><br />Finally, the successful introduction of non-canonical amino acids at multiple sites of KlenTaq DNA polymerase was exploited to study DNA synthesis by nuclear magnetic resonance (NMR) spectroscopy. As DNA polymerases actively contribute to the fidelity of DNA replication by discriminating against incorrect nucleotides, the mechanisms of DNA polymerases have been studied by various methods in the past.[88, 92, 93] However, NMR has only rarely been used to investigate substrate binding and conformational changes of DNA polymerases during the catalytic cycle of DNA synthesis.[135-138]<br /><br />In this work, TFM-KlenTaq DNA polymerase turned out to be well suited for studying the enzyme by 19F NMR. Additionally, a KlenTaq DNA polymerase was generated that displays [methyl-13C]methionine instead of natural methionine to investigate the DNA polymerase by two-dimensional 1H,13C NMR techniques. As KlenTaq´s methionine residues are located in both, the rigid palm, which participates in substrate binding and catalysis, and the flexible fingers domain that contributes to active site formation, methionine amino acids were well suited to monitor alterations during enzymatic catalysis.<br /><br />By tracking the resonances of the bioorthogonal NMR-active nuclei 13C and 19F, it was possible to specifically study local and global changes within the enzyme upon addition of different substrate combinations. In summary, DNA binding, nucleotide recognition and the conformational changes associated with active site closure could be detected by 19F NMR and 1H,13C-HSQC. Distinct differences in the NMR spectra of KlenTaq DNA polymerase were observed when encountering mismatching or abasic template sites in comparison to matched states. This indicates that the enzyme cycles through distinct paths in case of correct or incorrect nucleotide binding. Along with the observed differences in local dynamics or conformational heterogeneities, this might further explain how DNA polymerases achieve their high selectivity during DNA synthesis.<br /><br />Taken together, NMR turned out to be a potent method to study DNA polymerase mechanisms in solution. Further comparative studies along these lines should provide additional insight into these processes. First, the residual substrate combinations of match and mismatch states could be examined to further confirm the existing results. Furthermore, investigations of DNA lesions such as oxidized nucleobases and substrate combinations that are hardly accessible by crystallographic approaches would be of special interest.

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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