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No description available.

No description available.

DNA polymerases are found in a diverse range of organisms, prokaryotes, eukaryotes, viruses and bacteriophage. T7 DNA polymerase is a replicative enzyme from E. coli bacteriophage T7. It relies on the thioredoxin binding domain (TBD) of phage gene 5 protein (gp5) and E. coli thioredoxin (Trx) for processive replication of phage DNA. Although T7 DNA polymerase is processive, it is also thermolabile. In order to design a thermostable and processive DNA polymerase, the structural stabilities of the TBD and Trx were studied in respect to their binding affinity and affect on enzyme processivity. An artificial operon was designed for coexpression of subunits of T7 DNA polymerase. By means of a 9??His-tag at the amino terminus of gp5, T7 DNA polymerase complex was purified by one-step nickel-agarose chromatography, with subunits gp5 and Trx co-eluting in a one to one molar ratio. Purified T7 DNA polymerase was assayed for polymerase activity, processivity and residual activity and compared to the commercial T7 DNA polymerase. The two enzymes were not identical with commercial T7 DNA polymerase being less processive at 37??C. Mass spectrometry of the two enzymes identified a mutation of Phe102 to Ser in the Trx subunit (TrxS102) of commercial T7 DNA polymerase. The Ser102 mutation, was found near the carboxyl terminal helix of Trx. TrxS102 was less stable than wild type Trx. In the study of the TBD structural stability, a hybrid polymerase was constructed by inserting the TBD motif into the homologous position in the Stoffel fragment of Taq DNA polymerase. The hybrid enzyme was coexpressed with Trx from an artificial operon; however, the TBD inserted retained a mesophilic binding affinity to Trx. The chimeric polymerase required 100 molar excess of Trx for processive polymerase activity at 60??C. TBD structural deformation at elevated temperatures was hypothesized to be the cause of the change in the subunit stoichiometry. Mutagenesis of TBD would be required to increase its thermostability. An efficient, rapid high throughput mutagenesis method (SLIM) was invented and would be appropriate for further studies.

DNA polymerases are found in a diverse range of organisms, prokaryotes, eukaryotes, viruses and bacteriophage. T7 DNA polymerase is a replicative enzyme from E. coli bacteriophage T7. It relies on the thioredoxin binding domain (TBD) of phage gene 5 protein (gp5) and E. coli thioredoxin (Trx) for processive replication of phage DNA. Although T7 DNA polymerase is processive, it is also thermolabile. In order to design a thermostable and processive DNA polymerase, the structural stabilities of the TBD and Trx were studied in respect to their binding affinity and affect on enzyme processivity. An artificial operon was designed for coexpression of subunits of T7 DNA polymerase. By means of a 9??His-tag at the amino terminus of gp5, T7 DNA polymerase complex was purified by one-step nickel-agarose chromatography, with subunits gp5 and Trx co-eluting in a one to one molar ratio. Purified T7 DNA polymerase was assayed for polymerase activity, processivity and residual activity and compared to the commercial T7 DNA polymerase. The two enzymes were not identical with commercial T7 DNA polymerase being less processive at 37??C. Mass spectrometry of the two enzymes identified a mutation of Phe102 to Ser in the Trx subunit (TrxS102) of commercial T7 DNA polymerase. The Ser102 mutation, was found near the carboxyl terminal helix of Trx. TrxS102 was less stable than wild type Trx. In the study of the TBD structural stability, a hybrid polymerase was constructed by inserting the TBD motif into the homologous position in the Stoffel fragment of Taq DNA polymerase. The hybrid enzyme was coexpressed with Trx from an artificial operon; however, the TBD inserted retained a mesophilic binding affinity to Trx. The chimeric polymerase required 100 molar excess of Trx for processive polymerase activity at 60??C. TBD structural deformation at elevated temperatures was hypothesized to be the cause of the change in the subunit stoichiometry. Mutagenesis of TBD would be required to increase its thermostability. An efficient, rapid high throughput mutagenesis method (SLIM) was invented and would be appropriate for further studies.

DNA polymerases are found in a diverse range of organisms, prokaryotes, eukaryotes, viruses and bacteriophage. T7 DNA polymerase is a replicative enzyme from E. coli bacteriophage T7. It relies on the thioredoxin binding domain (TBD) of phage gene 5 protein (gp5) and E. coli thioredoxin (Trx) for processive replication of phage DNA. Although T7 DNA polymerase is processive, it is also thermolabile. In order to design a thermostable and processive DNA polymerase, the structural stabilities of the TBD and Trx were studied in respect to their binding affinity and affect on enzyme processivity. An artificial operon was designed for coexpression of subunits of T7 DNA polymerase. By means of a 9??His-tag at the amino terminus of gp5, T7 DNA polymerase complex was purified by one-step nickel-agarose chromatography, with subunits gp5 and Trx co-eluting in a one to one molar ratio. Purified T7 DNA polymerase was assayed for polymerase activity, processivity and residual activity and compared to the commercial T7 DNA polymerase. The two enzymes were not identical with commercial T7 DNA polymerase being less processive at 37??C. Mass spectrometry of the two enzymes identified a mutation of Phe102 to Ser in the Trx subunit (TrxS102) of commercial T7 DNA polymerase. The Ser102 mutation, was found near the carboxyl terminal helix of Trx. TrxS102 was less stable than wild type Trx. In the study of the TBD structural stability, a hybrid polymerase was constructed by inserting the TBD motif into the homologous position in the Stoffel fragment of Taq DNA polymerase. The hybrid enzyme was coexpressed with Trx from an artificial operon; however, the TBD inserted retained a mesophilic binding affinity to Trx. The chimeric polymerase required 100 molar excess of Trx for processive polymerase activity at 60??C. TBD structural deformation at elevated temperatures was hypothesized to be the cause of the change in the subunit stoichiometry. Mutagenesis of TBD would be required to increase its thermostability. An efficient, rapid high throughput mutagenesis method (SLIM) was invented and would be appropriate for further studies.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Kristi L. Kiick, Dept. of Materials Science & Engineering. Includes bibliographical references.

The aim of this project is to develop general approaches to the control of protein structures and functions. The project mainly consists of three aspects: (1) stabilizing folded protein structures, (2) reversible photo-control of protein folding and function, (3) design of a photo-switchable dominant negative protein to photo-control DNA binding. (1) Most proteins adopt specific folded structures to perform biological functions. Stabilizing the active folded forms of peptides or proteins is thus important for maintaining or enhancing the functions of these molecules. We hypothesized that a protein’s folded structure could be stabilized by introduction of a rigid cross-linker with its length matching the distance of the two attachment points in the folded structure. To test this, we synthesized a thiol-reactive alkyne-based rigid cross-linker and tested its effect. When it was introduced at i and i+11 positions of a model α-helical peptide, a significant promotion in the folded structure as well as strong resistance against thermal melting was observed. The rigid cross-linker was also applied to the Fyn SH3 domain, a protein with tertiary structure and a similar stabilization effect was obtained. This work demonstrates that a rigid cross-linker can be generally used to stabilize folded peptide/protein structures. (2) Reversible photo-switch of protein folding/unfolding offers exciting prospects for external manipulation of protein function because of its fast response, high spatial resolution and compatibility with living systems. I developed a general approach to the design of photo-switchable proteins based on the introduction of photo-switchable intramolecular cross-linkers. An azobenzene based photo-switch was used because the energy available from photoisomerization is higher than the free energy of protein folding. I chose the FynSH3 domain as a model protein. Taking the experimentally determined structure of the folded protein as a starting point, mutations were made to introduce pairs of Cys residues so that the distance between Cys sulfur atoms matches the ideal length of the cis form, but not the trans form, of the cross-linker. When the L3C-L29C-T47AFynSH3 mutant was cross-linked with the trans cross-linker, the protein was destabilized so that folded and unfolded forms coexisted. Irradiation of the cross-linker to produce the cis isomer recovered the folded state of the protein. Photo-control of FynSH3 binding to a proline-rich peptide was also demonstrated. This work shows that structure-based introduction of switchable cross-linkers is a feasible general approach for photo-control of global folding/unfolding of globular proteins, and thereby photo-control of their activity. (3) The third aspect of my PhD research is to apply the photo-switchable proteins to photo-control of Jun/Fos DNA binding activity. Fos and Jun are important transcription factors implicated in numerous cancers. They form a hetero-dimer that binds to specific DNA sequences. Dominant negative proteins are mutants of Fos or Jun that prevent native Jun/Fos DNA binding activity. I designed photo-switchable versions of these dominant negative proteins by covalently introducing photo-switchable cross-linkers. These proteins do not have any function in the dark due to their disrupted structures induced by the trans form cross-linkers. They only function as dominant negative proteins when the cross-linker is in the cis form after photo-irradiation. Several such proteins were synthesized and their effectiveness was tested. These photo-switchable dominant negative proteins are powerful tools for temporal control of Jun/Fos regulated gene expression.

The aim of this project is to develop general approaches to the control of protein structures and functions. The project mainly consists of three aspects: (1) stabilizing folded protein structures, (2) reversible photo-control of protein folding and function, (3) design of a photo-switchable dominant negative protein to photo-control DNA binding. (1) Most proteins adopt specific folded structures to perform biological functions. Stabilizing the active folded forms of peptides or proteins is thus important for maintaining or enhancing the functions of these molecules. We hypothesized that a protein’s folded structure could be stabilized by introduction of a rigid cross-linker with its length matching the distance of the two attachment points in the folded structure. To test this, we synthesized a thiol-reactive alkyne-based rigid cross-linker and tested its effect. When it was introduced at i and i+11 positions of a model α-helical peptide, a significant promotion in the folded structure as well as strong resistance against thermal melting was observed. The rigid cross-linker was also applied to the Fyn SH3 domain, a protein with tertiary structure and a similar stabilization effect was obtained. This work demonstrates that a rigid cross-linker can be generally used to stabilize folded peptide/protein structures. (2) Reversible photo-switch of protein folding/unfolding offers exciting prospects for external manipulation of protein function because of its fast response, high spatial resolution and compatibility with living systems. I developed a general approach to the design of photo-switchable proteins based on the introduction of photo-switchable intramolecular cross-linkers. An azobenzene based photo-switch was used because the energy available from photoisomerization is higher than the free energy of protein folding. I chose the FynSH3 domain as a model protein. Taking the experimentally determined structure of the folded protein as a starting point, mutations were made to introduce pairs of Cys residues so that the distance between Cys sulfur atoms matches the ideal length of the cis form, but not the trans form, of the cross-linker. When the L3C-L29C-T47AFynSH3 mutant was cross-linked with the trans cross-linker, the protein was destabilized so that folded and unfolded forms coexisted. Irradiation of the cross-linker to produce the cis isomer recovered the folded state of the protein. Photo-control of FynSH3 binding to a proline-rich peptide was also demonstrated. This work shows that structure-based introduction of switchable cross-linkers is a feasible general approach for photo-control of global folding/unfolding of globular proteins, and thereby photo-control of their activity. (3) The third aspect of my PhD research is to apply the photo-switchable proteins to photo-control of Jun/Fos DNA binding activity. Fos and Jun are important transcription factors implicated in numerous cancers. They form a hetero-dimer that binds to specific DNA sequences. Dominant negative proteins are mutants of Fos or Jun that prevent native Jun/Fos DNA binding activity. I designed photo-switchable versions of these dominant negative proteins by covalently introducing photo-switchable cross-linkers. These proteins do not have any function in the dark due to their disrupted structures induced by the trans form cross-linkers. They only function as dominant negative proteins when the cross-linker is in the cis form after photo-irradiation. Several such proteins were synthesized and their effectiveness was tested. These photo-switchable dominant negative proteins are powerful tools for temporal control of Jun/Fos regulated gene expression.

Directed evolution has been explored for a long time. Various ideas, methods, have been shown to be feasible and successful in the enzyme field. We were interested in evolving enzymes for applications. Therefore, we evolved human cystathionine gamma-lyase (hCGL) and E. coli biotin ligase for therapeutic and biotechnology applications. Wild-type human cystathionine gamma-lyase does not have any methionine-degrading activity, unlike the high methionine-degrading abilities of bacterial methionine gamma-lyase (MGL) found in Pseudomonas putida. The ability to engineer hCGL to breakdown methionine can be a potential cancer treatment by targeting the methionine-dependent cancer cells. However, the methionine-degrading activity of previously engineered hCGL has only shown 1% activity compared to MGL, too low to be useful in practical cancer therapeutics. By using a combination of protein design and phylogenetic analysis, we further evolved hCGL to achieve a higher methionine-degrading activity, with one variant displaying as much as 7% activity compared to bacterial MGL, making it a more likely candidate in cancer treatment.In addition, it has been shown that new orthogonal pairs of biotin protein ligase and biotin have many biotechnology applications. Therefore, we have developed selection scheme for directing the evolution of E. coli biotin protein ligase (BPL, gene: BirA) via in vitro compartmentalization, and have altered the substrate specificity of BPL towards the utilization of the biotin analogue desthiobiotin. Following just 6 rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA₆₋₄₀ and BirA₆₋₄₇, showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. Further characterization of BirA₆₋₄₀ and the single substitution variant BirA[subscript M157T] revealed that they had 2- to 3-fold higher kcat values for desthiobiotin, and 3- to 4-fold higher K[subscript M] values. The k[subscript cat]/K[subscript M] values for these enzymes were around 0.7-fold that of BirA[subscript wt-]. It is interesting the selections did not lower the K[subscript M] for desthiobiotin and actually led to a less efficient enzyme. This is an example of how "you get what you select for". Because peptide:DNA conjugates were distributed such that there was on average one template or less per emulsion compartment there was selection only for the catalytic rate (k[subscript cat]) of desthiobiotinylation and not for turnover. Given these conditions, it might be anticipated that the peptide substrate, rather than desthiobiotin, should be bound better by the winning variants, and in fact BirA₆₋₄₀ showed a reduced K[subscript M] value for BAP. / text