Halorhodospira halophila photoactive yellow protein (PYP) is a promising candidate to act as a photoswitching domain in engineered proteins due to the structural changes that occur during its photocycle. Absorption of a photon of wavelength 446 nm triggers trans to cis isomerization of its 4-hydroxycinnamic acid chromophore leading to large structural perturbations in the protein, particularly in the N-terminus. In the dark, a slower cis to trans reisomerization of the chromophore restores the protein’s native fold. The fusion of proteins to PYP’s N-terminus may therefore enable photomodulation of the activity of the attached protein. To test this hypothesis, this thesis descibes genetically encoded photoswitchable DNA-binding proteins that were developed by fusing the prototypical leucine-zipper type DNA-binding protein GCN4 bZIP to the N-terminus of PYP. Five different fusion constructs of full length or truncated GCN4 bZIP and full length PYP as well as fusion constructs of full length GCN4 bZIP and N-terminally truncated PYP mutants were designed in a structure-based approach to determine if the dimerization and DNA binding activities could be controlled by the PYP photocycle. Extensive biophysical characterization of the fusion constructs in the dark and under blue light irradiation using electronic absorption, circular dichroism and fluorescence spectroscopic techniques were performed. As all the fusion proteins could complete photocycles, the DNA binding abilities of the dark and light-adapted states of the proteins were characterized using spectroscopic techniques as well as by the electrophoretic mobility shift assay. All the fusion constructs maintained DNA-binding abilities, however they each differed in their affinities and the extent to which they were activated by blue light irradiation. The reasons for these differences in DNA-binding abilities and photoactivation are explored. Using the results from the characterization of these constructs, proposals are also made to develop more robust genetically encoded photoactivatable DNA-binding proteins of the same type.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19628号 / 医科博第66号 / 新制||医科||5(附属図書館) / 32664 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 髙折 晃史, 教授 浅野 雅秀, 教授 小柳 義夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In this thesis, emphasis has been placed on development of new approaches for high-throughput analysis of protein:DNA interactions in vitro and in vivo. In vitro strategies for detection of protein:DNA interaction require isolation of active and soluble protein. However, current methodologies for purification of proteins often fail to provide high yield of pure and tag-free protein mainly because enzymatic cleavage reactions for tag removal do not exhibit stringent sequence specificity. Solving this problem is an important step towards high-throughput in vitro analysis of protein:DNA interactions. As a result, parts of this thesis are devoted to developing new approaches to enhance the specificity of a proteolysis reaction. The first approach was through manipulation of experimental conditions to maximize the yield of the desired protein products from enterokinase proteolysis reactions of two His-tagged proteins. Because it was suspected that accessibility of the EK site was impeded, that is, a structural problem due to multimerization of proteins, focus was based on use of denaturants as a way to open the structure, thereby essentially increasing the stoichiometry of the canonical recognition site over noncanonical, adventitious sites. Promoting accessibility of the canonical EK target site can increase proteolytic specificity and cleavage yield, and general strategies promoting a more open structure should be useful for preparation of proteins requiring endoprotease treatment. One such strategy for efficient EK proteolysis is proposed: by heterodimerizing with a separate leucine zipper, the bZIP basic region and amino-terminus can become more open and potentially more accessible to enterokinase. In vivo strategies have the advantage over their in vitro counterparts of providing a native-like environment for assessing protein:DNA interactions, yet the most frequently used techniques often suffer from high false-positive and false-negative rates. In this thesis, a new bioprobe system for high-throughput detection of protein:DNA interactions in vivo is presented. This system offers higher levels of accuracy and sensitivity as well as accessibility and ease of manipulation in comparison with existing technologies.

In this thesis, emphasis has been placed on development of new approaches for high-throughput analysis of protein:DNA interactions in vitro and in vivo. In vitro strategies for detection of protein:DNA interaction require isolation of active and soluble protein. However, current methodologies for purification of proteins often fail to provide high yield of pure and tag-free protein mainly because enzymatic cleavage reactions for tag removal do not exhibit stringent sequence specificity. Solving this problem is an important step towards high-throughput in vitro analysis of protein:DNA interactions. As a result, parts of this thesis are devoted to developing new approaches to enhance the specificity of a proteolysis reaction. The first approach was through manipulation of experimental conditions to maximize the yield of the desired protein products from enterokinase proteolysis reactions of two His-tagged proteins. Because it was suspected that accessibility of the EK site was impeded, that is, a structural problem due to multimerization of proteins, focus was based on use of denaturants as a way to open the structure, thereby essentially increasing the stoichiometry of the canonical recognition site over noncanonical, adventitious sites. Promoting accessibility of the canonical EK target site can increase proteolytic specificity and cleavage yield, and general strategies promoting a more open structure should be useful for preparation of proteins requiring endoprotease treatment. One such strategy for efficient EK proteolysis is proposed: by heterodimerizing with a separate leucine zipper, the bZIP basic region and amino-terminus can become more open and potentially more accessible to enterokinase. In vivo strategies have the advantage over their in vitro counterparts of providing a native-like environment for assessing protein:DNA interactions, yet the most frequently used techniques often suffer from high false-positive and false-negative rates. In this thesis, a new bioprobe system for high-throughput detection of protein:DNA interactions in vivo is presented. This system offers higher levels of accuracy and sensitivity as well as accessibility and ease of manipulation in comparison with existing technologies.

Transcription factors are proteins that regulate transcription of genes by binding to specific DNA sequences proximal to the gene. The specificity and affinity of protein-DNA recognition is critical for proper gene regulation. This thesis explores the mechanisms of binding to the sequence 5’CACGTG, a common recognition sequence both in plants where it is known as the G-box and in mammalian cells where it is termed the E-box. This sequence is of clinical interest because it is the target of the transcription factor Myc, an oncogene linked to many cancers. A number of alpha-helical proteins with different dimerization elements, from the basic region-leucine zipper (bZIP), basic region helix-loop-helix leucine zipper (bHLHZ) and basic region helix-loop-helix-PAS (bHLH-PAS) protein families, are capable of binding to this sequence. The basic regions of all these protein families contain residues that contact DNA and determine DNA sequence specificity while the other subdomains are responsible for dimerization specificity. First, the influence of protein-DNA contacts on sequence specificity of the plant bZIP protein EmBP-1 was probed by point mutations in the basic region. Residues that contact the DNA outside the core G-box sequence and residues that contact the phosphate backbone were found to be important for sequence specificity. Second, the impact of the dimerization subdomains of bHLHZ protein Max, the required heterodimerization partner of the Myc protein, and bHLH-PAS protein Arnt was probed by mutation, deletion and inter-family subdomain swapping studies. All studied protein families are intrinsically disordered, forming structure upon dimerization and DNA binding. The dimerization domains were found to indirectly influence DNA binding by affecting folding, dimerization ability or proper orientation of the basic regions relative to DNA. Lastly, a new strategy for selection of G-box binding proteins in the Yeast One-hybrid system is explored. Together, these studies broaden our understanding of the structure-function relationship of the DNA-binding activities of these closely related families of transcription factors. The creation and characterization of mutants with altered specificity, affinity and dimerization specificity may also be useful for biotechnology applications.

Transcription factors are proteins that regulate transcription of genes by binding to specific DNA sequences proximal to the gene. The specificity and affinity of protein-DNA recognition is critical for proper gene regulation. This thesis explores the mechanisms of binding to the sequence 5’CACGTG, a common recognition sequence both in plants where it is known as the G-box and in mammalian cells where it is termed the E-box. This sequence is of clinical interest because it is the target of the transcription factor Myc, an oncogene linked to many cancers. A number of alpha-helical proteins with different dimerization elements, from the basic region-leucine zipper (bZIP), basic region helix-loop-helix leucine zipper (bHLHZ) and basic region helix-loop-helix-PAS (bHLH-PAS) protein families, are capable of binding to this sequence. The basic regions of all these protein families contain residues that contact DNA and determine DNA sequence specificity while the other subdomains are responsible for dimerization specificity. First, the influence of protein-DNA contacts on sequence specificity of the plant bZIP protein EmBP-1 was probed by point mutations in the basic region. Residues that contact the DNA outside the core G-box sequence and residues that contact the phosphate backbone were found to be important for sequence specificity. Second, the impact of the dimerization subdomains of bHLHZ protein Max, the required heterodimerization partner of the Myc protein, and bHLH-PAS protein Arnt was probed by mutation, deletion and inter-family subdomain swapping studies. All studied protein families are intrinsically disordered, forming structure upon dimerization and DNA binding. The dimerization domains were found to indirectly influence DNA binding by affecting folding, dimerization ability or proper orientation of the basic regions relative to DNA. Lastly, a new strategy for selection of G-box binding proteins in the Yeast One-hybrid system is explored. Together, these studies broaden our understanding of the structure-function relationship of the DNA-binding activities of these closely related families of transcription factors. The creation and characterization of mutants with altered specificity, affinity and dimerization specificity may also be useful for biotechnology applications.

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