京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第22797号 / エネ博第411号 / 新制||エネ||79(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 准教授 小瀧 努, 教授 森井 孝 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

No description available.

Rhomboids are a family of intramembrane serine proteases that cleave transmembrane protein substrates within the lipid membrane. They are involved in a wide range of biological processes, including signal transduction, parasite invasion, bacterial quorum sensing and apoptosis. While previous X-ray crystal structures and functional studies have provided some detailed insights into the mechanism of intramembrane hydrolysis, it is still not clear how the transmembrane substrate can gain access into the active site from the lipid environment. While several modes of action have been suggested, one hypothesis proposes a lateral movement of the fifth transmembrane helix, causing a displacement that would allow transmembrane substrates to enter the rhomboid active site. A powerful method that has the potential to yield insights into rhomboid dynamics is solution NMR; however, the large size of rhomboid protease samples has complicated conventional methods typically used to assess protein structure and dynamics. ¹⁹F NMR could allow the study of rhomboid conformational dynamics by providing a simplified spectrum with high sensitivity to changes in local chemical environments. In this thesis various methods of ¹⁹F incorporation were evaluated for utility in studying rhomboid conformational dynamics, focusing on the GlpG rhomboid from E. coli. First, GlpG samples were prepared with ¹⁹F incorporated into tryptophan sidechains, and 1D ¹⁹F NMR spectra were acquired. While spectra with decent spectral dispersion were obtained, the assignment process was complicated by low signal-to-noise, and multiple changes in the spectrum introduced by the mutation. Chemoselective labelling of cysteine residues with probes containing a trifluoromethyl group was also investigated and found to give rise to well resolved ¹⁹F NMR spectra with promising characteristics. In addition, protocols for incorporation of trifluoromethyl-phenylalanine using unnatural amino acid incorporation at introduced amber codon sites were also explored, since one of the long-term goals of this work is to study ¹⁹F-labelled GlpG in its native lipid environment. For this purpose, some protocol development was also performed to introduce GlpG into lipid nanoparticles using styrene maleic acid co-block polymers. However, low expression yields of trifluoromethyl-phenylalanine-labelled GlpG and the large size of the lipid nanoparticles are not yet compatible with solution NMR. Nonetheless, this thesis lays the groundwork for further development of these samples to allow the future study of conformational exchange of GlpG in native lipid membranes.

No description available.

Phenylalanine hydroxylase (PAH, EC: 1.14.16.1) is a non-heme iron tetrahydropterin-dependent monooxygenase that maintains phenylalanine (L-Phe) homeostasis via conversion of L-Phe to L-Tyr. PAH is an allosteric enzyme that converts from an inactive T-state to an active R-state upon addition of substrate, L-Phe. Allosteric activation is correlated with physical and structural changes within the enzyme and a large activation energy. Crystal structures of PAH have not identified the location of the allosteric effector binding site. Herein, we report computational protein mapping efforts using the FTmap algorithm and experimental site-directed mutagenesis studies designed to define and screen possible L-Phe allosteric binding sites. Mass spectroscopic analysis of PAH proteolytic fragments obtained after photo-crosslinking with 2-azido-3-phenylpropanoate overlapped with one computationally derived allosteric binding pocket containing residues 110-120 and 312-317. Ligand docking studies, fluorescence measurements, binding affinity and activity assays on wild-type and mutant enzymes further characterized the shape and specificity of this pocket. Thermodynamic studies using surface acoustic wave (SAW) biosensing determined the affinity of L-Phe for the allosteric site. Two L-Phe binding sites were observed upon SAW titrations, corresponding to the active and allosteric sites respectively ( K D,app^on 113 ± 12 µM active site, K D,app^on 680 ± 20 µM allosteric site). Site-directed mutagenesis was performed to prepare mutant enzymes containing a single tryptophan (L-Trp) residue. The fluorescence signatures of each of the three native L-Trp residues in PAH were determined by titrations with L-Phe. Trp187 primarily reports L-Phe induced allosteric conformational changes, while Trp120 reports active site L-Phe binding. Trp326 reports small signals of both active and allosteric site changes. Variable temperature stopped-flow fluorescence kinetic studies elucidated a working mechanism for L-Phe allosteric activation of PAH. Fluorescent signals from wild-type, single, and double L-Trp PAH mutants have been used to build kinetic mechanisms for the L-Phe binding in each subunit and subsequent active site reorganization or allosteric conformational change. In these mechanisms, the enzyme has reduced activity (1-2% of wtPAH) until both L-Phe induced active and allosteric site conformational changes have occurred. Failure of either activation step prevents enzyme turnover and is the chemical-based cause of the metabolic condition phenylketonuria.

Hedgehog (Hh) est un morphogène secrété qui contrôle la croissance et la différentiation cellulaire chez les métazoaires. La dérégulation de son activité entraine des maladies développementales et de nombreux cancers chez l’adulte. Chez la drosophile, la transduction du signal Hh est initiée par la fixation de Hh sur son récepteur Patched (Ptc), conduisant à la stabilisation de la protéine membranaire Smoothened (Smo) et à l’activation du complexe de transduction composé de 5 protéines : les kinases Fused (Fu), PKA, GprK2, la kinésine Costal 2 (Cos2), et le facteur de transcription Cubitus Interruptus (Ci). Ma thèse a porté sur l’étude de la régulation et des interactions moléculaires entre les composants du complexe de transduction. Par des approches complémentaires, j’ai montré qu’en absence d’Hh, les protéines PKA et Fu interagissent du côté C-terminal de Ci, alors que la présence d’Hh induit leur relocalisation vers le domaine N-terminal de Ci. J’ai pu prouver que l’élément déclencheur de ce remaniement protéique est Smo. En présence d’Hh, Smo s’incorpore dans le complexe de transduction, conduisant à l’activation et au déplacement de Fu vers la région N-terminale de Ci. Ce remaniement entraine la phosphorylation et l’activation de Ci. Ma thèse révèle l’importance des changements de conformation au sein du complexe de transduction de la voie Hh. Le mécanisme de transduction étant conservé entre invertébrés et invertébrés, mon doctorat apporte des éléments de recherche pour mieux comprendre le fonctionnement normal et pathologique des cellules. / Hedgehog (Hh) is a secreted morphogen that controls growth and differentiation in both vertebrates and invertebrates. The dysregulation of its activity leads to severe developmental defects, and the onset of cancer in adults. In Drosophila, the Hh signal transduction is initiated by the binding of Hh to its receptor Patched (Ptc). This induces the stabilization of the transmembrane protein Smoothened (Smo) and the subsenquent activation of a transduction complex consisting of 5 proteins: the kinases Fused (Fu), PKA and Gprk2, the kinesin Costal2 (Cos2), and the transcription factor of the pathway Cubitus Interruptus (Ci). The aim of my thesis was to study the regulation and molecular interactions between the different components of the transduction complex. Thanks to complementary techniques, I have shown that in absence of Hh the proteins Fu and PKA interact in C-terminal part of Ci, whereas on the presence of Hh induces their relocalization toward the N-terminal domain of Ci. I have proved that the trigger element of this moving is Smo. In presence of Hh, Smo goes into transduction complex, allowing the activation and the moving of Fu toward N-terminal domain of Ci. This relocalization is responsible of Ci phosphorylation and activation. My thesis reveals the importance of conformational changes inside the transduction complex of Hh pathway. As the mechanism of transduction is conserved between species, my PhD provides research elements in order to better understand the normal and abnormal functioning of cells.

Protein folding studies have provided important insights about the key role of non-covalent interactions in protein structure and conformational stability. Some of these interactions include salt bridges, cation-π, and anion-Ï€ interactions. Understanding these interactions is crucial to developing methods for predicting protein secondary, tertiary, quaternary structure from primary sequence and understanding protein-protein interactions and protein-ligand interactions. Several studies have described how the interaction between two amino acid side chains have a substantial effect on protein structure and conformational stability. This is under the assumption that the interaction between the two amino acids is independent of surrounding interactions. We are interested in understanding how salt bridges, cation-π, and anion-π interactions affect each other when they are in close proximity. Chapter 1 is a brief introduction on noncovalent interactions and noncovalent interaction cooperativity. Chapter 2 describes the progress we have made measuring the cooperativity between noncovalent interactions involving cations, anions and aromatic amino acids in a coiled-coil alpha helix model protein. Chapter 3 describes cooperativity between cation, anion, and nonaromatic hydrophobic amino acids in the context of a coiled-coil alpha helix. In chapter 4 we describe a strong anion-π interaction in a reverse turn that stabilizes a beta sheet model protein. In chapter 5 we measure the interaction between a cysteine linked maleimide and two lysines in a helix and show that it is a general strategy to stabilize helical structure.

PEGylation has been used for decades to enhance the pharmacokinetic properties of protein therapeutics. This method has been effective at increasing the serum half-life of these drugs, but the mechanism of how it does this is unclear. Chapter 1 is an introduction to the methods of PEGylation. In chapter 2 we show that the effect of PEGylation on the conformational stability of the WW domain differs based on amino acid linker and conjugation site. We show that all positions in the WW domain that were tested can be stabilized by at least one amino acid linker. The rate of proteolysis is proportional to the degree of conformational stability. Chapter 3 shows that PEG-based desolvation can increase the strength of the interaction between two salt bridge residues, though the effect of structural context is unclear. A crystal structure shows that PEG occupies the space between the PEGylation site and the salt bridge, displacing water. In Chapter 4 we discuss the effect that PEGylation has on the interaction strength of a solvent exposed hydrophobic patch. When the c Log P of the hydrophobic patch increases, PEG increases the conformational stability of the WW domain more dramatically. Chapter 5 is about the effect of PEG based desolvation on the strength of an NH-π hydrogen bond in the WW domain between Trp11 and Asn26. When Trp11 is mutated to Phe, Tyr and naphthylalanine (Nal), the melting temperatures correlate with the calculated interaction energies between the sidechain arene of the hydrogen bond acceptor and formamide. When Asn26 is PEGylated in the presence of each of these amino acids, the effect that PEG has on the conformational stability of the WW domain correlates with the melting temperature of the nonPEGylated variants, the calculated interaction energies, the arene molecular polarizability, and the arene molar volume.

<p>Phospholipase Cs (PLCs) are a family of enzymes that hydrolyze membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to generate inositol triphosphate (IP3) and diacylglycerol (DAG). These second messengers activate a variety of intracellular responses, including inflammation, vascular smooth muscle contraction, and cardiac hypertrophy. While much is known about how Gaq-mediated activation of PLCb occurs, the same cannot be said for Gbg-mediated activation. Residues within the PLCb-Gbg binding interface were previously identified in interior regions of the protein, suggesting the PH domain must undergo a conformational change to allow for Gbg-mediated activation. However, the role of PH domain conformational dynamics in Gbg-mediated activation of PLCb has yet to be determined. In this work, I discuss efforts to characterize conformational dynamics of the PLCb PH domain and its role in interactions of the enzyme with liposomes and Gbg. First, I generated a disulfide crosslink between the PH domain and EF hands1/2 of PLCb3, purified under oxidizing or reducing conditions, and conducted biochemical and structural tests to determine any differences in structure and/or function of the protein as compared to wild-type. Results of these studies provided the first direct structural evidence of PLCb PH domain dynamics in solution. Then, I discuss the rationale behind the generation of a surface cysteine-less PLCb for use in solvatochromic fluorescence assays in the presence and absence of liposomes and Gbg. Initial results of these studies suggest the PLCb PH domain favors a buried conformation alone and in the presence of Gbg or liposomes, and likely exists at an equilibrium between open and closed states.</p>

This paper describes our continuing studies on stabilization of peptide structures in supersecondary conformations that are designed to mimic conformational antigenic epitopes. In this work we have used the consensus Cys2His2 zinc-finger peptide motif as a template to engineer and synthesize antigenic loop peptide segments from two protein antigens, lactate dehydrogenase C4 isozyme (LDH-C4) and human chorionic gonadotropin β subunit (β-hCG). Confirmation that the engineered peptide constructs assumed a zinc-finger conformation was obtained by absorption spectroscopy of the Co2+ complexes. The circular dichroism (CD) spectra of the free peptides show random coil conformations, while the Zn2+-complexed peptides acquired the zinc-finger motif upon titration with Zn2+, as evidenced by the appearance of absorbances indicating α-helix and some β-conformation. No peptide aggregation was observed, as these peptides were monomeric under all conditions tested. In order to examine the immunogenicity of the zinc-finger constructs, one sequence from LDH-C4 (ZFLMVF) and two sequences from β-hCG (ZF2TT3 and ZF4TT3) were selected and chimeras were synthesized to incorporate promiscuous T-cell epitopes from either tetanus toxoid or measles virus. The ZFLMVF construct was highly immunogenic in rabbits, and the ZF2TT3 and ZF4TT3 peptides were highly immunogenic in both mice and rabbits, eliciting high-titer antipeptide antibodies specific for their immunogenic sequences. However, the antibodies raised to the zinc-finger constructs showed minimal reactivity against their respective native protein antigens as determined by ELISA. This is surprising in the case of β-hCG, since the ZF2 zinc-finger peptide was an effective inhibitor of binding of anti-β-hCG-loop(38-57) antibodies to whole hCG, as assessed by a competitive inhibition radioimmunoassay. This implies that, although the cyclized 40-52 sequence from βhCG and the zinc-finger peptide ZF2 exhibit similar conformations in solution, the zinc-finger engineered loop is apparently not in a sufficiently correct conformation for antibody recognition of native hCG. Our results with the LDH-C4 zinc finger loop imply that antibody recognition of antigen involves specific side-chain interactions that must be maintained by a precise conformation.