No description available.

Student Number : 0002482E - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Proteins in the glutathione transferase family share a common fold. The close packing of secondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobic core. This fold has a bababba topology and most proteins/domains with this fold have a topologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alpha glutathione transferases are one of 12 classes within the glutathione transferase family. To investigate the role of the conserved isoleucine residue in the structure, function and stability of glutathione transferases, homodimeric human glutathione transferase A1-1 (hGST A1-1) was used as a representative of the GST family. Ile71 was replaced with valine and the properties of I71V hGST A1-1 were compared with those of wildtype hGST A1-1. The spectral properties monitored using far-UV CD and tryptophan fluorescence indicated little change in secondary or tertiary structure confirming the absence of any gross structural changes in hGST A1-1 due to the incorporation of the mutation. Both wildtype and mutant dimeric proteins were determined to have a monomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130 mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71V hGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-fold increase in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests the mutation had a negative effect on substrate binding. The DDG for transition state stabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise the transition state of the SNAR reaction involving the conjugation of reduced glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value for I71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the most noticeable change due to the mutation occurs at the H-site of the active site. Conformational stability studies were performed to determine the contribution of Ile71 to protein stability. The non-superimposability of I71V hGST A1-1 unfolding curves and the decreased m-value suggest the formation of an intermediate state. The conformational stability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of the wildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathione to wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightly to wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). This indicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGST A1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall the results suggest that Ile71 conservation is for the stability of the protein as well as playing a pivotal indirect role in catalysis and substrate binding.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18331号 / 農博第2056号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4838(農学部図書室) / 31189 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 三上 文三, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The role of membrane-intrinsic enzymes of lipid metabolism in complex biological processes is being realized through comprehensive structure function studies. Detailed analysis of substrate-enzyme interactions occurring within the restrictive membrane environment has proved to be exceedingly challenging. Using detergent micelles, we describe a detailed model for substrate recognition and binding by the outer-membrane intrinsic enzyme PagP from Escherichia coli. PagP is an 8-stranded antiparallel β-barrel that transfers a palmitoyl group from a phospholipid molecule to lipid A, the endotoxin component of lipopolysaccharide. This simple modification provides bacterial resistance to host antimicrobial peptides and attenuates the inflammatory response signalled through the host toll-like receptor 4 pathway. We describe a molecular embrasure and a crenel, which display weakened transmembrane β-strand hydrogen bonding, to provide site-specific routes for lateral entry of substrates into the PagP active site. A Tyr147 localized to the L4 loop gates the entry of the phospholipid substrate through the crenel, while lipid A enters via the embrasure. The side chains of the catalytic residues that are located in the extracellular loops point towards the central axis of the enzyme, directly above the active site. An acyl-chain binding pocket known as the hydrocarbon ruler is buried within the transmembrane β-barrel structure, and is optimized to accommodate a 16-carbon saturated palmitate chain. The hydrocarbon ruler, therefore, accounts for PagP's stringent selectivity for a palmitate chain. Substituting Gly88 lining the floor of the hydrocarbon ruler with residues possessing linear, unbranched, aliphatic side chains changes the selectivity of PagP to utilize shorter acyl chains. The serendipitous discovery of an exciton interaction between Trp66 and Tyr26 at the floor of the hydrocarbon ruler provides an intrinsic spectroscopic probe to monitor the methylene unit acyl-chain resolution of PagP. A compromised acyl chain resolution of the Gly88Cys mutant is attributed to an unexpected decrease of the Cys sulfhydryl group pKa within the β-barrel interior, resulting in a burying of a charged thiolate within the PagP core. The structural perturbation associated with the Cys thiolate extinguishes the exciton and expands the acyl-chain selectivity. These molecular details of lateral lipid diffusion and acyl-chain selection provide the first such example for any membrane-intrinsic enzyme of lipid metabolism. / Thesis / Doctor of Philosophy (PhD)

No description available.

Das SurA-Protein ist ein wichtiger Bestandteil der periplasmatischen Faltungsmaschinerie aus Escherichia coli. Trotz zahlreicher Erkenntnisse sind die Mechanismen der Substraterkennung und -bindung noch nicht abschließend geklärt. Das SurA-Protein ist aus einem Chaperonmodul und zwei PPIase-Domänen aufgebaut. Die Bindestelle eines artifiziellen Peptides wurde zu Beginn der Arbeit in der PPIase-inaktiven Parvulin-Domäne I publiziert. Im Rahmen dieser Arbeit wurde untersucht, ob auch biologisch relevante, natürliche Peptide an dieser Bindestelle interagieren und ob es noch weitere Substratbindestellen innerhalb von SurA gibt. In ESR-spektroskopischen Versuchen wurde die Interaktion der isolierten Parvulin-Domäne I von SurA mit Peptiden aus einer LamB-Peptid-Bibliothek, sowie mit dem artifiziellen Peptid analysiert. Die Bindung des artifiziellen Peptides und eines Peptides aus der LamB-Peptid-Bibliothek an die isolierte Parvulin-Domäne I konnte nachgewiesen werden. Für weitere an SurA-bindende Peptide konnte an dieser Position keine Interaktion nachgewiesen werden. Mittels des genetischen Indikatorsystems ToxR wurden gezielt Kontaktpunkte zwischen dimerisierten SurA-Untereinheiten bzw. zwischen SurA und Peptid unterbunden, um deren Einfluss auf die wechselseitige Interaktion zu untersuchen. Hierbei wurden einzelne Positionen in isolierten SurA-Domänen identifiziert, die an einer Interaktion beteiligt sind. Die Mutation dieser Interaktionsstellen führten zu keinem signifikanten Verlust der in vivo-Funktion, welche mittels der Fähigkeit der SurA-Varianten zur Komplementation des synthetisch letalen Phänotypen einer surA skp-Doppelmutante untersucht wurde. Die Grundlagen für die Methodik der photoaktivierbaren, ortsspezifischen Quervernetzung von OMP-Polypeptiden an SurA- bzw. SurAI-Proteine wurden etabliert. / The SurA protein is an important part of the periplasmic folding machinery in Escherichia coli. Despite numerous findings are the mechanisms of substrate recognition and folding not yet completely resolved. The SurA protein consists of a chaperone module and two parvulin domains. In the beginning of this work a peptide binding site was published which was located in the PPIase inactive parvulin domain I. It was investigated in this thesis whether biological relevant, natural peptides would also bind with this binding site and if additional substrate binding sites exist within the SurA protein. In ESR-spectroscopy experiments both the interaction of the isolated parvulin domain I of SurA with peptides of a LamB peptide library and with the artificial peptide were examined. Binding of the artificial peptide and one peptide of the LamB peptide library to the isolated parvulin domain I could be detected. For the remaining tested peptides, which are confirmed to be SurA binders, no interaction could be verified at this position. By use of the genetic indicator system ToxR the contact points between dimerized SurA subunits respectively between SurA and peptide were prevented site-specifically to examine their influence on the mutual interaction. Here single positions in isolated SurA-domains were identified, which are part of an interaction. The mutation of these interaction sites lead to no significant loss of the in vivo function, which was analyzed by the capability of the SurA variants to complement the synthetic lethal phenotype of a surA skp double mutant. The fundamentals for the method of photoactivated site-specific crosslinking of OMP polypeptides to SurA respectively SurAI were established.