E. coli SecA is an essential component for protein translocaiton across membrane. SecA can be deleted from its N- and/or C-terminal ends without losing complementation activity. In this study, we determined the dispensity of both ends of SecA molecule. The minimal length at the SecA C-terminus is dependent on the length of the N-terminal region. SecA10-826 and SecA22-829 are the two minimal length SecAs. One more amino acid deleted at the C-terminal end completely abolished their complementation activity. A hydrophobic amino acid is required at the 826th amino acid in the minimal-length SecAs. Both SecA22-828 and SecA22-829 could form a dimer, and have decreased ATPase and protein translocation activities. The active truncated SecA mutants tended to have more soluble form than membrane-bound form, but were stably embedded in membrane. In contrast, the inactive truncated SecA mutants tended to have more membrane-bound form, but were not stable in membrane. Thus, the loss of complementation is not related to dimerization, ATPase and translocation activity but to certain extent related to their biased subcelluar localization and conformation in membrane. Isolated membranes of E coli strains were solubilized and fractionated by sucrose gradient fractionation. These membranes fractions were depleted of SecY and YidC, but contained SecD, SecF and GroEL. Proteoliposomes reconstituted from these fractionated membrane proteins were active in pOmpA translocation which required SecA and ATP. Membrane fractions from strain CK1801 in which the unc gene is deleted were reconstituted into liposomes and also showed translocation activities. Moreover, proteoliposomes reconstituted with Bacteriorodopsin alone were not active in translocation, while proteoliposomes reconstituted with Bacteriorodopsin and CK1801 membrane fractions showed elevated translocation efficiency. These data suggested that proton motive force is not obligatory for, but stimulatory to translocation of pOmA. Purified GroEL was reconstituted into lipsomes and the reconstituted proteoliposomes were active in pOmpA translocation although at lower efficiency. This translocation also required SecA and ATP. These results together suggested that translocation of pOmpA is active in the absence of SecY and YidC. In the absence of SecYEG, translocation of pOmpA requires SecA and ATP. GroEL, SecD and SecF may participate in the SecY-independent translocation.

Understanding how protein sequence, structure and function coevolve is at the core of functional genome annotation and protein engineering. The fundamental problem is to determine whether sequence variation contributes to functional differences or if it is a consequence of evolutionary divergence that is unrelated to functional specificity. To address this problem, we cannot merely analyze sequence variation between homologous proteins that have different functions. For comparison, we need to understand the factors that determine sequence variation in proteins that have the same function, such as a set of orthologous enzymes. Here, we address this problem by analyzing the evolution of functionally important residues in the o-succinylbenzoate synthase (OSBS) family. The OSBS family consists of several hundred enzymes that catalyze a step in menaquinone (Vit. K2) synthesis. Based on phylogeny, the OSBS family can be divided into eight major subfamilies. We assayed wild-type OSBS enzyme activities. The results show that the enzymes from γ-Proteobacteria subfamily 1 and Bacteroidetes have relatively low values, the enzyme from Cyanobacteria subfamily 1 is intermediate, and the values for the proteins from the Actinobacteria and Firmicutes subfamilies are relatively high. We are using computational and experimental methods to identify functionally important amino acids in each subfamily. Our data suggest that each subfamily has a different set of functionally important residues, even though the enzymes catalyze the same reaction. These differences may have accumulated because different mutations were required in each subfamily to compensate for deleterious mutations or to adapt to changing environments. We assessed the roles of these amino acids in enzyme structure and function. Our method achieved 70% successful rate to identify positions that play important roles in one family but not another. The residues P119 and A329 play important role in D. psychrophila but not in T.fusca OSBS. We also observed two class switch mutations in T.fusca, P11 and P22. The mutations at these two position have a similar kinetic parameters as wild-type D. psychrophila OSBS.

Calmodulin (CaM) is a ubiquitous cytosolic Ca2+-binding protein involved in the binding and regulation of more than three-hundred intracellular target proteins. CaM consists of two globular domains joined by a central linker region. In the archetypical model of CaM binding to a target protein, the Ca2+-replete CaM wraps its two domains around a single α-helical target peptide; however, other conformations of CaM bound to target peptides and proteins have recently been discovered. Due to its ability to bind and affect many different intracellular processes, there is significant interest in a better understanding of the structural and conformational basis of CaM’s ability to bind and recognize target proteins. The mammalian nitric oxide synthase (NOS) enzymes are bound and activated by CaM. The NOS enzymes catalyze the production of nitric oxide (•NO), a free radical involved in numerous intercellular processes such as neurotransmission, vasodilation, and immune defense. There are three different isoforms of nitric oxide synthase (NOS) found in mammals – neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All three enzymes are homodimeric with each monomer consisting of an N-terminal oxygenase domain and a multidomain C-terminal reductase domain. A CaM-binding domain separates the oxygenase and reductase domains. There is a unique opportunity to investigate CaM’s control over •NO production by the NOS enzymes since each isoform shows a different mode of activation and control by CaM. At elevated cellular Ca2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca2+. The focus of this thesis is to better our present understanding of the conformational and structural basis for CaM’s ability to bind and activate the three mammalian NOS isozymes with particular emphasis on the interactions between CaM and iNOS. To further investigate the differences in the association of CaM to the Ca2+-dependent and Ca2+-independent NOS isoforms, a variety of CaM mutants including CaM-troponin C chimeras, CaM EF hand pair proteins, and CaM mutants incapable of binding to Ca2+ were employed. The inherent differences in binding and activation observed using these CaM mutants is described. Differences in the binding of the N- and C-terminal domains, as well as the central linker of CaM to peptides corresponding to the CaM-binding domain of each NOS enzyme and holo-NOS enzymes was investigated. The conformation of CaM when bound to NOS peptides and holo-NOS enzymes was also studied using fluorescence (Förster) resonance energy transfer (FRET). A preliminary three-dimensional structural study of Ca2+-replete and Ca2+-deplete CaM in complex with an iNOS CaM-binding domain peptide is also described. Combining the cumulative results in this thesis, a working model for iNOS’s regulation by CaM is proposed. Future suggested experiments are described to further the characterization of CaM binding to the NOS enzymes and other CaM-target proteins. The studies described in this thesis have expanded and improved the present understanding of the CaM-dependent binding and activation of the NOS isozymes, particularly the interactions between CaM and iNOS.

Adenosine triphosphate (ATP) is the energy currency of all living cells and its production is a key reaction in the energy metabolism of living organisms. Cells produce most of the ATP they require through ATP synthase, a unique molecular rotary motor driven by the movement of protons across the lipid membrane. In E.coli, ATP synthase is composed of a soluble domain called F1, which houses the catalytic sites, and a transmembrane domain called F0 that shuttles protons across the membrane to drive ATP production in the F1 sector. The F0 domain is built of three subunit types: subunit a and a dimer of subunit b form the stator of the motor, while a decameric c ring forms the rotor. The dynamic interface between a and c10 forms the proton channel. The ultimate goal of this work is to determine the structure of the proton transport machinery and understand the molecular mechanism of proton translocation in ATP synthase. We have characterized some of the key events in the stepwise assembly of the F0--complex. We have designed and validated a model protein, consisting of genetically fused subunits a and c, for structural studies. We have made progress towards determining the structure of the proton channel, including the development of a novel procedure for purification of subunit a and the a/c fusion protein, and crystallization of subunit a. Medical applications of this work include the potential development of novel antibiotic compounds, as well as the characterization and potential treatment of three human diseases caused by disruptions in proton transport through F0.

Hemocytes are important as storage and producers of proteins of the innate immune defence, as well as actors of the cellular immune response. Therefore the hematopoietic process is critical for survival of most invertebrates. In order to search for molecules of importance for hemocyte development in crayfish we investigated proteins in crayfish plasma, which were increased after microbial challenge. As a result we were able to identify, purify and characterize a new invertebrate cytokine named astakine, and could clearly show that this protein is important for hematopoietic development in vivo as well as in an in vitro cell culture system. Astakine contains a prokineticin (PK) domain shown for the first time in an invertebrate, however, unlike the vertebrate PKs, astakine binds to a cell surface F1 ATP synthase β subunit located on the hematopoietic tissue (hpt) cell membranes. Extracellular ATP synthases as receptors have earlier been reported in different vertebrate cells and here we show that extracellular ATP synthase β subunit acts as a receptor for an invertebrate cytokine and is involved in hematopoiesis. We also found two other groups of proteins, which were increased in plasma after microbial challenge and they were further characterized. A great number of different Kazal type proteinase inhibitors were produced by the hemocytes and this type of proteinase inhibitors have variable reactive sites determining the specificity of their inhibition. In crayfish Kazal inhibitors with similar reactive sites were found as a response to specific microorganisms suggesting that the crayfish Kazal proteinase inhibitors may provide enough variability to participate in diverse innate immune reactions against different pathogens. Antimicrobial peptides were synthesized by the hemocytes and were likewise released in high amount upon microbial infection and we have characterized the main group of cystein-rich crustin-like antimicrobial peptides and investigated their tissue distribution and expression pattern.

Calmodulin (CaM) is a ubiquitous cytosolic Ca2+-binding protein involved in the binding and regulation of more than three-hundred intracellular target proteins. CaM consists of two globular domains joined by a central linker region. In the archetypical model of CaM binding to a target protein, the Ca2+-replete CaM wraps its two domains around a single α-helical target peptide; however, other conformations of CaM bound to target peptides and proteins have recently been discovered. Due to its ability to bind and affect many different intracellular processes, there is significant interest in a better understanding of the structural and conformational basis of CaM’s ability to bind and recognize target proteins. The mammalian nitric oxide synthase (NOS) enzymes are bound and activated by CaM. The NOS enzymes catalyze the production of nitric oxide (•NO), a free radical involved in numerous intercellular processes such as neurotransmission, vasodilation, and immune defense. There are three different isoforms of nitric oxide synthase (NOS) found in mammals – neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All three enzymes are homodimeric with each monomer consisting of an N-terminal oxygenase domain and a multidomain C-terminal reductase domain. A CaM-binding domain separates the oxygenase and reductase domains. There is a unique opportunity to investigate CaM’s control over •NO production by the NOS enzymes since each isoform shows a different mode of activation and control by CaM. At elevated cellular Ca2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca2+. The focus of this thesis is to better our present understanding of the conformational and structural basis for CaM’s ability to bind and activate the three mammalian NOS isozymes with particular emphasis on the interactions between CaM and iNOS. To further investigate the differences in the association of CaM to the Ca2+-dependent and Ca2+-independent NOS isoforms, a variety of CaM mutants including CaM-troponin C chimeras, CaM EF hand pair proteins, and CaM mutants incapable of binding to Ca2+ were employed. The inherent differences in binding and activation observed using these CaM mutants is described. Differences in the binding of the N- and C-terminal domains, as well as the central linker of CaM to peptides corresponding to the CaM-binding domain of each NOS enzyme and holo-NOS enzymes was investigated. The conformation of CaM when bound to NOS peptides and holo-NOS enzymes was also studied using fluorescence (Förster) resonance energy transfer (FRET). A preliminary three-dimensional structural study of Ca2+-replete and Ca2+-deplete CaM in complex with an iNOS CaM-binding domain peptide is also described. Combining the cumulative results in this thesis, a working model for iNOS’s regulation by CaM is proposed. Future suggested experiments are described to further the characterization of CaM binding to the NOS enzymes and other CaM-target proteins. The studies described in this thesis have expanded and improved the present understanding of the CaM-dependent binding and activation of the NOS isozymes, particularly the interactions between CaM and iNOS.

MosA is a protein found in <i>Sinorhizobium meliloti</i> L5-30 and has been suggested to be responsible for the biosynthesis of the rhizopine 3-O-methyl-scyllo-inosamine (3-MSI) from scyllo-inosamine (SI). However, we have shown MosA is a dihydrodipicolinate synthase (DHDPS) catalyzing the condensation of pyruvate with aspartate-β-semialdehyde (ASA). Since the DHDPS reaction occurs through a Schiff base aldol-type mechanism it was proposed that MosA could be an O-methyltransferase utilizing 2-oxo-butyrate (2-OB) as a novel methyl donor. This interesting yet unlikely possibility would explain MosA's role in the biosynthesis of 3-MSI without ignoring its similarity to DHDPS. Alternatively, MosA may have two catalytic domains one of which possesses a novel binding motif for S-Adenosyl methionine (SAM) to account for methyltransfer activity. In vitro demonstration of MosAs methyltransferase activity is required to resolve this apparent contradiction.<p>This dissertation describes the chemical synthesis of the rhizopines, investigation into whether MosA has a direct role in rhizopine biosynthesis and the thermodynamic characterization of compounds interacting with MosA as observed by isothermal titration calorimetry. <p>Initial investigation into MosAs methyltransferase activity began with 2-OBs interaction with the enzyme. Inhibition experiments determined 2-OB is a competitive inhibitor with respect to pyruvate of the DHDPS reaction of MosA. Furthermore, protein mass spectrometry of MosA in the presence of 2-OB and sodium borohydride indicated that a Schiff base enzyme intermediate was indeed being formed providing evidence that the proposed mechanism may exist. However, neither of the rhizopines had any effect on the DHDPS activity and HPLC assays determined that no 3-MSI was being produced by MosA in the presence of SI and 2-OB. Furthermore, HPLC assays failed to detect methyl transfer activity by MosA utilizing the SAM as a methyl donor. <p>Isothermal titration calorimetry provided thermodynamic characterization of the pyruvate and 2-OB Schiff base intermediates formed with MosA. In addition, ITC provided insight into the nature and thermodynamics of (S)-lysines inhibition of MosA. ITC failed to detect any interactions between the rhizopines or SAM with MosA. These results indicate that MosA is only a DHDPS and does not catalyze the formation of 3-MSI from SI as hypothesized in the literature.

Sepsis, a great challenge to the physician, is characterized with massive oxidative stress of tissue, cytokine inflammation and increases in nitric oxide (NO) production. Meanwhile, free radical induced by oxidative stress also injures cell membrane or DNA. The way to terminate free radical chain reaction is to administer antioxidant. The commonly used anesthetic, propofol, was thought to be with antioxidant capacity. In the first part of this thesis, we investigated the different role of oxidative injury and NO via systemic injection of LPS in rats. We demonstrated oxidative injury is associated with both early and late stage whereas NO is engaged primarily in late stage cardiovascular depression. Propofol, a rapid onset and fast recovery anesthetic, is attributed to protect anainst cardiovascular depression via attenuating the late stage NO surge in aorta by inhibition of iNOS upregulation. We also examine the influence of propofol on temporal changes in power density of frequency components of systemic arterial pressure (SAP) variability in rat with sepsis and the role of inducible NO synthase (iNOS). We have the conclusions that iNOS-induced NO might be involved in the manifestation of high-frequency and low-frequency components of the SAP spectrum during endotoxemia when low-dose propofol is used and the effect of NO is blunted when high-dose propofol is administered. Due to further investigation was needed to the cellular protective mechanisms of propofol, we delineate the effect of propofol to free radical related enzymel involved in sepsis via both in vivo and vitro studies with rats subjected to LPS (15 mg/kg) and H9C2, L2, NR8383 (derived from rat cardiac myocyte, lung, macrophage, respectively), respectively. Our results demonstrated that propofol may play the major protective role on iNOS, superoxide dismutase and p47 phox oxidative enzymes on lung epithelial cells. Propofol also provided protective effects on cardiac myocyte and macrophage with suppression of iNOS only although free radical production were all significantly suppressed. Ischemia-reperfusion (IR) injury may also produce a lot of free radical and cytokines to cause tissue damage and is common in clinical. We investigated the effect of propofol on free radical and cytokine production via this different model and compared with another rapid recovery anesthesitc, sevoflurane. Aortic decalmping surgery in porcine and their monocyte, aortic and coronary smooth muscle cells were applied for in vivo and in vitro model, respectively. We also demonstrated that propofol but not sevoflurane suppressed the production of free radical and cytokine in monocyte and smooth muscle cells but not in vivo model. In sepsis and IR model that produced a lot free radical and cytokines, propofol eliminated the free redical and cytokines via suppressed different kinds of oxidative enzymes in different cells of different organs to express its protective role. However, as an anesthetic, propofol must be used carefully to perform its maximal benefit.

Many inflammatory diseases are growing increasing common in the aging society of Taiwan. Inflammation cascades can cause diseases such as rheumatoid arthritis, osteoarthritis, chronic asthma, multiple sclerosis, and so on. The clinically used anti-inflammatory drugs have many side effects and are expensive. Therefore, it is imperative that we find alternatives to these drugs. Marine natural compounds offer great hope in the development of drugs for treating inflammatory diseases. In the present study, we found that Chao-10, which is a marine-derived compound isolated from Formosan soft coral, significantly inhibited the expression of the pro-inflammatory protein, inducible nitric oxide synthase (iNOS), in the lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophage cell line. We suggest that Chao-10 may serve as a potential new anti-inflammatory agent. However, the mechanism by which the anti-inflammatory effects of Chao-10 are mediated is yet unclear. Therefore, we performed two-dimensional electrophoresis (2-DE) to investigate the regulatory mechanism for the anti-inflammatory effect of Chao-10. We isolated some proteins that may be involved in the anti-inflammatory mechanism of Chao-10. In addition, we used immunoprecipitation to find that nucleophosmin (NPM) could interact with nuclear factor kappa B (NF-£eB). Therefore, we hypothesize that nucleophosminmay be involved in the regulation of NF-£eB to enhance the down-regulation of iNOS proteins. In summary, the anti-inflammatory effects of Chao-10 are probably mediated through the some other signaling pathway. Importantly, Chao-10 not only offers some new biomarkers of inflammation but also provides an encouraging outlook on therapeutic approaches.

In recent years, studies have increasingly recognized that many natural products with biological activity have been isolated from marine organisms, while the chemical structures are very different from those of land-based organisms. Therefore, the ocean is a natural drug source. Regarding drug screening, anti-inflammatory activity has become a key point, and many studies confirm that inflammation plays an important role in many human diseases. Many different compounds are now in the clinical evaluation stage. However, the inflammation-related diseases being closely linked, there is an urgent need to study the anti-inflammatory effects as well as screen the therapeutic drugs for research and development. In this study, we isolated and purified compounds from Formosan gorgonian (Briareum excavatum) and Formosan soft coral (Lobophytum sarcophytoides) and investigated biological activities. We confirmed that the natural compound Brei from B. excavatum and the compounds Sac-1 and Sac-2 from L. sarcophytoides produced significant inhibition of the proinflammatory proteins inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-induced murine macrophages (RAW 264.7) cell model. We examined in vivo whether the B. excavatum Brei has anti-inflammatory and antinociceptive effects by using the carrageenan-induced inflammation model. Using the paw-edema assay, we performed several important investigations such as the plantar analgesia test, mechanical hyperalgesia test (allodynia), and weight-bearing analysis of animal behavior to evaluate the degree of pain and inflammation. Our results demonstrate that the natural product Brei can reduce paw-pad swelling, thermal hyperalgesia, threshold latency, and improve the affected limb in the carrageenan-induced inflammatory model. In the histopathology analysis, we showed that Brei significantly inhibited the aggregation and infiltration of inflammation-related blood cells and improved the inflammatory status of the tissues. Therefore, the marine natural compound Brei has anti-inflammatory activity and it can be used as a therapeutic compound for acute inflammation in the near future.