Li, Man Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-119). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VI / Abbreviations --- p.VIII / Abbreviations of Chemicals --- p.X / List of Tables --- p.XI / List of Figures --- p.XII / Table of Contents --- p.XIII / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- General amino acid control in yeast --- p.1 / Chapter 1.2 --- Mammalian eIF2α kinases --- p.7 / Chapter 1.2.1 --- Heme-regulated inhibitor kinase (EIF2AK1/HRI) --- p.7 / Chapter 1.2.2 --- Protein kinase dsRNA-dependent (EIF2AK2/PKR) --- p.8 / Chapter 1.2.3 --- PKR-like ER kinase (EIF2AK3/PERK) --- p.9 / Chapter 1.2.4 --- General control non-repressible 2 (EIF2AK4/GCN2) --- p.10 / Chapter 1.2.5 --- Activating transcription factor 4 (ATF4) --- p.11 / Chapter 1.3 --- Plant General Amino Acid Control --- p.12 / Chapter 1.3.1 --- Studies of the homolog of GCN2 in Arabidopsis thaliana --- p.12 / Chapter 1.3.2 --- Studies of the homolog of other eIF2a kinase in plant --- p.14 / Chapter 1.3.3 --- Studies of the homolog of other GAAC components --- p.14 / Chapter 1.4 --- Previous works in our lab --- p.15 / Chapter 1.5 --- Hypothesis and Objectives --- p.17 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.18 / Chapter 2.1.1 --- "Bacterial cultures, plant materials and vectors" --- p.18 / Chapter 2.1.2 --- Primers --- p.21 / Chapter 2.1.3 --- Commercial kits --- p.25 / Chapter 2.1.4 --- "Buffer, solution, gel and medium" --- p.25 / Chapter 2.1.5 --- "Chemicals, reagents and consumables" --- p.25 / Chapter 2.1.6 --- Enzymes --- p.25 / Chapter 2.1.7 --- Antibodies --- p.25 / Chapter 2.1.8 --- Equipments and facilities --- p.25 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- Growth conditions of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.1 --- Surface sterilize of Arabidopsis thaliana seed --- p.26 / Chapter 2.2.1.2 --- Growing of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.3 --- Treatment of Arabidopsis seedling --- p.26 / Chapter 2.2.2 --- Basic molecular techniques --- p.27 / Chapter 2.2.2.1 --- Liquid culture of Escherichia coli --- p.27 / Chapter 2.2.2.2 --- Preparation of plasmid DNA --- p.27 / Chapter 2.2.2.3 --- Restriction digestion --- p.27 / Chapter 2.2.2.4 --- DNA purification --- p.28 / Chapter 2.2.2.5 --- DNA gel electrophoresis --- p.28 / Chapter 2.2.2.6 --- DNA ligation --- p.29 / Chapter 2.2.2.7 --- CaCl2 mediated E. coli transformation --- p.29 / Chapter 2.2.2.8 --- Preparation of DNA fragment for cloning --- p.29 / Chapter 2.2.2.9 --- PCR reaction for screening positive E. coli transformants --- p.30 / Chapter 2.2.2.10 --- DNA sequencing --- p.30 / Chapter 2.2.2.11 --- RNA extraction from plant tissue with tRNA --- p.31 / Chapter 2.2.2.12 --- Extraction of RNA without tRNA --- p.31 / Chapter 2.2.2.13 --- cDNA synthesis --- p.32 / Chapter 2.2.2.14 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.2.2.15 --- Western blotting --- p.33 / Chapter 2.2.3 --- Sub-cloning of AtGCN2 --- p.34 / Chapter 2.2.3.1 --- Sub-cloning full length AtGCN2 into pMAL-c2 --- p.36 / Chapter 2.2.3.2 --- Sub-cloning of the N-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.3.3 --- Sub-cloning of the C-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.4 --- Cloning of the eIF2α candidates for the in vitro assay --- p.41 / Chapter 2.2.4.1 --- Cloning of At2g40290 (putative eIF2α candidate) --- p.41 / Chapter 2.2.4.2 --- Cloning of At5g05470 (putative eIF2α candidate) into pBlueScript KS II + --- p.43 / Chapter 2.2.4.3 --- Sub-cloning of At5g05470 into pGEX-4T-1 --- p.43 / Chapter 2.2.4 --- Expression and purification of fusion proteins --- p.45 / Chapter 2.2.5 --- Expression of fusion proteins in E. coli --- p.45 / Chapter 2.2.5.2 --- Extraction of E. coli soluble proteins --- p.45 / Chapter 2.2.5.3 --- Purification of GST tagged fusion protein --- p.46 / Chapter 2.2.5.4 --- Purification of MBP tagged fusion protein --- p.46 / Chapter 2.2.5.5 --- Concentration of purified fusion proteins --- p.46 / Chapter 2.2.5.6 --- MS/MS verification of purified fusion proteins --- p.47 / Chapter 2.2.6 --- Gel mobility shift assay --- p.47 / Chapter 2.2.6.1 --- Synthesis of short biotinylated RNA --- p.47 / Chapter 2.2.6.2 --- Ligation of short biotinylated RNA with tRNA --- p.48 / Chapter 2.2.6.3 --- Gel mobility shift assay --- p.48 / Chapter 2.2.6.4 --- Blotting of the sample on to nitrocellulose membrane --- p.48 / Chapter 2.2.6.5 --- Detection of the tRNA on the membrane --- p.49 / Chapter 2.2.6.6 --- Detection of the MBP fusion proteins on the membrane --- p.49 / Chapter 2.2.7 --- In vitro kinase assay of AtGCN2 --- p.49 / Chapter 2.2.8 --- In vitro translation inhibition assay --- p.50 / Chapter 2.2.8.1 --- In vitro transcription of HA mRNA --- p.50 / Chapter 2.2.8.2 --- In vitro translation --- p.51 / Chapter 2.2.8.3 --- Detection of the protein dot blot --- p.51 / Chapter 2.2.9 --- Gene expression analysis by real time PCR --- p.52 / Chapter 2.2.10 --- Total seed nitrogen analysis --- p.53 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Blast search results suggested that AtGCN2 may be the sole eIF2α kinase in Arabidopsis thaliana --- p.54 / Chapter 3.2 --- Existence of two eIF2α candidates in Arabidopsis thaliana genome --- p.59 / Chapter 3.3 --- Fusion proteins were successfully expressed and purified --- p.63 / Chapter 3.4 --- C-terminal of AtGCN2 has a higher affinity toward tRNA than rRNA --- p.67 / Chapter 3.5 --- Both eIF2α candidates can be phosphorylated by full length AtGCN2 in vitro --- p.70 / Chapter 3.6 --- AtGCN2 can inhibit translation in vitro --- p.72 / Chapter 3.7 --- Overexpression of AtGCN2 did not affect expression of selected genes --- p.74 / Chapter 3.8 --- Overexpression of AtGCN2 did not affect seed nitrogen content and C:N ratio under normal growth conditions --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Existing evidence supported that AtGCN2 is the sole eIF2α kinase in Arabidopsis thaliana --- p.85 / Chapter 4.2 --- Kinase activities of AtGCN2 and its two substrates in Arabidopsis --- p.86 / Chapter 4.3 --- C-terminal binds tRNA in the gel mobility shift assay --- p.88 / Chapter 4.4 --- Overexpression of AtGCN2 did not affect gene expression of the transgenic lines under nitrogen starvation and azerserine treatment --- p.90 / Chapter 4.5 --- Overexpression of AtGCN2 did not alter the seed nitrogen content --- p.91 / Chapter 4.6 --- Existence of GCN4 and ATF4 in plant --- p.92 / Chapter 4.7 --- Alternative model without GCN4 and ATF4 homolog --- p.93 / Chapter 4.8 --- Possible application of the in vitro kinase assay --- p.94 / Chapter 4.9 --- Possible application of the in vitro translation inhibition analysis platform in future study --- p.95 / Chapter Chapter 5 --- Conclusion and Future Prospective --- p.97 / Appendices / Appendix I Commercial kits used in this project --- p.98 / "Appendix II Buffer, solution, gel and medium" --- p.99 / "Appendix III Chemicals, reagents and consumables" --- p.102 / Appendix IV Enzymes --- p.103 / Appendix V Antibodies --- p.104 / Appendix VI Equipments and facilities --- p.105 / Appendix VII Supplementary Data --- p.106 / Appendix VIII Amplification efficiency of real time primers --- p.108 / References --- p.109

Post-translational modification (PTM) of proteins by enzymes such as methyltransferases, kinases and deacetylases play a crucial role in the regulation of many metabolic pathways. Determining the substrate scope of these enzymes is essential when studying their biological role. However, the combinatorial nature of possible protein substrate sequences makes experimental screening assays intractable. To predict new substrates for proteins, various computational approaches have been developed. Our method relies on crystallographic data and a novel multistate computational protein design algorithm. We previously used our method to successfully predict four new substrates for SMYD2 (Lanouette S & Davey J.A., 2015), doubling the number of known targets for this PTM enzyme that has been difficult to characterize using other methods. This was possible by first extracting a specificity profile of Smyd2 using our algorithm and subsequently screening a peptide library for matching sequences. However, our method did not yield successful results when attempting to reproduce specificity profiles of other proteins (64% accuracy on average). Different protein environments have demonstrated limitations in the methodology and lead us to further develop the algorithm on a more thorough dataset. Using our new optimized method, specificity profile predictions increase by roughly 20% (84% accuracy on average), independent of the structural template used. The algorithm was then used to blindly predict a specificity profile for the methyltransferase Smyd3, an enzyme for which limited data is currently available. A library of 2550 peptides was screened with the predicted profile, yielding 123 matching sequences. We randomly chose 64 for experimental validation (SPOT peptide array) of methylation by Smyd3 and found 45 methylated and 19 non-methylated peptides (70% success rate). Finally, we released to the community a web version of the algorithm, which can be accessed as http://viper.science.uottawa.ca.

An effective and safe vaccine against Hepatitis B virus already exists, yet morbidity and mortality of this illness are still high. The key to developing a reliable treatment is a deep knowledge of the virus' life cycle and functions of all its components. In the presented work we explored an interactome of the Core protein of the Hepatitis B virus. Using proximity-dependent biotin identification technique (BioID) coupled to mass spectrometry we have identified a list of potential candidates that are either significantly enriched (in total 105 proteins) or less abundant in the presence of the HBV Core protein in the cell (40 proteins). The list also includes known HBV Core interacting proteins SRPK1 and SRPK2, and p53 protein whose expression is known to be repressed due to the HBV Core interaction with the E2F1 transcription factor. Many of the newly identified possible HBV Core interacting proteins are involved in biological processes already known or are suspected to be influenced by the HBV such as translational and transporting processes or gene expression and macromolecule production. Overall, this work comprehensively characterizes the interaction landscape of the HBV Core protein in the live cells and might thus serve as a reliable start for in depth HBV-host interaction analysis. Key...

Proteine bestehen aus einer spezifischen Sequenz verschiedener Aminosäuren, die ihre charakteristische Funktion bestimmt. Die große Variabilität an Aminosäuresequenzen ermöglichte die Evolution einer nahezu unbegrenzten Anzahl an Proteinen. Meistens nehmen diese Schlüsselpositionen ein, von robusten Baustoffen bis hin zu molekularen Maschinen. Daher kann eine Fehlfunktion gravierende Auswirkungen auf das Leben haben, z.B. Krankheiten wie Alzheimer oder Epilepsi. Um die Funktionen und Fehlfunktionen zu verstehen, ist eine umfassende Kenntnis der Proteinfaltung, der Protein-Protein Assoziation, sowie den Dynamiken innerhalb von Proteinen erforderlich. Diese Vorgänge wurden in dieser Arbeit an drei isolierten Proteindomänen durch die Anwendung der Fluoreszenzlöschmechanismen der H-Dimerbildung und des photoinduzierten Elektronentransfers untersucht. Der entfaltete Zustand der Bindungsdomäne BBL, das Teil des 2-oxo-acid Dehydrogenasekomplexes ist, wurde unter physiologischen Bedingungen mit Zirkulardichroismus (CD) und einer Kombination aus photoinduziertem Elektronentransfer und Fluoreszenzkorrelationsspektroskopie analysiert. Beide Methoden zeigten übereinstimmend anhand von 20 in BBL einzeln eingefügten konservativen Punktmutationen, dass Seitenketteninteraktionen keine Auswirkungen auf die Sekundärstruktur des denaturierten Zustandes, den Ausgangspunkt der Faltung, haben. Mit Hilfe der Dekonvolation der CD-Spektren wurde zudem gezeigt, dass die Reststruktur im denaturierten Zustand der helikalen Proteindomäne von β-Strängen und β-Kehren dominiert wird, die eine entscheidende Funktion bei der Faltung in den nativen Zustand haben könnten. Die N-terminale Domäne (NTD), der für die Materialforschung hochinteressanten Spinnen-seidenfaser, ist für die Polymerisation des Spinnenseidenfadens auf den pH-Wechsel von pH 7 auf pH 6 hin verantwortlich. Dieser für die Proteinfunktion wichtige Prozess wurde durch die Einbringung eines extrinsischen Fluoreszenzschalters, basierend auf der H-Dimerbildung, mit der Stopped-Flow-Technik untersucht. Es wurde gezeigt, dass die NTDs 104 mit einer Rate von 3 x 10^8 M-1 s-1 assoziieren und somit nahezu das Geschwindigkeitslimit der Protein-Protein Assoziation erreicht wird. Zwei geladenen Seitenketten, der D39 und D40, kommt eine entscheidende Funktion in dem Prozess zu, da eine Mutation dieser die Assoziation verhindert. Des Weiteren wurde gezeigt, dass sich die NTD auf eine Erhöhung der Ionenstärke entgegengesetzt zu anderen Proteinen verhält: die Dissoziation wird beschleunigt, die Assoziation nicht beeinflusst. Gleiches Verhalten wurde auf den einzelnen Austausch der übrigen protonierbaren Aminosäureseitenketten hin beobachtet, ausgenommen die Mutation der E119, welche die Dissoziation verlangsamt. Daher scheint der makromolekulare Dipol, der auf Grund der Ladungsverteilung in der NTD entsteht, die Assoziation maßgeblich zu beeinflussen. Glutamatrezeptoren sind an der schnellen synaptischen Signalweiterleitung im Nervensys-tem von Vertebraten beteiligt. Die Konformationen der Ligandenbindungsdomäne (LBD) haben dabei entscheidende Auswirkungen auf die Funktion des Gesamtrezeptors. Diese wurden mit einer Kombination aus photoinduziertem Elektronentransfer und Fluoreszenzkorrelationsspektroskopie untersucht. Mit dieser Methode wurde ein dynamisches Bild der gebundenen sowie ungebundenen Form der AMPA-spezifischen Glutamatrezeptor 2-LBD gezeigt. Es wurde zudem gezeigt, dass sich die Dynamiken in Abhängigkeit der Bindung von den Agonisten Glutamat und AMPA, dem partiellen Agonisten Kainate oder Cyclothiazid (CTZ), welches eine Dimerisierung der LBDs bewirkt, unterschiedlich verändern. Dies könnte eine Auswirkung auf die Funktion der Rezeptoren haben. Die Anwendung der Fluoreszenzlöschmechanismen der H-Dimerbildung und des photoinduzierten Elektronentransfers in dieser Arbeit hat gezeigt, dass diese die Möglichkeit bieten, unterschiedlichste Fragestellungen zu beantworten und so Einblicke in dynamische Funktionsweisen von Proteinen eröffnen. Kombiniert mit etablierten Fluoreszenzmethoden ist es so möglich quantitativ Kinetiken auf unterschiedlichen Zeitskalen zu untersuchen. / Proteins are composed of a specific sequence of variable amino acids that specify their specific function. The vast variability of possible amino acid sequences allowed for the evolution of a nearly infinite number of different proteins. Mostly these will have key positions, reaching from strong building material to molecular machines. Therefore a malfunction will have an immense effect on life, i.e. diseases like Alzheimer disease or epilepsy. In order to understand the function and malfunction an intense understanding of the protein folding, the protein-protein association, as well as the protein dynamics is essential. These processes have been investigated in this thesis with three isolated protein domains by the application of the fluorescence quenching mechanisms of the H-dimer-formation and by the photoinduced electron transfer. The unfolded state of the binding domain BBL, which is part of the 2-oxo-acid-dehydrogenase-complex, was analysed under physiological conditions by circular dichroism (CD), and a combination of photoinduced electron transfer and fluorescence correlation spectroscopy. Both methods showed accordingly, using 20 singly implanted conservative point mutations in BBL, that side-chain interactions have no effect on the secondary struc-ture of the denatured condition, the initial state of the folding. Using deconvolution on the CD spectra it was shown in addition, that the remaining structure of the helical protein domain in the denatured state is dominated by β-strands and β-turns. These may have a decisive function during the folding in the native state. The N-terminal domain of the spider silk fibre, which is of high interest for the material research, is responsible for the polymerisation of the spider silk fibre during the pH-change from pH 7 to pH 6. This important process for the protein function was investigated with the Stopped-Flow technique by the application of an extrinsic fluorescence switch, based on the H-dimer formation. It was shown, that NTDs associate with a rate of 3 x 10^8 M-1 s-1, and so nearly reach the speed limit of the protein-protein association. In this process two charged side chains, the D39 and D40, have a decisive function, as a mutation of these will 106 avoid the association. Furthermore it was shown, that the NTD will react in opposition to other proteins on the increase of the ionic strength: the dissociation will be speeded up, and the association is not influenced. The identical behaviour was observed with the single ex-change of the other protonable amino acid side chains, except for the mutation of E119, which retards dissociation. Therefore the macromolecular dipol, that is formed by the charge distribution of the NTD, is obviously influencing the association. Glutamate receptors participate in the fast synaptic signal transfer in vertebrates. Here dif-ferent conformations of the ligand binding domain (LBD) have decisive effects on the function of the entire receptor. These have been investigated with a combination of photoin-duced electron transfer and fluorescence correlation spectroscopy. Using this method a dynamic behaviour of the bound and unbound form of the AMPA specific glutamate receptor 2 LBD was shown. Furthermore it was shown, that the dynamics will change differently in dependence of the binding of the agonist glutamate and AMPA, the partial agonist kainate or Cyclothiazide (CTZ), which effects a dimerization of the LBDs. This may have an effect on the function of the receptors. The use of the fluorescence quenching mechanisms of the H-dimer formation and the pho-toinduced electron transfer in this thesis has shown, that these offer the opportunity to an-swer a large range of questions and open a view to the dynamic functionality of proteins. Combined with established fluorescence methods it is possible to quantitatively investigate kinetic rates at different time scales.

Protein-protein interactions play a crucial role in the development of drug delivery devices for the increasingly important biologicals, including antibodies, growth factors and cytokines. The understanding thereof might offer opportunities for tailoring carriers or drug proteins specifically for this purpose and thereby allow controlled delivery to a chosen target. The possible applications range from trigger-dependent release to sustained drug delivery and possibly permanently present stimuli, depending on the anticipated mechanism. Silk fibroin (SF) is a biomaterial that is suitable as a carrier for protein drug delivery devices. It combines processability under mild conditions, good biocompatibility and stabilizing effects on incorporated proteins. As SF is naturally produced by spiders and silkworms, the understanding of this process and its major factors might offer a blueprint for formulation scientists, interested in working with this biopolymer. The natural process of silk spinning covers a fascinating versatility of aggregate states, ranging from colloidal solutions through hydrogels to solid systems. The transition among these states is controlled by a carefully orchestrated process in vivo. Major players within the natural process include the control of spatial pH throughout passage of the silk dope, the composition and type of ions, and fluid flow mechanics within the duct, respectively. The function of these input parameters on the spinning process is reviewed before detailing their impact on the design and manufacture of silk based drug delivery systems (DDS). Examples are reported including the control of hydrogel formation during storage or significant parameters controlling precipitation in the presence of appropriate salts, respectively. The review details the use of silk fibroin to develop liquid, semiliquid or solid DDS with a focus on the control of SF crystallization, particle formation, and drug-SF interaction for tailored drug load. Although we were able to show many examples for SF drug delivery applications and there are many publications about the loading of biologics to SF systems, the mechanism of interaction between both in solution was not yet extensively explored. This is why we made this the subject of our work, as it might allow for direct influence on pharmaceutical parameters, like aggregation and drug load. In order to understand the underlying mechanism for the interaction between SF and positively charged model proteins, we used isothermal titration calorimetry for thermodynamic characterization. This was supported by hydrophobicity analysis and by colloidal characterization methods including static light scattering, nanoparticle tracking analysis and zeta potential measurements. We studied the effects of three Hofmeister salts – NaCl (neutral), NaSCN (chaotropic) and Na2SO4 (cosmotropic) – and the pH on the interaction of SF with the model proteins in dependence of the ratio from one to another. The salts impacted the SF structure by stabilizing (cosmotropic) or destabilizing (chaotropic) the SF micelles, resulting in completely abolished (cosmotropic) or strongly enhanced (chaotropic) interaction. These effects were responsible for different levels of loading and coacervation when varying type of salt and its concentration. Additionally, NaCl and NaSCN were able to prolong the stability of aqueous SF solution during storage at 25°C in a preliminary study. Another approach to influence protein-protein interactions was followed by covalent modification. Interleukin-4 (IL-4) is a cytokine driving macrophages to M2 macrophages, which are known to provide anti-inflammatory effects. The possibility to regulate the polarization of macrophages to this state might be attractive for a variety of diseases, like atherosclerosis, in which macrophages are involved. As these cases demand a long-term treatment, this polarization was supposed to be maintained over time and we were planning to achieve this by keeping IL-4 permanently present in an immobilized way. In order to immobilize it, we genetically introduced an alkyne-carrying, artificial amino acid in the IL-4 sequence. This allowed access to a site-specific click reaction (Cu(I)-catalyzed Huisgen azide-alkyne cycloaddition) with an azide partner. This study was able to set the basis for the project by successful expression and purification of the IL-4 analogue and by proving the availability for the click reaction and maintained bioactivity. The other side of this project was the isolation of human monocytes and the polarization and characterization of human macrophages. The challenge here was that the majority of related research was based on murine macrophages which was not applicable to human cells and the successful work was so far limited to establishing the necessary methods. In conclusion, we were able to show two different methods that allow the influence of protein-protein interactions and thereby the possible tailoring of drug loading. Although the results were very promising for both systems, their applicability in the development of drug delivery devices needs to be shown by further studies. / Die Wechselwirkungen zwischen Proteinen spielen eine entscheidende Rolle in der Entwicklung von Freigabesystemen für die immer wichtiger werdenden Protein-Therapeutika, wie Antikörper, Wachstumsfaktoren und Zytokine. Das Verständnis dieser Mechanismen würde die Möglichkeit eröffnen, sowohl die Träger, als auch die zu verabreichenden Proteine so zu verändern und zu steuern, dass sie auf kontrollierte Weise an einem bestimmten Ort freigesetzt werden. Die Anwendungen hierfür reichen von Trigger gesteuerter Freisetzung, über verzögerte Freigabe bis zur permanenten Präsentation von Stimuli, abhängig davon was für die jeweilige Applikation gewünscht ist. Seidenfibroin (SF) ist ein Biomaterial, welches verschiedene positive Eigenschaften für die Anwendung als Trägermaterial in sich vereint, indem es unter sehr milden Bedingungen verarbeitet werden kann, gut biokompatibel ist und stabilisierend auf eingebettete Proteine wirken kann. Da SF in der Natur von Spinnen und Seidenraupen produziert wird, könnte das Verständnis dieses Prozesses, sowie seiner wichtigsten Faktoren eine Vorlage für die Formulierung dieses Biopolymers geben. Der natürliche Prozess des Seidenspinnens vereint eine faszinierende Vielfalt von Aggregatszuständen, die von kolloidalen Lösungen über Hydrogele bis hin zu festen System reichen. Die Übergänge zwischen diesen Zuständen sind in vivo sehr sorgfältig kontrolliert. Die Hauptfaktoren dieses Prozesses sind der pH-Wert während der Passage der Spinnlösung durch die Drüse, sowie die Art und Zusammensetzung der Ionen und die herrschenden Scherkräfte. Die Funktion dieser einzelnen Faktoren auf den Spinnprozess wurde recherchiert und wird beschrieben, bevor ihr Einfluss auf die Entwicklung und Herstellung von seidenbasierten Freigabesystemen untersucht wird. Es werden Beispiele vorgestellt, die die Kontrolle der Hydrogelbildung während der Lagerung untersuchen oder signifikante Parameter für die kontrollierte Präzipitation in Gegenwart bestimmter Salze zeigen. Der Review betrachtet den Einsatz von Seidenfibroin in der Entwicklung von flüssigen, halbfesten oder festen Freigabesystemen und legt besonderen Fokus auf die Kontrolle der SF Kristallisation, Partikelbildung und Interaktion mit dem Arzneistoff für steuerbare Beladung. Obwohl wir viele Beispiele für die Anwendung von SF in Freigabesystemen zeigen konnten und viele Publikationen die Beladung von Proteinen auf SF-Systeme behandeln, wurde der Mechanismus der Interaktion zwischen beiden bisher nicht detailliert untersucht. Es gibt wenige Studien die einige Aspekte abdecken, aber keines beschäftigte sich spezifisch mit dieser Fragestellung. Darum machen wir dies zum Gegenstand unserer Arbeit, da dies einen direkten Einfluss auf pharmazeutische Parameter, wie Aggregation und Beladung, erlauben würde. Um den zugrundeliegenden Mechanismus der Wechselwirkung zwischen SF und einem positiv geladenen Modellprotein zu verstehen, nutzten wir isotherme Titrationskalorimetrie für eine thermodynamische Charakterisierung. Diese wurde durch kolloidale Charakterisierungsmethoden wie Statische Lichtstreuung, nanoparticle tracking analysis und Zeta-potentialmessungen, sowie Hydrophobitätsbestimmungen unterstützt. Wir untersuchten die Effekte von drei verschiedenen Hofmeister Salzen - NaCl (neutral), NaSCN (chaotrop) und Na2SO4 (kosmotrop) – und des pH Wertes auf die Interaktion von SF mit dem Modellprotein in Abhängigkeit vom Verhältnis der beiden zueinander. Die Salze beeinflussten die SF Struktur, indem sie die SF Mizellen entweder stabilisierten (kosmotrop) oder destabilisierten (chaotrop) und dadurch die Interaktion entweder vollständig unterbanden (kosmotrop) oder verstärkten (chaotrop). Diese Effekte waren verantwortlich für verschiedene Level des Loadings und der Koazervation, wenn Salzart und –konzentration variiert wurden. Außerdem waren NaCl und NaSCN in der Lage die Stabilität einer wässrigen SF-Lösung während der Lagerung bei 25°C zu verlängern. Ein andere Ansatz um die Wechselwirkung zwischen Proteinen zu beinflussen wurde mit kovalenter Modifikation verfolgt. Interleukin-4 (IL-4) ist ein Zytokin und kann Makrophagen zu M2 Makrophagen polarisieren, welche dann anti-inflammatorische Wirkungen haben. Die Möglichkeit diese Polarisation zu regulieren wäre für verschiedene Krankheiten, wie Arteriosklerose, bei denen Makrophagen eine Rolle spielen interessant. Da in diesen Fällen eine Langzeitbehandlung von Nöten ist sollte die Polarisation über die Zeit erhalten bleiben. Wir planten dies durch die Immobilisation von IL-4 zu erreichen, die für eine permanente Präsenz sorgen würde. Um IL-4 zu immobilisieren haben wir eine künstliche Aminosäure in die Sequenz eingeführt, die eine Alkingruppe trägt. Diese ermöglicht den Zugang zu einer Kupfer vermittelten, spezifischen Click-Reaktion (Cu(I)-catalyzed Huisgen azide-alkyne cycloaddition) mit einem Azid-Partner. Diese Studie war in der Lage die Basis für dieses Projekt zu erstellen, indem wir eine erfolgreiche Expression und Aufreinigung des IL-4 Analogons leisten konnten und dieses sowohl erhaltene Bioaktivität als auch Verfügbarkeit für die Clickreaktion zeigte. Die andere Seite dieses Projekts bestand aus der Isolation von humanen Monozyten und der Polarisation und Charakterisierung von humanen Makrophagen. Die Herausforderung hierbei lag darin dass die meiste Forschung auf diesem Gebiet an murinen Makrophagen durchgeführt wurde und dies nicht auf humane Zellen übertragbar war, und die erfolgreiche Arbeit bisher, beschränkte sich auf die Etablierung der nötigen Methoden. Zusammenfassend lässt sich sagen, dass wir in der Lage waren zwei verschiedene Methoden zur Beeinflussung der Protein-Protein Wechselwirkungen und damit der Beladung zu zeigen. Obwohl die Ergebnisse für beide Systeme vielversprechend waren muss ihre Anwendbarkeit in der Entwicklung von Freigabesystemen noch durch weitere Studien belegt werden.

Protein-Protein Interactions (PPIs) play a very important role in biological functions and therefore the inhibition of specific Protein-Protein Interactions has a huge therapeutic value. The most successful small molecular PPIs inhibitors do not fit with the prevalent `Rule of Five' drug profile. To overcome the disadvantages of small molecular PPIs inhibitors, peptide based PPIs inhibitors were developed. Herein we describe the development of a new class of peptidomimetics AA-peptides. The AApeptides were designed based on chiral PNA backbone. Substitution of nucleobases yields AApeptides that are resistant to proteolysis and capable of mimicking peptides. Two types of AApeptides were discussed in this dissertation "α-AApeptides" and "γ-AApeptides". The AApeptides were shown to disrupt p53/MDM2 protein-protein interaction and tomimic fMLF tripeptide to target G protein-coupled formyl peptide receptors (FPRs). Moreover, the lipidated α-AApeptides can mimic the structure and function of natural antimicrobial lipopeptides and show broad-spectrum activity against both Gram-positive and Gram-negative bacteria. Lastly I have designed and synthesized a serials of phosphopeptides to disrupt cancer related STAT3-STAT3 dimerization.

Water has long been assumed to be essential for biological function. To understand the molecular basis of the role of water in protein function, several studies have established a correlation between enzyme activity and hydration level. While a threshold of hydration of 0.2 h (grams of water per gram of dried protein) is usually accepted for the onset of enzyme activity, recent works show that enzyme activity is possible at water contents as low as 0.03 h (Lind et al., 2004). Diffusion limitation in these experiments was avoided by monitoring enzyme-catalyzed hydrolysis of gas-phase esters. However, since water is also a substrate for the enzyme used in these experiments, they cannot be used to probe the possibility of activity at zero hydration. However, the pig liver esterase and C. rugosa lipase B are able to catalyse alcoholysis reactions in which an acyl group is transferred between an ester and an alcohol. Therefore, by following this reaction and using a gas phase catalytic system, we have been able to show that activity can occur at 0 g/g. These results led to the question of the accuracy of determinations of very low water concentrations; i.e., how dry is 0 g/g? Although gravimetric measurements of the hydration level do not allow us to define the anhydrous state of the protein with sufficient sensitivity, using 18O-labeled water, we have been able to quantify the small number of water molecules bound to the protein after drying, using a modification of the method of Dolman et al. (1997). Testing different drying methods, we have been able to determine a level of hydration as low as 2 moles of water per mole of protein (equivalent to 0.0006 h in the case of pig liver esterase) and have shown that in the case of the pig liver esterase, activity can occur at this hydration level. At the molecular level, if the hydration level affects activity, we can expect an effect on the protein dynamics. Neutron scattering spectra of hydrated powders, for instance, show that diffusive motions of the protein increase with the hydration (Kurkal et al., 2005) To address the question of the protein motions involved in the onset of enzyme activity at low hydration, we performed neutron scattering experiments on a pico-second time scale on dried powders. Preliminary results show a dynamical transition at hydration levels as low as 3 h. Molecular dynamic simulations have also been used in this study to access the dynamics of the active site. Overall, the results here show that pig liver esterase can function at zero hydration, or as close to zero hydration as current methods allow us to determine. Since the experimental methodology restricts this work to a small number of enzymes, it is unlikely that it will ever be possible to determine if all enzymes can function in the anhydrous state: however, the results here indicate that water is not an obligatory requirement for enzyme function.

Thesis (Ph.D.)--McMaster University, 2005. / Supervisor: Dr. Christian Baron. Includes bibliographical references (leaves 94-101).

Fueled by ever-growing genomic information and rapid developments of proteomics–the large scale analysis of proteins and mapping its functional role has become one of the most important disciplines for characterizing complex cell function. For building functional linkages between the biomolecules, and for providing insight into the mechanisms of biological processes, last decade witnessed the exploration of combinatorial and chip technology for the detection of bimolecules in a high throughput and spatially addressable fashion. Among the various techniques developed, the protein chip technology has been rapid. Recently we demonstrated a new platform called “Spacially addressable protein array” (SAPA) to profile the ligand receptor interactions. To optimize the platform, the present study investigated various parameters such as the surface chemistry and role of additives for achieving high density and high-throughput detection with minimal nonspecific protein adsorption. In summary the present poster will address some of the critical challenges in protein micro array technology and the process of fine tuning to achieve the optimum system for solving real biological problems. / Singapore-MIT Alliance (SMA)

Thesis (M.S. in Cancer Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.