High-throughput genomics projects have resulted in a rapid accumulation of protein sequences. Therefore, computational methods that can predict protein functions and functional sites efficiently and accurately are in high demand. In addition, prediction methods utilizing only sequence information are of particular interest because for most proteins, 3-dimensional structures are not available. However, there are several key challenges in developing methods for predicting protein function and functional sites. These challenges include the following: the construction of representative datasets to train and evaluate the method, the collection of features related to the protein functions, the selection of the most useful features, and the integration of selected features into suitable computational models. In this proposed study, we tackle these challenges by developing procedures for benchmark dataset construction and protein feature extraction, implementing efficient feature selection strategies, and developing effective machine learning algorithms for protein function and functional site predictions. We investigate these challenges in three bioinformatics tasks: the discovery of transmembrane beta-barrel (TMB) proteins in gram-negative bacterial proteomes, the identification of deleterious non-synonymous single nucleotide polymorphisms (nsSNPs), and the identification of helix-turn-helix (HTH) motifs from protein sequence.

No description available.

Forschungsgegenstand war ein neuentwickeltes modulares Kassettensystem mit Hedera helix 'Woerner', das im Folgenden als hydroponische Nutzwand bezeichnet wurde. Die Lebensqualität in Ballungsräumen sinkt aufgrund von steigender Verdichtung. Stadtgrün verbessert die Lebensqualität und sorgt für lokale Klima- und Luftverbesserung. Allerdings besteht ein Nutzungskonflikt mit anderen Bebauungsvorhaben. Hier verspricht der Einsatz der hydroponischen Nutzwand ein hohes Potential, da das wandgebundene Fassadenbegrünungssystem weitgehend bodenunabhängig ist. Es erfolgte eine Studie zur Quantifizierung des Leistungspotenzials. Neben einer detaillierten Beschreibung des Kassettensystems und der Versuchsanlage, die eine nach Norden und eine nach Süden exponierte Nutzwand darstellte, erfolgte die Erstellung einer Wasserbilanz und eines abgeleiteten Bewässerungsplanes. Im Substrat wurden Untersuchungen zum Sauerstoffgehalt durchgeführt. Ebenso war auch die Wirkung auf das Bestandsklima ein wesentliches Kriterium, um das Kassettensystem zu beschreiben. Für die Leistungsabschätzung wurden Wachstumsanalysen zur Beschreibung der Pflanzenproduktion durchgeführt. Für einige Kassettenelemente wurde dabei im Wurzelraum ein Pflanzenstärkungsmittel mit Bacillus subtilis angewendet, mit dem Ziel, eine Wachstumssteigerung zu erreichen. In einem Austrocknungsversuch im Gewächshaus wurde der Effekt verschiedener Konzentrationen des Pflanzenstärkungsmittels auf eine erhöhte Stresstoleranz von Hedera helix 'Woerner' untersucht. Der Wasserhaushalt stellte einen gesonderten Schwerpunkt dar, bei dem zwei Ansätze zur Bewässerungssteuerung verfolgt wurden. Es erfolgte eine Modellierung der Evapotranspiration über Daten aus meteorologischen Messungen und Messungen zur Transpiration der Pflanzen im Bestand. In einem zweiten Ansatz wurden die Möglichkeiten der pflanzenbasierten Sensorik untersucht, wofür ein elektronischer Blattdickensensor zum Einsatz kam. Die Erkenntnisse aus der Dissertation sollten zeigen, welche Praxistauglichkeit eine hydroponische Nutzwand besitzt und ob sie lokal in der Lage ist, dem Problem sinkender Lebensqualität im städtischen Raum entgegenzuwirken.

No description available.

Pour mimer le repliement des structures tridimensionnelles des biomolécules, les chimistes ont développé des oligomères artificiels capables d’adopter des formes repliées et bien définie en solution : les foldamères. Néanmoins, la variété des structures secondaires isolées que l’on rencontre au sein des foldamères n’atteint pas encore celle des biomolécules. La combinaison de différentes séquences d’oligoamides aromatiques ayant des structures secondaires distinctes a permis le développement d’architectures de type « hélice-feuillet-hélice » définie dans lesquelles chaque sous-composant secondaire conserve son intégrité respective. Ces objets uniques en forme de panier possèdent une fenêtre ouverte modulable inscrite dans le squelette du foldamère par laquelle une molécule invitée peut être accueillie. Comme preuve de concept, la liaison et le relargage d’une molécule invitée à l’une de ces structures se sont révélées rapides à l’échelle de la RMN 1H. Ensuite, le développement de brins oligomériques composés de monomères codant pour de faibles rayons de courbure a permis l’obtention d’hélices doubles. Ces structures auto-assemblées de haut-poids moléculaires possèdent un diamètre de l’ordre du nanomètre. Enfin, des segments hélicoïdaux codant pour des diamètres larges ont été couplés à des pseudo-coudes artificiels dans le but d’obtenir des architectures possédant une large cavité polaire inspirés de la structure des tonneaux β. Ces approches ouvrent la voie vers la conception d’objets moléculaires toujours plus complexes au-delà la chimie des biomolécules. / To mimic the particular folding of the biomolecules’ three-dimensional structures, chemists have developed artificial oligomers that fold into a compact and well-defined structures in solution: foldamers. Nevertheless, the variety of isolated secondary structures of foldamers is not equal to those of biomolecules. The association of different sequences of aromatic oligoamide having distinct secondary structures allowed the development of well-defined helix-sheet-helix architectures in which subcomponents conserve their respective integrity. These unique basket-like objects possess an open-window within the foldamer backbone in which a molecular guest can be accommodate. As a proof of concept, guest binding to one of these structures was found to be fast on the NMR time scale. Then, the development of oligoamide aromatic strands made of monomer encoding for low curvature has allowed to obtain double helices structures. These self-assembled structures showing high molecular weights present a nanometer scale diameter. Eventually, these oligomeric strands were coupled to artificial turn units to obtain β-barrels-like architectures having a large polar cavity. These approaches open the access to the design of ever more complex molecular objects beyond the chemistry of biomolecules.

Nesse trabalho, são investigadas as propriedades dinâmicas de modelos da mecânica estatística na criticalidade. Inicialmente, trabalhando com modelos de spin e utilizando os conceitos de persistência global e de dimensão anômala da magnetização inicial, mostramos que o modelo de Baxter-Wu não está na mesma classe de universalidade dos modelos de Potts com quatro estados e de Ising com interação de três spins em uma direção, todos bidimensionais. Na segunda parte da tese, estudamos o fenômeno de crescimento da superfície gerada pela deposição segundo as regras que definem os autômatos celulares probabilísticos propostos por Grassberger (modelos A e B). Esses dois autômatos não pertencem à classe de universalidade de Domany-Kinzel e apresentam novos expoentes críticos, cuja origem se deve à conservação de paridade. Determinamos o expoente de crescimento beta w, válido em tempos curtos, assim como os outros expoentes críticos associados ao crescimento de superfície (alfa e z). Nossas estimativas se comparam bem com os resultados obtidos a partir de razões de inteiros propostas por Jensen para os expoentes beta, ni paralelo e ni perpendicular. Finalmente, investigamos a transição de fase entre o estado helicoidal e o estado desordenado (random coil) da polialanina e do fragmento peptídico PTH(1-34), que corresponde aos resíduos 1 a 34 da região aminoterminal do hormônio das paratireóides. Nosso cálculo, que leva em conta as interações entre todos os átomos da molécula, está baseado em uma abordagem de tempos curtos. Os resultados dessa análise indicam que a transição helix-coil das polialaninas e do PTH(1-34) é de segunda ordem e apontam para uma classe de universalidade para a transição helix-coil em homopolímeros e proteínas (partindo de um estado helicoidal). / In this work we investigated dynamic properties of statistical mechanical models at criticality. At first, using the concepts of global persistence and anomalous dimension of initial magnetization, we showed that the Baxter-Wu model does not belong to the same universality class as 4-state Potts model and Ising with multispin interaction in one direction. In the sequence, we studied the roughening behavior generated by deposition governed by rules defined by probabilistic cellular automata proposed by Grassberger (A and B models). Those models are known do not belong to the Domany-Kinzel universality class. They are characterized by different exponents which are related to the parity conserving (PC). We estimated the growth exponent beta w, in short-time regimen, such as, other critical exponents associated to the surface growth (alpha and z). Our results are in good agreement with those expected for parity conserving universality class. At last we studied the phase transition between the completely helical state and the random coil of the polyalanine, such as, for the 34-residue human parathyroid fragment PTH(1-34). Our short-time simulations of the helix-coil transition are based on a detailed all-atom representation of proteins. The results indicate that helix-coil transition in polyalanine and PTH(1-34) is a second-order phase transition and suggest a universality class to the helix-coil transition in homopolymer and (helical) proteins.

A sequential series of mutational events is necessary for the development of leukemia. The misexpression of TAL1, a basic helix-loop-helix (bHLH) transcription factor, is the most common mutation in T cell acute lymphoblastic leukemia (T-ALL). Tal1 transgenic mice develop leukemia with a long latency and incomplete penetrance indicating additional mutations are necessary to develop disease. To investigate additional mutational events that potentially contribute to TAL1-expressing T-ALL patients, we sought to identify cooperating mutations in Tal1 transgenic mice. Clinical studies implicated the loss of the INK4a/ARF locus, which encodes two tumor suppressors, p16INK4a and p14ARF, in the majority of T-ALL patients. We demonstrated disease acceleration in tal1/ink4a/arf+/-, tal1/pl6ink4a+/- and tal1/p19arf+/- mice, thereby providing genetic evidence that Tal1 cooperates with loss of either p16Ink4a or p19Arf in leukemogenesis. The cooperation of Tal1 with the loss of or p16Ink4a or p19Arf, is consistent with our observation that Tal1 alters cell cycle regulation in leukemia by promoting S phase induction and apoptosis in vivo. An additional mutational event common in tal1 tumors is activation of the Notch1 signaling pathway. We provide evidence that the majority of tal1 tumors express increased levels of Notch1, and exhibit activating notch1 mutations. Additionally, tal1 tumors display sensitivity to the pharmacologic inhibition of γ-secretase activity in vitro, indicating that γ-secretase inhibitors may prove an efficacious treatment for TAL1-expressing T-ALL patients. Furthermore, we developed a doxycycline-regulated NotchIC T-ALL cell line, which will allow the identification of important Notch1IC target genes in leukemogenesis.

我們假設，將scleraxis (Scx)基因轉導入肌腱來源的幹細胞（TDSC），製成TDSC-Scx細胞系。TDSC-Scx會促進肌腱修復。本研究的目的在於探索Scx促進TDSC成肌腱分化的作用，以及TDSC-Scx對肌腱修復的促進作用。 / 使用慢病毒載體將Scx轉導入TDSCs，不含Scx的空載作為對照也轉導入TDSCs，用載體上帶有的抗性基因，殺稻瘟菌素對細胞進行篩選。分別建成TDSC-Scx和TDSC-Mock細胞系。Scx 的表達分別用定量PCR以及免疫螢光在mRNA和蛋白水準進行鑒定。TDSC-Scx成肌腱，成軟骨和成骨方向的分化能力用定量PCR檢驗。用大鼠臏腱視窗損傷模型進行了細胞移植試驗，測試TDSC-Scx對肌腱損傷的修復作用。實驗分為三組：（1）支架組，（2）空載體組，（3）Scx組。在細胞移植後的第二，四，八周，收集正在修復中的肌腱樣品，進行植入細胞的存留狀態，鈣化，組織學和生物力學測驗。 / TDSC-Scx比TDSC-Mock有更強的成肌腱分化能力。但是，在成軟骨-成骨分化方面，沒有結論。在動物試驗，植入的細胞在第二周仍然可見，但自第四周起，就不見了。在第八周，各組均有個別樣品輕微異位鈣化，但各組別間並無顯著差異。在早期，TDSC-Scx組比空載體組和支架組合得來更好的修復肌腱的能力。 / TDSC-Scx可能促進肌腱損傷的早期修復。 / We hypothesized that transduction of tendon-derived stem cell (TDSC) with scleraxis (Scx) might promote its tenogenic differentiation and promote better tendon repair compared to TDSC without Scx transduction. This study thus aimed to investigate the effect of Scx transduction on the tenogenic differentiation of TDSC and the effect of the resulting cell line in the promotion of tendon repair. / TDSCs were transduced with lentivirus-mediated Scx or empty vector and selected by blasticidin. The mRNA and protein expression of Scx were checked by qRT-PCR and Immuno fluorescence, respectively. The expression of different lineage markers were examined by qRT-PCR. A rat patellar tendon window injury model was used. The operated rats were divided into 3 groups: (1) scaffold-only group, (2) TDSC-Mock group and (3) TDSC-Scx group. At week 2, 4 and 8 post-transplantation, the repaired patellar tendon was harvested for ex vivo fluorescent imaging, vivaCT imaging, histology, or biomechanical test. / TDSC-Scx consistently showed higher expression of tendon-related markers compared to TDSC-Mock. However, the effect of Scx transduction on the expression of chondro-osteogenic markers was less conclusive. The transplanted TDSCs could be detected in the window wound at week 2 but not at week 4. Ectopic ossification was detected in some samples at week 8 but there was no difference among different groups. The TDSC-Scx group promoted early tendon repair histologically and biomechanically compared to the scaffold-only group and the TDSC-Mock group. / TDSC-Scx might be used for the promotion of early tendon repair. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tan, Chunlai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 66-70). / Abstracts also in Chinese. / Chapter Thesis/Assessment Committee --- p.i / Acknowledgment --- p.ii / Publication --- p.vi / Abstract --- p.vii / 摘要 --- p.viii / Chapter Chapter 1 --- Tendon injury and tendon tissue engineering --- p.1 / Chapter 1.1 --- Anatomy of tendon --- p.1 / Chapter 1.2 --- Epidemiology of tendon injury --- p.3 / Chapter 1.3 --- Process and problems of tendon healing --- p.4 / Chapter 1.4 --- Current treatment and cell-based therapy for tendon repair --- p.5 / Chapter 1.5 --- Transcriptional factor Scleraxis and tendon --- p.8 / Chapter 1.5.1 --- Helix-loop-helix (HLH) and bHLH proteins --- p.8 / Chapter 1.5.2 --- Scleraxis --- p.9 / Chapter 1.6 --- Research focus and implications --- p.11 / Chapter 1.7 --- Hypotheses and objectives of this study --- p.12 / Chapter 1.8 --- Clinical significance --- p.13 / Chapter Chapter 2 --- Materials and Methods --- p.14 / Chapter 2.1 --- Study Design --- p.14 / Chapter 2.2 --- Establishment of TDSC-Scx cell line --- p.15 / Chapter 2.2.1 --- Isolation of TDSC and cell culture --- p.15 / Chapter 2.2.2 --- Establishment of TDSC-Scx cell line --- p.17 / Chapter 2.2.2.1 --- Construction of plasmid --- p.17 / Chapter 2.2.2.2 --- Transfection --- p.23 / Chapter 2.2.2.3 --- Infection --- p.24 / Chapter 2.2.2.4 --- Selection --- p.24 / Chapter 2.2.2.5 --- Characterization of TDSC-Scx and TDSC-Mock --- p.25 / Chapter 2.2.2.5.1 --- qRT-PCR --- p.25 / Chapter 2.2.2.5.2 --- Immune-fluorescent (IF) --- p.26 / Chapter 2.2.2.6 --- Lineage marker expression --- p.26 / Chapter 2.2.3 --- Data analysis --- p.29 / Chapter 2.3 --- The effect of TDSC-Scx on healing in a patellar tendon window injury model --- p.30 / Chapter 2.3.1 --- Animal surgery --- p.30 / Chapter 2.3.2 --- Ex Vivo Fluorescence Imaging --- p.32 / Chapter 2.3.3 --- vivaCT --- p.33 / Chapter 2.3.4 --- Histology --- p.33 / Chapter 2.3.5 --- Biomechanical test --- p.35 / Chapter 2.3.6 --- Data analysis --- p.37 / Chapter Chapter 3 --- Results --- p.38 / Chapter 3.1 --- Generation of TDSC-Scx and TDSC-Mock cell lines --- p.38 / Chapter 3.1.1 --- Plasmid --- p.38 / Chapter 3.1.2 --- Cell morphology --- p.38 / Chapter 3.1.3 --- Expression of Scx --- p.38 / Chapter 3.1.4 --- Expression of chondro-/osteo-/tenogenic markers --- p.39 / Chapter 3.2 --- The healing effect of TDSC-Scx on a patella tendon window injury model --- p.40 / Chapter 3.2.1 --- The fate of transplanted cells --- p.40 / Chapter 3.2.2 --- Ossification --- p.40 / Chapter 3.2.3 --- Histology --- p.40 / Chapter 3.2.3.1 --- Fiber arrangement --- p.41 / Chapter 3.2.3.2 --- Cellularity --- p.41 / Chapter 3.2.3.3 --- Cell alignment --- p.42 / Chapter 3.2.3.4 --- Cell rounding --- p.42 / Chapter 3.2.3.5 --- Vascularity --- p.43 / Chapter 3.2.3.6 --- Fiber structure --- p.43 / Chapter 3.2.3.7 --- Hyaline degeneration --- p.43 / Chapter 3.2.3.8 --- Inflammation --- p.44 / Chapter 3.2.3.9 --- Ossification --- p.44 / Chapter 3.2.4 --- Biomechanical properties --- p.44 / Chapter Chapter 4 --- Discussion --- p.55 / Chapter 4.1 --- In vitro --- p.55 / Chapter 4.1.1 --- Scx transduction did not lead to morphological change in TDSCs --- p.55 / Chapter 4.1.2 --- Scx transduction led to higher expression of Scx mRNA --- p.55 / Chapter 4.1.3 --- TDSC-Scx expressed higher levels of tenogenic markers --- p.56 / Chapter 4.2 --- In vivo --- p.58 / Chapter 4.2.1 --- The fate of transplanted cells --- p.58 / Chapter 4.2.2 --- Ossification was vague --- p.58 / Chapter 4.2.3 --- TDSC-Scx promoted tendon healing --- p.58 / Chapter 4.2.4 --- Biomechanical properties --- p.59 / Chapter 4.2.5 --- Clinical consideration --- p.60 / Chapter 4.3 --- Similar studies --- p.61 / Chapter Chapter 5 --- Limitations --- p.63 / Chapter 5.1 --- Direct Scx protein expression and function information unavailable --- p.63 / Chapter 5.2 --- Differentiation assay --- p.64 / Chapter Chapter 6 --- Conclusion --- p.65 / Reference --- p.66 / Appendix --- p.71

Pektatlyase (Pel-15) aus dem alkalophilen Bodenbakterium Bacillus spec. KSM-P15 ist mit 197 Aminosäuren eines der kleinsten, bekannten β-3-Solenoidproteine. Sie spaltet Polygalakturonsäurederivate in einem Ca2+-abhängigen β-Eliminierungsprozess. Wie bei allen Proteinen dieser Enzymfamilie ist auch die Polypeptidkette von Pel-15 zu einer einsträngigen, rechtsgängigen, parallelen β-Helix aufgewunden. In diesem Strukturmotiv enthält jede Windung drei β-Stränge, die jeweils durch flexible Schleifenbereiche miteinander verbunden sind. Insgesamt acht Windungen stapeln sich in Pel-15 übereinander und bilden entlang der Helixachse flächige, parallele β-Faltblätter aus. Im Bereich dieser β-Faltblätter existiert ein ausgedehntes Netzwerk von Wasserstoffbrückenbindungen, durch das der hydrophobe Kern, der sich im Inneren der β-Helix befindet, vom umgebenden Lösungsmittel abgeschirmt wird. Besondere Abschlussstrukturen an beiden Enden der β-Helix, wie sie typischerweise bei anderen Ver-tretern dieser Strukturklasse ausgeprägt werden, sind in Pel-15 nicht zu beobachten. Stattdessen sind die terminalen Bereiche der β-Helix über Salzbrücken und hydrophobe Seitenkettenkontakte stabilisiert. In der vorliegenden Dissertation wurde die Pektatlyase Pel-15 hinsichtlich ihres Faltungsgleichgewichtes, ihrer enzymatischen Aktivität und der Kinetik ihrer Strukturbildung charakterisiert. In eine evolutionär konservierte Helixwindung wurden destabilisierende Mutationen eingeführt, und deren Auswirkungen mittels spektroskopischer Methoden analysiert. Die Ergebnisse zeigen, dass Pel-15 in Gegenwart des Denaturierungsmittels Guanidiniumhydrochlorid einen hyperfluoreszenten Gleichgewichtsustand (HF) populiert, der nach Messungen von Faltungs- und Entfaltungskinetiken ein konformationelles Ensemble aus den Zuständen HFslow und HFfast darstellt. Diese HF-Zustände sind durch eine hohe Aktivierungsbarriere voneinander getrennt. In Rückfaltungsexperimenten populieren nur etwa 80 % der faltenden Moleküle den Zwischenzustand HFslow, der mit einer Zeitkonstante von ca. 100 s zu HFfast weiterreagiert. Die Denaturierungsmittelabhängigkeit dieser Reaktion ist sehr gering, was eine trans-/cis-Prolylisomerisierung als geschwindigkeitslimitierenden Schritt nahelegt. Die Existenz eines cis-Peptides in der nativen Struktur macht es erforderlich, den denaturierten Zustand als ein Ensemble kinetisch separierter Konformationen, kurz: DSE, zu betrachten, das durch die Spezies Ufast und Uslow populiert wird. Nach dem in dieser Arbeit aufgestellten „Minimalmodell der Pel-15 Faltung“ stehen die HF-Spezies (HFslow, HFfast) mit den Konformationen des DSE in einem thermodynamischen Kreisprozess. Das Modell positioniert HFfast und die native Konformation N auf die „native Seite“ der Aktivierungsbarriere und trägt damit der Tatsache Rechnung, dass die Gleichgewichtseinstellung zwischen diesen Spezies zu schnell ist, um mit manuellen Techniken erfasst zu werden. Die hochaffine Bindung von Ca2+ (Kd = 10 μM) verschiebt sich das Faltungsgleichgewicht bereits in Gegenwart von 1 mM CaCl2 soweit auf die Seite des nativen Zustandes, das HFfast nicht länger nachweisbar ist. Entgegen anfänglicher Vermutungen kommt einer lokalen, evolutionär konservierten Disulfidbrücke im Zentrum der β-Helix eine wichtige Stabilisierungsfunktion zu. Die Disulfidbrücke befindet sich in einem kurzen Schleifenbereich der β-Helix nahe dem aktiven Zentrum. Obwohl ihr Austausch gegen die Reste Val und Ala die freie Stabilisierungsenthalpie des Proteins um ca. 10 kJ/mol reduziert, lässt die Struktur im Bereich der Mutationsstelle keine gravierende Veränderung erkennen. Auch die katalytisch relevante Ca2+-Bindungsaffinität bleibt unbeeinflusst; dennoch zeigen Enzymaktivitätstests für VA-Mutanten eine Reduktion der enzymatischen Aktivität um fast 50 % an. Die evolutionär konservierte Helixwindung im Allgemeinen und die in ihr enthaltene Disulfidbrücke im Besonderen müssen nach den vorliegenden Ergebnissen also eine zentrale Funktion sowohl für die Struktur des katalytischen Zentrums als auch für die Strukturbildung der β-Helix während der Faltungsreaktion besitzen. Die Ergebnisse dieser Arbeit finden in mehreren Punkten Anklang an Faltungseigenschaften, die für andere β -Helixproteine beschrieben wurden. Vor allem aber prädestinieren sie Pel-15 als ein neues, β-helikales Modellprotein. Aufgrund seiner einfachen Topologie, seiner niedrigen Windungszahl und seiner hohen thermodynamischen Stabilität ist Pel-15 sehr gut geeignet, die Determinanten von Stabilität und Strukturbildung des parallelen β-Helix-Motivs in einer Auflösung zu studieren, die aufgrund der Komplexität bestehender β-helikaler Modellsysteme bislang nicht zur Verfügung stand. / Pectate lyase Pel-15 was isolated from alcaliphlic Bacillus spec. strain KSM-P15. Like all pectate lyases Pel-15 binds and subsequently cleaves polygalacturonic acid, the main pectic compound in plant cell walls and middle lamellae, in a Ca2+ dependent beta-elimination reaction. With 197 amino acids and a molecular mass of only 21 kDa the protein is one of the smallest right-handed parallel beta-helical proteins known today. Polypeptide chains that are classified into this structural family adopt super-helical folds in which each “solenoid stack” consists of three beta-structured regions that are connected by flexible turn segments. Along its longitudinal axis the right-handed parallel beta-helix thus comprises three elongated parallel beta-sheets that are stabilized by an extensive network of hydrogen bonds wrapping around the densely packed hydrophobic core. Together with the shield-like arrangement of hydrogen bonds this hydrophobic core is considered as the main contributor to an exceptionally high stability that is a common feature of all beta-helical proteins. In contrast to most right-handed parallel beta-helices, Pel-15 is devoid of any terminal capping domains and laterally associated secondary structure. Therefore, this protein is considered to be a promising model protein of a pure beta-helix which will help to understand the determinants of both parallel beta-sheet formation and stability. In the dissertation at hand optical spectroscopic methods were used to assess the enzymatic activity, the folding/unfolding equilibrium and the kinetic mechanism of structure formation in neutral buffered solutions. Results indicate that Pel-15 populates a hyper-fluorescent equilibrium intermediate (HF) that is effectively populated in presence of the denaturing agent guanidinium hydrochloride (GdmCl). According to kinetic folding and unfolding experiments HF is not only an essential on-pathway intermediate but has to be considered as a conformational ensemble in which several hyperfluorescent states are in thermodynamic equilibrium with each other. According to their existence in kinetic folding trajectories these different HF-species were termed HFslow and HFfast. The activation energy between both states is remarkably high leading to a time constant of about 100 seconds for the reaction HFslow ⇆ HFfast. Since native Pel-15 contains an energetically disfavoured cis-prolyl peptide between A59 and P60 it is proposed that HFslow and HFfast differ in their prolyl peptide conformations. Two main results emerge from this dissertation. First, an extensive study of the Pel-15 folding- and unfolding behaviour facilitated the proposal of a “minimal folding model”. According to this model the HF-states and the according denatured species Uslow and Ufast are aligned into a thermodynamic circle. This implies that unfolded polypeptide chains reach the HF-ensemble via parallel folding trajectories. Since the native conformation N together with HFfast are on the same side of the activation barrier, it is the reaction HFslow ⇆ HFfast that is the rate limiting step in the folding reaction of Pel-15. Second, the importance of an evolutionarily conserved disulfide bond in the central region of Pel-15 was tested by site directed mutagenesis and subsequent spectroscopic characterization. The exchange of the disulfide against a hydrophobic pair of alanine and valine decreases the folding free energy by about 10 kJ/mol. Although this value is unexpectedly high, structural perturbations around both mutational positions are small as was deduced from X-Ray crystallography. Interestingly, the stability decrease is accompanied by a major loss of enzymatic activity while the Ca2+ binding affinity is not significantly affected. It is therefore concluded that the allosterically relevant disulfide bond stabilizes long-range interactions that stabilize several adjacent solenoid turns near the N-terminus of the protein. Indeed, planar stacking interactions are perturbed and flexibility of N-terminal loops is increased once the disulfide bond is removed. This dissertation establishes Pel-15 as a novel beta-helical model protein and – even more important – smoothes the way for a generally accepted perspective on the formation and stability of parallel beta-sheet proteins.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008.