Дисертації з теми "Backbone dynamic"

The remarkable evolution of wireless networks into the next generation to provide ubiquitous and seamless broadband applications has recently triggered the emergence of Wireless Mesh Networks (WMNs). The WMNs comprise stationary Wireless Mesh Routers (WMRs) forming Wireless Backbone Mesh Networks (WBMNs) and mobile Wireless Mesh Clients (WMCs) forming the WMN access. While WMCs are limited in function and radio resources, the WMRs are expected to support heavy duty applications : that is, WMRs have gateway and bridge functions to integrate WMNs with other networks such as the Internet, cellular, IEEE 802.11, IEEE 802.15, IEEE 802.16, sensor networks, et cetera. Consequently, WMRs are constructed from fast switching radios or multiple radio devices operating on multiple frequency channels. WMRs are expected to be self-organized, self-configured and constitute a reliable and robust WBMN which needs to sustain high traffic volumes and long "online" time. However, meeting such stringent service expectations requires the development of decentralized dynamic transmission power control (DTPC) approaches. This thesis addresses the DTPC problem for both single and multiple channel WBMNs. For single channel networks, the problem is formulated as the minimization of both the link-centric and network-centric convex cost function. In order to solve this issue, multiple access transmission aware (MATA) models and algorithms are proposed. For multi-radio multi-channel (MRMC) WBMNs, the network is modelled as sets of unified channel graphs (UCGs), each consisting of interconnected active network users communicating on the same frequency channel. For each UCG set, the minimization of stochastic quadratic cost functions are developed subject to the dynamic Link State Information (LSI) equations from all UCGs. An energy-efficient multi-radio unification protocol (PMMUP) is then suggested at the Link-Layer (LL). Predictive estimation algorithms based on this protocol are proposed to solve such objective functions. To address transmission energy and packet instabilities, and interference across multiple channels, singularly-perturbed weakly-coupled (SPWC) control problems are formulated. In order to solve the SPWC transmission power control problem, a generalized higher-order recursive algorithm (HORA) that obtains the Riccati Stabilizing Solutions to the control problem is developed. The performance behaviours of the proposed models and algorithms are evaluated both analytically and through computer simulations. Several simulations are performed on a large number of randomly generated topologies. Simulation and analytical results confirm the efficacy of the proposed algorithms compared to the most recently studied techniques

Large-amplitude vibration of thin plates and shells has been critical design issues for many engineering structures. The increasingly more stringent safety requirements and the discovery of new materials with amazingly superior properties have further focused the attention of research on this area. This thesis deals with the vibration problem of rectangular, circular and angle-ply composite plates. This vibration can be triggered by an initial vibration amplitude, or an initial velocity, or both. Four types of boundary conditions including simply supported and clamped combined with in-plane movable/immovable are considered. To solve the differential equation generated from the vibration problem, Lindstedt's perturbation technique and Runge-Kutta method are applied. In previous works, this problem was solved by Lindstedt's Perturbation Technique. This technique can lead to a quick approximate solution. Yet based on mathematical assumptions, the solution will no longer be accurate for large amplitude vibration, especially when a significant amount of imperfection is considered. Thus Runge-Kutta method is introduced to solve this problem numerically. The comparison between both methods has shown the validity of the Lindstedt's Perturbation Technique is generally within half plate thickness. For a structure with a sufficiently large geometric imperfection, the vibration can be represented as a well-known backbone curve transforming from soften-spring to harden-spring. By parameter variation, the effects of imperfection, damping ratio, boundary conditions, wave numbers, young's modulus and a dozen more related properties are studied. Other interesting research results such as the dynamic failure caused by out-of-bound vibration and the change of vibration mode due to damping are also revealed.

Il principale obiettivo di questo lavoro di tesi è lo studio del ruolo giocato da un ponte disolfuro sulle proprietà di legame di una proteina citosolica, la Liver Bile Acid Binding Protein (L-BABP), nella quale è naturalmente presente. In particolare si vuole far luce sulle capacità della proteina di legare Acidi Biliari (BA) e sulle sue proprietà funzionali. Gli acidi biliari circolano tra il fegato e l’intestino attraverso un meccanismo definito “circolazione enteroepatica”, il quale è fortemente regolato dagli stessi acidi biliari. Gli acidi biliari sono infatti in grado di influenzare l’espressione di numerosi geni coinvolti nella loro sintesi e nel loro trasporto, mediante un legame con recettori di acidi biliari intracellulari primari, quali il recettore farnesoide X (FXR). La comprensione del meccanismo che regola l’interazione di trasportatori intracellulari con acidi biliari è un passaggio chiave per la costruzione di un modello rappresentativo del trasferimento di BAs dal citoplasma al nucleo e potrebbe essere utilizzato per lo studio di agenti terapeutici applicabili nel trattamento di disordini metabolici, quali l’obesità, il diabete di tipo 2, l’iperlipidemia e l’aterosclerosi. Per raggiungere una dettagliata descrizione dal punto di vista molecolare e della dinamica coinvolta nella formazione di un complesso ternario, tra L-BABP e due molecole di acidi biliari, è stata utilizzata la spettroscopia NMR (Nuclear Magnetic Resonance), parallelamente ad un’analisi cinetica e termodinamica, specificatamente implementata per questi studi. Nello specifico, mediante la Risonanza Magnetica Nucleare, sono state studiate le proprietà strutturali, di interazione e di dinamica di due forme di L-BABP di pollo, diverse tra loro per la presenza/assenza di un ponte disolfuro. Le interazioni proteina/ligando caratteristiche del complesso sono state studiate arricchendo alternativamente la proteina ed il ligando, con isotopi NMR attivi. La proteina è stata titolata aggiungendo concentrazioni sempre crescenti dell’acido glico-colico (GCA) e glico-chenodeossicolico (GCDA), arricchiti in 15N, in modo da poter seguire la variazione delle loro risonanze attraverso l’acquisizione e l’analisi di numerosi spettri NMR (HSQC, DOSY). I dati ottenuti hanno permesso di determinare la stechiometria di legame e i fenomeni di scambio, ma non sono risultati sufficienti per ricavare informazioni dettagliate sull’affinità, la cooperatività e i meccanismi di legame. Si è quindi deciso di analizzare la variazione dei segnali NMR in funzione della concentrazione di ligando per fare maggiore chiarezza sul meccanismo di interazione tra L-BABP e gli acidi biliari. A questo scopo, sono stati recentemente riportati, nuovi approcci NMR per lo studio delle interazioni proteina/ligandi che avvengono nella scala dei tempi dei micro- e millisecondi, che sfruttano l’analisi delle larghezze di riga ed esperimenti di “relaxation dispersion”. In particolare la combinazione di questi due approcci di indagine si sono rivelati utili per la comprensione della relazione esistente tra dinamica e funzione della proteina. Studi di rilassamento 15N, effettuati sulla proteina apo, hanno rivelato la presenza di moti lenti, nella scala dei tempi de micro- millisecondi. La principale domanda a cui si vuole rispondere è se tali moti sono essenziali per il legame con gli acidi biliari, se portano a conformazioni competenti all’inserimento dei ligandi e se sono influenzati dalla presenza del ponte disolfuro. L’analisi delle larghezze di riga, estratte dagli esperimenti di titolazione, effettuati sulla proteina arricchita isotopicamente in 15N, con successive aggiunte di GCDA, e gli esperimenti di “relaxation dispersion” hanno permesso di individuare un meccanismo di legame a più stadi e di ricavare alcune delle costanti cinetiche coinvolte.
The aim of this thesis is to understand the role played by a naturally occurring disulphide bridge on the bile acid (BA) binding and functional properties of cytosolic Liver Bile Acid Binding Protein (L-BABP). Bile acids circulate between liver and intestine through a mechanism defined as “enterohepatic circulation”, which is a tightly regulated process, particularly by BAs themselves. Indeed BAs are able to influence the expression of numerous genes involved in their synthesis and transport by binding to the primary intracellular nuclear bile acid receptor, farnesoid X receptor (FXR). Understanding the mechanism regulating the interactions of intracellular carriers with bile acid is a key step to provide a model for the transfer of BAs from cytoplasm to the nucleus and can be used to inspire design of therapeutic agents in the treatment of metabolic disorders, such as obesity, type 2 diabetes, hyperlipidaemia and atherosclerosis. To achieve a detailed molecular and dynamical description of the binding mechanism driving to the formation of the ternary complex of L-BABPs with two BA molecules, spectroscopic methods together with kinetic and thermodynamic analysis have been applied and implemented. In particular structural, dynamical and interaction properties of two forms of chicken L-BABP (cL-BABP), differing by the presence/absence of a naturally occurring disulphide bridge, have been investigated through nuclear magnetic resonance (NMR) approaches. The study of protein-ligand interactions by NMR was performed analysing complexes where, alternatively, either the protein or the ligand were isotopically labelled. 15N enriched glycocholic (GCA) and glycochenodeoxycholic acid (GCDA), two of the most important members of bile salts pool, were employed for protein titrations and their resonances followed through the acquisition and analysis of several NMR experiments (HSQC, DOSY). The obtained results shed light on binding stoichiometry and ligand exchange phenomena but were not sufficient to derive detailed information on affinity, cooperativity and binding mechanism. Thus NMR lineshape analysis as a function of ligand concentration was chosen as an appropriate tool to investigate the complex interaction mechanism within the cL-BABP/BA system. In this line, new NMR approaches have been recently described which allow a reliable and sensitive investigation of ligand binding events occurring on microsecond to millisecond (μs-ms) time scales using lineshape and relaxation dispersion experiments[1]. Particularly, the combination of these NMR methods can be useful in the study of complex multi-step mechanisms, allowing the correlation between protein dynamics and function[2]. 15N relaxation studies, performed on the apo-protein, revealed the presence of slow motions occurring on the microseconds-milliseconds timescale. The central question to be addressed is here whether these motions are essential for ligand uptake, how they can eventually lead to conformations competent for binding and how they are influenced by the presence of the disulfide bridge. The analysis of titration experiments of 15N labelled protein with unlabelled GCDA through lineshape analysis and relaxation dispersion allowed to define a multi-step binding mechanism for bile salt binding to liver BABPs and to provide an estimate of the kinetics involved.

Eukaryotic cells rely on a complex system of membrane-enclosed compartments that are maintained by the trafficking of shuttling vesicles. The fusion of these vesicles with the target compartment relies on multiprotein complexes that have been conserved throughout eukaryotic evolution. SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are considered the engine of membrane fusion in all trafficking pathways of the cell. Upon specific protein-protein interactions, SNARE proteins that are localized in opposing membranes form a four helix bundle that releases free energy and induces membrane fusion. The SNARE motif is the elementary unit of this bundle and defines all SNARE proteins. SNARE proteins possess other regulatory domains that contribute in modulating the specificity of the fusion event. One of these accessory elements is the Longin Domain (LD). Other than being well conserved among all eukaryotes, the LD is not limited to SNAREs only and is present in many molecular processes related to the life cycle of vesicles. LD-containing SNAREs are called Longins and are divided in three main subfamilies: Ykt6p, Sec22b, VAMP7. The Longin Domain (LD) is usually composed of about 120 amino acids arranged in a globular structural fold that consists of five ? strands (?1- ?5) sandwiched by one ? helix (?1) on one side and two helices (?2 and ?3) on the other side. The LD can fold back onto the SNARE motif in Ykt6p and Sec22b but not in Nyv1p – a fourth minor longin subfamily. This intramolecular interaction involves a surface-exposed hydrophobic pocket contributed by the ?1-?3 structural elements, which is bound by the SNARE motif. This mechanism eventually inhibits and prevents unspecific formation of the SNARE complex, thus regulating the vesicle fusion process. However, very little is known about the dynamic properties of such mechanism. The present study uses VAMP7 as a model system to reveal these characteristics. Our interest in VAMP7 relies on the fact that not only VAMP7 lacks any direct evidence of the LD-SNARE interaction, but it also offers a system of natural variations to the usual LD-SNARE domain arrangement that can prove extremely useful in our study. The present work reveals unknown dynamic properties of the LD-SNARE interaction supporting a dominantly “closed conformation” for Longins, with heterogeneous characteristics. The results shown in this research complement well with what we already know about a similar auto-inhibitory mechanism observed in the Syntaxin subfamily of SNAREs. Therefore, we provide here new bases for a better understanding of the regulatory mechanisms involved in vesicle fusion.
Le cellule eucariote sono caratterizzate da un complesso sistema di membrane, che offre svariate compartimentazioni con diverse condizioni chimico-fisiche. Se da una parte tale sistema permette la realizzazione di un’ampia gamma di processi biochimici, dall’altra richiede un altrettanto complesso sistema di interscambio atto al suo mantenimento. Tale interscambio è assicurato dal trafficking di vescicole che originano da un compartimento donatore e riversano il loro contenuto in un compartimento accettore attraverso un processo che richiede la fusione delle membrane lipidiche. Tale processo si fonda sull’organizzazione di complessi macromolecolari a cui contribuiscono varie famiglie proteiche ben conservate attraverso l’evoluzione eucariotica. La famiglia delle SNARE è una di queste. Le SNAREs sono considerate i motori della fusione di membrane. La loro capacità di formare complessi specifici in trans tra le due memrane su cui risiedono fornisce il contributo energetico necessario a indurre la fusione degli strati lipidici. Tali complessi consistono in un intreccio di quattro eliche chiamate SNARE motifs, domini di circa 60-70 amino acidi che definiscono tutte le SNAREs. Oltre allo SNARE motif, le SNAREs contengono spesso domini accessori a funzione regolativa. Uno di questi è il Longin Domain (LD). Il LD non è limitato alle sole SNAREs e anzi si ritrova in altre famiglie proteiche tutte coinvolte in processi molecolari riguardanti il ciclo vitale di una vescicola. Nelle SNAREs, il LD definisce una famiglia chiamata Longins, suddivisa a sua volta nelle proteine Ykt6, Sec22b e VAMP7. Il LD consiste di circa 120 aminoacidi organizzati in una struttura spaziale globulare che comprende un piano di cinque foglietti ? (?1- ?5), complessati da un’alfa elica (?1) su un lato e da altre due eliche (?2-?3) sull’altro. In Ykt6 e Sec22b si è dimostrata la possibilità che il LD si ripieghi sullo SNARE motif e lo coordini su una sua superficie idrofobica compresa tra ?1 e ?3. Questo meccanismo si è dimostrato in grado di prevenire la formazione di complessi SNARE non specifici. Tuttavia ben poco si conosce ad oggi sulla natura di questa interazione in termini dinamici, a differenza di quanto invece si sa per un analogo meccanismo osservato nella famiglia SNARE delle Sintaxine. In altri temrini non è dato sapere se nelle Longine questo meccanismo implica una conformazione stabilmente “chiusa” di LD e SNARE, o se piuttosto esso si realizza come un equilibrio dinamico tra conformazioni aperte e chiuse. Una serie di motivi, tra cui l’assenza di dati diretti per questo fenomeno in VAMP7 e la possibilità di usufruire di sue varianti naturali, ci hanno spinto a scegliere VAMP7 come sistema modello per fornire le risposte ai suddetti interrogativi. I nostri dati suggeriscono per le Longine una conformazione stabilmente chiusa, ma non omogenea e capace di cambi conformazionali molto rapidi. Questo lavoro complementa bene quanto già noto per le sintaxine e fornisce dunque la possibilità di comprendere meglio i meccanismi regolativi gneralmente adottati nella fusione vescicolare.

Thesis (M.S.)--University of Maryland, College Park, 2004.
Thesis research directed by: Dept. of Chemistry and Biochemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Thesis (Ph. D.)--University of Kentucky, 2007.
Title from document title page (viewed on April 25, 2007). Document formatted into pages; contains: xii, 145 p. : ill. (some col.). Includes abstract and vita. Includes bibliographical references (p. 131-143).

The Cre sequence (ACGT) is a site responsible for the binding of specific transcription factors that determine the activation of genes. Due to its major role in gene transcription, it has become a subject of immense research. The binding of transcription factors to the Cre binding site has been determined to be dependent on DNA conformation. In this study, the effects of flanking sequence around the Cre binding site on the conformation and the dynamics of DNA were investigated. The Cre binding site was studied in its native form with differing flanking sequences to determine the BI/BII profile (conformation) and the magnitude of the energy transition barrier (dynamics) between the BI and BII conformations of each phosphate step of the following three dodecamer sequences: CreACAG, CreGGAG, and CreTATA. In order to obtain the BI/BII profile of each phosphate step, 2D 31P-NMR NOESY and HSQC experiments at various temperatures were utilized. Based of the basic principles of kinetics, the lower the energy barrier between the two conformations, the easier the transition between the BI and BII conformation. Therefore, it was hypothesized that low and high %BII character lead to a large energy barrier (high ∆G‡ values), whereas average %BII character leads to a small energy barrier (low ∆G‡ values). The results of the 2D 31P-NMR experiments of the three dodecamer sequences confirmed this relationship between the %BII character and the magnitude of the energy barrier (∆G‡). However, further conformation and dynamics studies must be conducted to further understand the correlation.

Comprendre la dynamique locale des protéines dans leur état natif (structure repliée etfonctionnelle) est essentiel pour comprendre leur dynamique globale et leur fonction biologique. Aucours de cette thèse, nous avons étudié la dynamique locale de plusieurs petites protéines enmesurant les fluctuations de sondes locales le long de la séquence d’acide aminé de ces protéines.Nous avons essayé de comprendre la dynamique de ces sondes locales, comment celles-ci serelaxaient entre leurs différentes conformations, comment leurs fluctuations étaient corrélées lesunes aux autres et comment peuvent-elles être reliées à la fonction biologique des protéines.Dans les trois premiers chapitres, nous introduisons les concepts du mouvement Browniende rotation libre, de la spectroscopie par Résonance Magnétique Nucléaire (RMN) et de ladynamique moléculaire (DM). Dans les chapitres 4 et 5, nous avons étudié la dynamique desliaisons amides de la chaine principale (backbone) des protéines sur leurs paysages d’énergie libre.Dans le chapitre 4, nous avons démontré que les fluctuations des liaisons amide dubackbone de la protéine VA3 sont décrites par une diffusion rotationnelle anormale plutôt que parune diffusion rotationnelle libre généralement utilisée pour interpréter les données RMN enrelaxation de spins et en couplage résiduel dipolaire. [...] Dans le chapitre 5, nous avons démontré la diffusion rotationnelle anormale de ces liaisons jusqu’à une échelle de temps de 100 ns en utilisant dix simulations de DM de 1 μs de la protéineUbiquitine. Nous avons aussi étudié la convergence des paramètres RMN extraits des trajectoiresde DM en fonction de leur durée. [...] Dans le chapitre 6, nous avons réalisé une l’étude de la corrélation entre les mouvements du backbone et des chaines latérales des protéines. [...] Dans la première partie du dernier chapitre de cette thèse, nous avons étudié l’évolution de la corrélation dynamique entre les chaines latérales et la chaine principale d’une protéine durant des évènements de dépliement/repliement. Pour cette étude préliminaire, nous avons utilisé unesimulation de DM d’un « ultra-fast folder » nommé Trp-cage réalisée à 380K. Nous avons confirméles résultats précédemment trouvés pour les protéines dans leur état natif. Nous avons observél’augmentation de la corrélation entre les séries temporelles yn(t) and δn(t) pendant un évènementde dépliement caractérisé par la sortie du tryptophane de sa cage. Un paramètre stérique s aégalement été défini afin de quantifier les intéractions des chaines latérales avec leurenvironnement. Dans une seconde partie de ce dernier chapitre, nous présentons une étudepréliminaire du dépliement d’un « downhill folder » nommé gpW sous contrainte d’une force. Pourcaractériser le dépliement de la protéine gpW, nous avons calculé les chemical shifts des atomes Cª et Hⁿ du backbone le long de sa séquence en fonction d’une coordonnée réactionnelle choisie comme étant la distance entre les Cª de résidus C- et N- terminaux. Nous avons démontré qu’il était difficile de discerner un comportement particulier à partir des tous les chemical shifts en fonction de la distance. Cependant, en moyennant la valeur des chemical shifts en sur tous lesrésidus de la protéine nous trouvons que l’évolution de cette valeur moyenne en fonction de ladistance permettait de décrire les évènements du dépliement de la protéine en fonction de lacoordonnée de réaction durant la simulation de DM
Understand the local dynamics of proteins in their native state, i.e. in their folded functionalstructure, is a prerequisite to understand their global dynamics and their biological function. In thepresent thesis, we investigated the local dynamics of several small proteins by recording thefluctuations of local probes along the amino-acid sequence of those proteins. We tried tounderstand the dynamics of the local probe, i.e. how they relax between their differentconformations, how their fluctuations are correlated to each other, how their fluctuations arerelated to the function of the proteins. In the first three chapters, we introduced the concepts of the free rotational Brownian motion, of the Nuclear Magnetic Resonance spectroscopy and of the Molecular Dynamics (MD)simulations. In chapters 4 and 5, we studied the dynamics of the backbone amide bonds of theproteins on their free-energy landscape. In chapter 4, we demonstrated that the fluctuations of the backbone amide bonds of the protein VA3 are described by a rotational anomalous diffusion rather than by a free rotationaldiffusion, as often assumed in the interpretation of the raw NMR-measured data (Spin relaxation(SR) data and Residual Dipolar Coupling (RDC) data. [...] In chapter 5, we demonstrated the anomalous diffusion of backbone amide bonds up to 100 ns by using ten MD trajectories of 1 μs of duration for the protein ubiquitin. We also studied the convergence of the NMR-derived parameters extracted from the MD trajectories in function of their duration. [...] In chapter 6, we addressed the question of the correlation between the motions of the side chains and main chain of a protein. [...] In the first part of the final chapter of the present thesis, we investigated the evolution of the correlation between the side-chain and the main-chain motions of a protein during unfolding/folding events. In this preliminary work, we used a single MD simulation of the ultrafast folder Trp-cage performed at 380 K. We confirmed the results found for proteins in theirnative state. We observed an increase of the correlation between the two time series yn(t) and δn(t) during an unfolding event characterized, here, by the exit of the TRP residue of its “cage”.A steric parameter s was also defined in order to quantify interactions of the amino-acid side chainwith its environment. In a second part of the last chapter, we present a preliminary study of theunfolding of the downhill folder gpW under a mechanical force. To characterized the unfolding ofgpW, we computed the chemical shift of the Cª and of the Hⁿ atoms along the amino-acidsequence of the protein in function of a reaction coordinate: the distance, rCªCª , between the Cª atoms of the N and C terminal residues. We demonstrated that it is hard to distinguish a typical behavior of all the chemical shift of all the residues along the amino-acid sequence in function of the distance rCªCª . However, by averaging the chemical shift over all the residues of the protein we found that the evolution of the average value of the chemical shift described the unfolding eventsof the protein during the MD simulations

D. Tech. Engineering: Electrical.
The remarkable evolution of wireless networks into the next generation to provide ubiquitous and seamless broadband applications has recently triggered the emergence of Wireless Mesh Networks (WMNs). The WMNs comprise stationary Wireless Mesh Routers (WMRs) forming Wireless Backbone Mesh Networks (WBMNs) and mobile Wireless Mesh Clients (WMCs) forming the WMN access. While WMCs are limited in function and radio resources, the WMRs are expected to support heavy duty applications: that is, WMRs have gateway and bridge functions to integrate WMNs with other networks such as the Internet, cellular, IEEE 802.11, IEEE 802.15, IEEE 802.16, sensor networks, et cetera. Consequently, WMRs are constructed from fast switching radios or multiple radio devices operating on multiple frequency channels. WMRs are expected to be self-organized, self-configured and constitute a reliable and robust WBMN which needs to sustain high traffic volumes and long "online" time. However, meeting such stringent service expectations requires the development of decentralized dynamic transmission power control (DTPC) approaches. This thesis addresses the DTPC problem for both single and multiple channel WBMNs. For single channel networks, the problem is formulated as the minimization of both the link-centric and network-centric convex cost function. For multi-radio multichannel (MRMC) WBMNs, the network is modelled as sets of unified channel graphs (UCGs), each consisting of interconnected active network users communicating on the same frequency channel.

碩士
國立中央大學
化學學系
106
Sulfolobus solfataricus can be found in volcanoes and hot spring. It grows best at 80-85°C and at the pH level of 2-4. Sso7c4, a member of 7-kDa families of Sulfolobus solfataricus, is a histone-like dimer protein; it can resist to heat and acid. In this study, we employed molecular dynamics simulations to investigate the interactions between Sso7c4 dimers and a double-stranded DNA. We observed that an arginine pair (R22/R11′) and C-terminus of Sso7c4 have significant interactions with the phosphates on the DNA backbone, which are consistent with results of binding assay. In addition, we also observed that six positively charged lysine residues (K8，K20，K24，K28，K50，K54) have interactions with the phosphates on the DNA backbone. Furthermore, the whole Sso7c4 proteins sliding on DNA backbone is observed. Contact map analysis shows the two Sso7c4 dimers have important intermolecular interactions through hydrophobic residues pulling two Sso7c4 dimers closer. These interactions and the sliding of proteins on DNA backbone deform the DNA structure such as bending and shortening the DNA length.

碩士
國立清華大學
生物科技研究所
95
Chemkines play an important role in the neutrophil-mediated inflammation in human pathophysiology. ELR-CXC chemkines belong to the CXC subfamily of chemkines with ELR-motif at the N-terminal region. The ELR-CXC chemokines play an important role in inflammatory responses to many pathogenic insults as their ability to chemoattract and activate neutrophils via CXC receptor. The analogue of CXCL8 (IL-8) in which two residues were mutated (IL8K11R/G31P) can effectively block neutrophil responses via CXCR1 and CXCR2. We measured the spin-lattice relaxation rate R1, spin-spin relaxation rate R2 and 15N {H} nuclear overhauser enhancement (NOE) of IL8K11R/G31P at 14.09T. The programs pdbinteria and R2R1_diffusion were used to estimate the diffusion tensor and generate a new pdb file. Then, Model-free formalism was performed to determine the order parameter (S2), the correlation time of local motion (τe) and the chemical exchange contribution (Rex). The reduced spectral density functions at different frequencies were also used to obtain the relaxation information. These analysis provided information that backbone dynamics of IL8K11R/G31P around Arg11 and Pro31 are more flexible than other region.

Les interactions entre les squelettes sucre-phosphate de nucléotides jouent un rôle important dans la stabilisation des structures tertiaires de larges molécules d’ARN. Elles sont régies par des règles particulières qui gouverne leur formation mais qui jusque là demeure quasiment inconnues. Un élément structural d’ARN pour lequel les interactions sucre-phosphate sont importantes est le motif d’empaquetage de deux doubles hélices d’ARN le long du sillon mineur. Ce motif se trouve à divers endroits dans la structure du ribosome. Il consiste en deux doubles hélices interagissant de manière à ce que le squelette sucre-phosphate de l’une se niche dans le sillon mineur de l’autre et vice versa. La surface de contact entre les deux hélices est majoritairement formée par les riboses et implique au total douze nucléotides. La présente thèse a pour but d’analyser la structure interne de ce motif et sa dépendance de stabilité résultant de l’association optimale ou non des hélices, selon leurs séquences nucléotidiques. Il est démontré dans cette thèse qu’un positionnement approprié des riboses leur permet de former des contacts inter-hélices, par l’entremise d’un choix particulier de l’identité des pairs de bases impliquées. Pour différentes pairs de bases participant à ce contact inter-hélices, l’identité optimale peut être du type Watson-Crick, GC/CG, or certaines pairs de bases non Watson-Crick. Le choix adéquat de paires de bases fournit une interaction inter-hélice stable. Dans quelques cas du motif, l’identité de certaines paires de bases ne correspond pas à la structure la plus stable, ce qui pourrait refléter le fait que ces motifs devraient avoir une liberté de formation et de déformation lors du fonctionnement du ribosome.
Although backbone-backbone interactions play an important role in stabilization of the tertiary structure of large RNA molecules, the particular rules that govern the formation of these interactions remain basically unknown. One RNA structural element for which the backbone-backbone interactions are essential is the along-groove packing motif. This motif is found in numerous locations in the ribosome structure; it consists of two double helices arranged such that the backbone of one helix is packed in the minor groove of the other helix and vice versa. The contact area between the two helices is mostly formed by riboses and totally involves twelve nucleotides. Here we analyze the internal structure of the along-groove packing motif and the dependence of stability of the association of the helices on their nucleotide sequences. We show that the proper positioning of the riboses that allows them to form inter-helix contacts is achieved through the particular choice of the identities of the base pairs involved. For different base pairs participating in the inter-helix contacts the optimal identities can be Watson-Crick, GC/CG, or certain non-Watson-Crick base pairs. The proper choice of the base pairs provides for the stable inter-helix interaction. In some cases of the motif, the identities of certain base pairs do not correspond to the most stable structure, which may reflect the fact that these motifs should break and form during the ribosome function.

博士
國立清華大學
生物資訊與結構生物研究所
103
We investigated the structural and dynamic characters of two proteins in the study: Hepatoma-derived growth factor (HDGF) and receiver domain of ethylene receptor 1 (ETR1-RD). In the first case, HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic PWWP motif. The HATH domains have drawn attention because of the binding with heparin/heparan sulfate, DNA and methylated histone peptide. Depending on the sequence of the PWWP motif, HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro). A-type HATH is highly unstable in solution and P-type HATH has available structure. We evaluated the difference on structure, dynamics and ligand binding. Analysis of NMR backbone 15N relaxations revealed additional backbone dynamics in the interface between the b-barrel and the C-terminal helix bundle. The β1/β2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction. A-type HATH, therefore, shows a tendency toward higher-order aggregation when binding with heparin and DNA oligomers. In the second case, ETR1, in response to ethylene, plays versatile roles in plant physiology. Although the downstream regulators have been identified, the molecular recognition remains unknown. It has been speculated that the cytoplasmic signaling of ETR1 adopts a two-component system involving the conserved receiver domain (RD). We used NMR method to investigate the structure and dynamics of ETR1-RD. Combining NMR backbone chemical shifts into the structural calculation, we defined the solution ETR1-RD structure similar to X-ray structure, but ETR1-RD is a monomer, not the dimer observed in X-ray crystal. Notably, NMR investigation reported no phosphorylation for ETR1-RD. Comparing the backbone dynamics to other receiver regulators, we suspect the backbone flexibility is critical to determine the phosphorylation property. ETR1-RD is an atypical receiver regulator.

Thesis (Ph. D.)--Florida State University, 2007.
Advisor: Timothy M. Logan, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Mar. 10, 2008). Document formatted into pages; contains xviii, 204 pages. Includes bibliographical references.

博士
國立清華大學
化學系
90
In this study, the thermodynamic and backbone dynamics properties of human acidic fibroblast growth factor (hFGF-1) are described. hFGF-1 is a ~ 16 kDa, all b-barrel protein which plays key roles in several important cellular processes related to morphogenesis, development and angiogenesis. The thermodynamic parameters characterizing the conformational stability of the human acidic fibroblast growth factor (hFGF-1) have been determined by isothermal urea denaturation and thermal denaturation at fixed concentrations of urea using fluorescence and far-UV CD circular dichroism (CD) spectroscopy. Temperature denaturation experiments in the absence and presence of urea show that hFGF-1 has a tendency to undergo cold denaturation. Two-dimensional 1H-15N HSQC spectra of hFGF-1 acquired at sub zero temperatures clearly show that hFGF-1 unfolds under low-temperature conditions. To describe the internal motions of hFGF-1 ranging from pico- to millisecond, 15N NMR relaxation data have been used to characterize the backbone dynamics of hFGF-1 in its free and sucrose octasulfate (SOS) bound states. Significant conformational exchange (Rex) is observed for several residues in the free form of the protein. However, the conformational exchange behavior of hFGF-1 is tremendously reduced upon SOS binding. Also, the receptor-binding segment comprising residues 103-111 shows increased flexibility in the presence of SOS. To investigate the internal motions of hFGF-1 range from millisecond to days, hydrogen-deuterium exchange experiments in the absence and low concentrations of denaturant were performed. In contrast to the equilibrium unfolding events monitored by optical probes, native-like state hydrogen exchange data shows that the beta-trefoil architecture of hFGF-1 does not behave as a single cooperative unit. There are at least two structurally independent units with differing stabilities in hFGF-1. Beta-strands I, II, III, VI, VII, X, XI and XII fit into the global unfolding isotherm. By contrast, residues in beta-strands IV, V, VIII and IX exchange by the sub-folding isotherm and could be responsible for the occurrence of high-energy partially unfolded state(s) in hFGF-1. The work described here elucidates the correlation between the structural dynamics with biological function of hFGF-1. This work also proves that the slow exchanging residues in hFGF-1 do not represent the folding nucleus of the protein.

碩士
國立臺灣大學
物理學系研究所
86
Onconase 係一兼具細胞毒性及抗癌性之核醣核酸脢, 我們利用一系列的 2 維及 3 維光譜來決定蛋白質 onconase 的結構及動性, 此具細胞毒性的蛋白質能抑制癌細胞的生 長.我們使用 3 個改良過的 2 維異核核磁共振脈衝序列, 來量測 onconase 的動力行為 參數, 包含 15N 之自旋-晶格弛緩參數 (T1), 自旋-自旋弛緩參數 (T2), 以及 15N-1H 異核交互作用增益參數 (NOE). 來分析這些參數, 我們利用 Lipari 及 Szabo 的自由 模型法則, 以得到次序參數 (S2), 有效相關時間 (te), 化學交換速率 (Rex), 以及蛋白 質整體轉動相關時間 (tm). 我們發現 onconase 基本上係一結構相當緊緻的蛋白質, 特 別是它的二級結構區分子振動範圍似乎相當小. 相反地, 活性中心之胺基酸有較特殊的動 性. 一般而言, 這些動性較大, 以便能調整與受子作用. 較特殊的是其中之一 Lysine 胺 基酸之次序參數接近於1. 代表此與鄰近之分子結成氫鍵. 這些參數的結果將提供我們更 深切地了解, onconase 骨架的動力行為. 但其生化上之意義有待進一步的了解. we have employed 2- and 3-dimensional NMR techniques to determinethe three -dimensional solution structure of onconase, a cytotoxic ribonuclease that inh ibits tumor cell growth. Using three modified 2-dimensional heteronuclear NMR pulse sequences, we have measured the spin-lattice relaxation time (T1), spin- spin relaxation time (T2) and through space interaction (so-called Nuclear Ove rhause Enhancement-NOE) of the backbone amide nitrogen nuclei of onconase. The se data were analyzed by using a model-free approach to determine the generali zed order parameter (S2), the effective correlation time for internal motion ( te), chemical exchange broadening (Rex), and the overall molecular rotational c orrelation time (tm). The results showed that the loop regions are more flexib le than the secondary structural regions. Furthermore, the active residues, wi th the exception of Lys31, are much more flexible, suggesting that the active site residues are capable of adapting to substrate conformation. The order par ameter of Lys31 is close to unity, suggesting that this residue is hydrogen bo nded to adjacent groups. More works are needed to understandthe functional imp lication of our results.

碩士
淡江大學
化學學系碩士班
101
Mastoparan B (MPB) is an antimicrobial peptide that was isolated from the hornet (Vespa basalis) venom. It’s composed of 14 amino acids, containing multiple positive charge residues and amidated C-terminus. Studies have suggested that the lysine at position 2 and the tryptophan at position 9 are important for the activities of MPB. To probe their role for structure and activity, we synthesized three peptides, MPB, Y9 (mutated at position 2 with asparagines) and N2Y9 (mutated at position 2 and 9 with asparagines and tyrosine, respectively). We investigated their antimicrobial activity, structure and dynamics at 310 K in both 30%/70% TFE/H2O and SDS micell solutions. Antimicrobial activity of the peptides, in sequence from the highest to the lowest, is showed as MPB, Y9 and N2Y9. Circular dichroism (CD) spectra indicated that the peptides adopt random coil conformation in water and α-helical structure in TFE and SDS micell solutions. In TFE, the N-terminal structures of MPB and Y9 are demonstrated more diverged and flexible than that of N2Y9. However, MPB and Y9 form longer and more stable helical structures in SDS environment. It is suggested that the affinity of binding with SDS for MPB and Y9 is higher than for N2Y9. The diffusion studies showed that the oligomerized behaviors of Y9 and N2Y9 are similar in TFE. It indicates that these two peptides with similar energetics in intermolecular interactions. As changed from TFE to SDS environment, the change profile of NH chemical shifts in N-terminal is quite different between Y9 and N2Y9.It may contribute to the consequence of N-terminal charge interacted with membrane. Antibacterial activity of MPB and its binding ability with membrane are affected by the positive charge residue Lys at N-terminal and C-terminal. The flexible N-terminal when contact with membrane will form active conformation to facilitate the insertion of hydrophobic residues into membrane. Trp as well as the hydrophobic residues on one side of the amphipathic helix can affect the capacity of peptide into hydrophobic membrane core.

碩士
淡江大學
化學學系碩士班
99
Mastoparan-B(MP-B), is a tetradecapeptide isolated from the venom of the hornet Vespa basalis (LKLKSIVSWAKKVL-NH2).Structure of MP-B is random coil in aqueous solution and folded into an amphiphilic α-helix in the presence of TFE. However, as temperature increase, random coil structure is dominant. In this study, we are interest to insight into the structure transition of coil-helix as temperature varied. We investigate the structure, diffusion and dynamics of MP-B as temperature varied in aqueous TFE and SDS micelle by NMR and CD. The characteristic differences, which may imply the folding process of MP-B, are reported here. Role of tryptophan residue on stabilization of helical structure of MP-B is also characterized.

No
The conformational propensities of amino acids are an amalgamation of sequence effects, environmental effects and underlying intrinsic behavior. Many have attempted to investigate neighboring residue effects to aid in our understanding of protein folding and improve structure prediction efforts, especially with respect to difficult to characterize states, such as disordered or unfolded states. Host-guest peptide series are a useful tool in examining the propensities of the amino acids free from the surrounding protein structure. Here, we compare the distributions of the backbone dihedral angles (φ/ψ) of the 20 proteogenic amino acids in two different sequence contexts using the AAXAA and GGXGG host-guest pentapeptide series. We further examine their intrinsic behaviors across three environmental contexts: water at 298 K, water at 498 K, and 8 M urea at 298 K. The GGXGG systems provide the intrinsic amino acid propensities devoid of any conformational context. The alanine residues in the AAXAA series enforce backbone chirality, thereby providing a model of the intrinsic behavior of amino acids in a protein chain. Our results show modest differences in φ/ψ distributions due to the steric constraints of the Ala side chains, the magnitudes of which are dependent on the denaturing conditions. One of the strongest factors modulating φ/ψ distributions was the protonation of titratable side chains, and the largest differences observed were in the amino acid propensities for the rarely sampled αL region.
NIH

In the first part of the dissertation, spider dragline silk is studied by solid state NMR techniques. The dependence of NMR frequency on molecular orientation is exploited using the DECODER experiment to determine the orientation of the protein backbone within the silk fibre. Practical experimental considerations require that the silk fibres be wound about a cylindrical axis perpendicular to the external magnetic field, complicating the reconstruction of the underlying orientation distribution and necess-itating the development of numerical techniques for this purpose. A two-component model of silk incorporating static b-sheets and polyglycine II helices adequately fits the NMR data and suggests that the b-sheets are well aligned along the silk axis (20 FWHM) while the helices are poorly aligned (68 FWHM). The effects of fibre strain, draw rate and hydration on orientation are measured. Measurements of the time-scale for peptide backbone motion indicate that when wet, a strain-dependent frac-tion of the poorly aligned component becomes mobile. This suggests a mechanism for the supercontraction of silk involving latent entropic springs that undergo a local straindependent phase transition, driving supercontraction. In the second part of this dissertation a novel method is developed for exciting NMR and nuclear quadrupole resonance (NQR) by rf irradiation at multiple frequencies that sum to (or differ by) the resonance frequency. This is fundamentally different than traditional NMR experiments where irradiation is applied on-resonance. With excitation outside the detection bandwidth, two-photon excitation allows for detection of free induction signals during excitation, completely eliminating receiver dead-time. A theoretical approach to describing two-photon excitation is developed based on average Hamiltonian theory. An intuition for two-photon excitation is gained by analogy to the coherent absorption of multiple photons requiring conservation of total energy and momentum. It is shown that two-photon excitation efficiency impro-ves when the two applied rf frequencies deviate from half-resonance. For two-photon NQR, it is shown that observable magnetization appears perpendicular to the excita-tion coil, requiring a second coil for detection, and that double quantum coherences are also generated. Several model systems and experimental geometries are used to demonstrate the peculiarities of two-photon excitation in NMR and NQR.
Science, Faculty of
Physics and Astronomy, Department of
Graduate

碩士
國立清華大學
生物資訊與結構生物研究所
94
Human hepatoma-derived growth factor (hHDGF) is a member of HRPs (HDGF-related proteins) family of proteins. HRPs belong to a new protein family that has been known in nephrogenesis, tumorigenesis, vascular development, cell proliferation and transcriptional activation. All the HRPs have the conserved N-terminal homologous to the amino terminal of HDGF (HATH) domain, but vary in the C-terminal domain. hHDGF is a 240-amino acid protein, which can be divided into two parts: the first is the well-structured HATH domain, from residues 1-100 and the structure has been determined by NMR; the second is the C-terminal domain, from residues 101-240 , which is disorder. HATH domain has been shown to bind to heparin and heparin sulfate located outside the surface of cell membrane and facilitated internalization of the protein into cell. The C-terminal domain may help translocate the protein from cytoplasm to nucleus, and serve as a signal to stimulate cell growth. Previous studies showed that HATH domain monomer shares a portion of structure with each other to form the particular dimer called the domain-swapped dimer. Dimer has much higher heparin-binding affinity to heparin than that of the monomer. My thesis work is aimed at determining the difference in dynamics by NMR relaxation method between monomer and the homo-dimer of the HATH domain. 15N spin relaxation rates and heteronuclear NOE determined at 600 MHz field were analyzed by Modelfree approach to extract the dynamic parameter: S2 (order parameter), τe (effective correlation time) and Rex (chemical exchange rate). I also use Reduced Spectral Density Mapping to determine the spectral density functions: J(ω0.87H), J(0) and J(ωN). The results showed that the domain swapped semi-dimer packs similar in spatial and orientation compares to the monomer by order parameters and spectral density function, J(0.87H). Besides fast local fluctuation loop motions act similar in both monomer and dimer, there were much more micro- to millisecond motions in monomer. Both in monomer and dimer, the L2 loop region acts more complicated dynamic motions with fitting to effective correlation times and exchange rates. We demonstrated the dynamic similarity and differences in monomer and dimer of HATH domain in wide-ranged time scale by using NMR relaxation experiments.

碩士
國立臺灣師範大學
物理學系
93
Thioesterase I (TEP-I) of Esherichia coli catalyzes the hydrolytic cleavage of fatty acyl-coenzyme A (CoA) thioesters. In addition to be a thioesterase, TEP-I has been shown to be a serine protease of the SGNH-hydrolase family. The residues involve in the catalytic process include the catalytic triad of Ser10, Asp154 and His157, and the oxyanion hole groups, which have been identified as the amide groups of Ser10 and Gly44 and the side chain of Asn73. The binding process of TEP-I with its inhibitor DENP (diethyl p-nitrophenyl phosphate) involves a fast formation of the Michaelis-Menten complex (MC) and a subsequent slow formation of the tetrahedral complex (TC). This slow kinetic makes TEP-1 an excellent model system for investigating the molecular structures and dynamics of the catalytic intermediate states. We have determined the backbone 15N NMR spin relaxation rates of the three catalytic states of TEP-I, namely the free enzyme, the TEP-1/DENP Michaelis complex, and the TEP-1/DENP tetrahedral complex at 600MHz (1H frequency). We used the Model-free approach to calculate generalized order parameters, S2, the effective correlation times, e, and a chemical exchange rate, Rex. We found that significant number of NH bonds exhibit observed s-ms time scale motion in the MC state. Changes in the generalized order parameters, characteristics of internal motion, along the catalytic pathway were also observed. His157 and Tyr15, which are located in active site pocket and which were shown by X-ray crystal structure to form - stacking in apo-form, showed significant disorder in the MC state. Furthermore, the mobility of the loop around the binding pocket is also affected by the DENP binding. We have also conducted molecular dynamics simulation of TEP-I of apo-form, MC, and TC. To analyze the overall motion and atomic fluctuation in the two-step catalytic process, we have calculated B-factor, dipolar nuclear relaxation order parameters, and the hydrogen bond network in the neighborhood of the oxyanion hole groups. The B-factor profile of each residue is in generally in good accord with the X-ray result. The 15N NMR nuclear relaxation order parameter indicated that the loop near the catalytic triad Ser10 is mostly disordered in T.S. in nano-pico second time scale. The dynamical characteristic was also confirmed by molecular dynamics simulation. The analysis of hydrogen bond network is aimed at revealing the inter-block motions of different catalytic states. We found that the hydrogen bonds among the neighboring residues of the Asn73 oxyanion hole are rarely formed in the apo-form. Thus, motion within blocks is less mobile in T.C. than apo-form of enzyme and this result is in very good agreement with NMR experiments. In conclusion, we showed that the mobility of catalytic triad plays a crucial role in the catalytic process.

STRESZCZENIE PRACY W JĘZYKU POLSKIM Wyznaczenie struktury i dynamiki białka CsPin oraz jego oddziaływania z modelowymi peptydami metodami spektroskopii NMR Łukasz Jaremko Opiekunowie: prof. dr hab. Andrzej Ejchart Instytut Biochemii i Biofizyki PAN w Warszawie, Środowiskowe Laboratorium NMR e-mail: aejchart@ibb.waw.pl, tel. (22) 659 20 37 prof. dr hab. Karol Jackowski Wydział Chemii, Uniwersytet Warszawski e-mail: kjack@chem.uw.edu.pl, tel. (22) 822 02 11 w 315 WSTĘP TEORETYCZNY Izomerazy peptydylowo-prolylowe (PPI) są obecne we wszystkich organizmach żywych. Katalizują one proces rotacji wokół wiązania peptydowego (izomeryzację cis - trans) w białkach w układzie Xaa-Pro. W skład tej grupy białek wchodzą cyklofiliny, białka wiążące FK506 oraz parwuliny. Parwuliny są przeważnie małymi białkami bardzo konserwatywnymi ewolucyjnie, występują one zarówno u przedstawicieli królestwa Procaryota jak i Eucaryota. Jak wykazały przeprowadzone dla przedstawicieli królestwa Eucaryota badania, białka z grupy parwulin zaangażowane są między innymi w regulację cyklu komórkowego i kontrolę jakości powstałych w wyniku biosyntezy białek. Proces ten ma szczególne znaczenie, gdyż istnieją uzasadnione podejrzenia, że niektóre niewielkie parwuliny jak białka Pin (Protein Interacting with NINA-kinase) mogą uczestniczyć w pierwszych etapach zwijania białek, odpowiadając za kontrolę ich jakości. Nieprawidłowo zwinięte białka znajdują się obecnie w centrum zainteresowania nauki, gdyż powodują one szereg groźnych chorób zwierząt i ludzi. Przykładem mogą być choroby neurodegeneracyjne: choroby prionowe, Alzheimera czy Parkinsona. W literaturze w przeciągu ostatniej dekady niejednokrotnie wskazywano na powiązania pomiędzy występowaniem ludzkiej parwuliny hPin1 a różnymi chorobami neurodegeneracyjnymi, w tym tauopatiami. Parwulina CsPinA pochodząca z zimnolubnych organizmów, żyjących w zimnych morskich wodach należących do archeonów Cenarchaeum symbiosum, które są symbiontami morskich gąbek z gatunku Axinella mexicana, z powodu swojego uderzającego podobieństwa do ludzkich białek Pin1 oraz Pin14 jest interesującym obiektem do badań funkcjonalnych oraz strukturalnych. Odpowiada ona za izomeryzację wiązania peptydowego w niskich temperaturach, co jest wyjątkowe w świecie przyrody, gdyż wszystkie dotychczas poznane białka z rodziny parwulin aktywne są w znacznie wyższych temperaturach. Parwulina CsPinA katalizuje proces izomeryzacji wiązania peptydowego w temperaturze 10oC, natomiast optymalna temperatura pracy pozostałych obecnie poznanych parwulin wynosi około 30oC i więcej. Dodatkowo niewielkie rozmiary parwuliny CsPinA (92 aa) oraz dobrze zbadane homologiczne białka z innych organizmów ciepłolubnych, sprawiają iż jest ona doskonałym modelowym układem do badania adaptacji niskotemperaturowych oraz wpływu temperatury na strukturę oraz dynamikę funkcjonalną enzymu. ZNACZENIE PROJEKTU Przedstawiony projekt ma przede wszystkim znaczenie poznawcze i pozwoli opisać molekularne różnice między parwulinami wysoko (>30oC) oraz niskotemperaturowymi (~10oC). Otrzymane wyniki nie ograniczą się do jednej klasy enzymów. Z pewnością zainteresują one wielu badaczy zajmujących się reakcjami enzymatycznymi, niejednokrotnie wrażliwymi na niewielkie zmiany temperatury a stanowiącymi podstawę procesów życiowych. Proponowane badania zapewnią wgląd na poziomie molekularnym na zależność między wydajnością pracy enzymu a temperaturą zarówno od strony strukturalnej jak i dokładnej dynamiki procesów katalitycznych. Podsumowując, podstawowym znaczeniem projektu jest zrozumienie często subtelnych różnic w strukturze i dynamice enzymu w warunkach optymalnych dla jego działania oraz odległych od tych warunków. Porównanie wyników uzyskanych dla parwuliny CsPinA aktywnej w niskiej temperaturze z jej bakteryjnymi i ludzkimi odpowiednikami aktywnymi w wyższych temperaturach umożliwi lepsze poznanie sposobów w jaki różne organizmy przystosowały się do życia w skrajnych warunkach termicznych. Wiedza ta może posłużyć do projektowania enzymów pracujących w pożądanych warunkach termicznych, co jest niezmiernie interesujące zarówno dla czystej nauki jak i zastosowań przemysłowych. CELE Cele pracy zostały przedstawione poniżej: I. Badania strukturalne pierwszej parwuliny z Archea z wykorzystaniem wielowymiarowych technik magnetycznego rezonansu jadrowego (NMR): - przypisania sygnałów 1H, 13C oraz 15N łańcucha głównego oraz łańcuchów bocznych CsPinA; - określenie motywów drugorzędowych oraz zwinięcia białka CsPinA; - określenie wysoko rozdziejczej struktury CsPinA w roztworze; II. opis dynamiki łańcucha głównego białka CsPinA w temperaturze fizjologicznej z wykorzystaniem pomiarów magnetycznej relaksacji jąder 15N; III. określenie reszt odpowiedzialnych za wiązanie modelowego peptydu; IV. wpływ temperatury na dynamikę globalną oraz lokalną białka CsPinA; V. przyspieszenie badań dynamiki łańcucha głównego białka formalizmem MFA/EMFA z wykorzystaniem wyłącznie stałych prędkości relaksacji R1 oraz R2 przy wyeliminowaniu czasochłonnych pomiarów {1H}-15N NOE; VI. wyznaczenie parametrów strukturalnych rN-H oraz Δσ(15N) z danych magnetycznej relaksacji jąder 15N białka CsPinA w temperaturze fizjologicznej. STRESZCZENIE Z REALIZACJI CELÓW Wymienione cele zostały zrealizowane. Poniżej zamieszczony jest skrócony opis z realizacji poszczególnych celów pracy. Ad. I. Rysunek 1 | Przypisane widmo 2D 1H-15N HSQC białka CsPinA. Przeprowadzone badania metodami cieczowego magnetycznego rezonansu jądrowego (NMR) pozwoliły rozwiązać i udokładnić wysoko rozdzielczą strukturę pierwszej parwuliny z archeonów. Parwulina CsPinA składa się z 92 aminokwasów. Widma 1H-15N HSQC (Rys. 1) oraz 1H-13C HSQC charakteryzują się doskonałą dyspersją sygnałów co w połączeniu z wielkością białka sprawia, że jest ono idealnym obiektem do badań technikami cieczowego NMR. Wysoko rozdzielcza struktura NMR parwuliny CsPinA wykazała, że białko posiada zwinięcie typu β-α3-β-α-β2 charakterystyczne dla wszystkich znanych dotychczas parwulin. Jedyna różnica dotyczy helisy3, będącej w tym przypadku krótką helisą typu 310 (Rys. 2). Figure 2 | Zwinięcie białka CsPinA z zaznaczonymi strukturami drugorzędowymi. Porównanie pierwszej parwuliny CsPinA z archeonów z jej bakteryjnymi oraz eukariotycznymi odpowiednikami wykazało znacząco większy rozmiar miejsca aktywnego wiązania substratu (Rys. 3). Można to tłumaczyć jako adaptację do pełnienia funkcji izomerazy w niskich temperaturach. Rysunek 3 | Porównanie powierzchni hydrofobowej oraz rozmieszczenia reszt kluczowych dla aktywności enzymatycznej w centrum katalitycznym parwulin: A) C. symbiosum PinA, CsPinA (PDB 2RQS); B) E. coli Par10 (PDB 1JNS); C) H. sapiens Pin1 (PDB 1PIN); oraz D) H. sapiens Par14 (PDB 1EQ3). Promienie wejścia do centrum katalitycznego zostały policzone w programie MolMol; średni błąd wynosi ± 0.35 Å. Ad. II. Pomiary magnetycznej relaksacji 15N (R1, R2, {1H}-15N NOE) wolnego białka CsPinA zostały wykonane dla próbce pojedynczo 15N-znakowanego białka CsPinA o stężeniu ~0.9 mM na spektrometrach o częstotliwości podstawowej 400, 500, 600 i 700 MHz. W przypadku pomiarów {1H}-15N NOE przy 600 MHz oraz 700 MHz przerwy relaksacyjne d1 wynoszące 8 s oraz odpowiednio 12 i 14 s okazały się za krótkie, co zmusiło nas do odrzucenia danych {1H}-15N NOE otrzymanych dla tego pola. Pomiary dla czterech częstotliwości (R1, R2 oraz {1H}-15N NOE, częstotliwości 400, 500, 600 i 700 MHz) zapewniły komplet danych relaksacyjnych, które pozwoliły opisać z wystarczającą dokładnością dynamikę łańcucha głównego z wykorzystaniem modelu Extended Model-Free Approach (EMFA) w 10oC. Rysunek 4 | Reszty wykazujące dynamikę w skali czasowej μs-ms. Badania dynamiki łańcucha głównego białka CsPinA wykazały, że reszty helisy3 (oznaczonej na Rys. 2 jako α3), będącej krótką helisą 310 oraz sąsiedniej pętli wykazują w temperaturze 10°C wyraźną dynamikę w skali mikro do milisekund (Rys. 4 – na rysunku numeracja jest przesunięta o -5 reszt) oraz heterogenność w rodzinie struktur wyznaczonej z danych NMR. Wyniki analizy EMFA otrzymane dla białka CsPinA pozwoliły zidentyfikować reszty charakteryzujące się ruchami w skali mikro do milisekund. Są to następujące reszty aminokwasowe: H14, Q25, F36, G37, D46, S55, V65, K66, E87 oraz F88 (Rys 4). Pokrywają się one z resztami wykazującymi zmiany przesunięć chemicznych po związaniu modelowego peptydu HQSPWNN do białka (Rys. 5). Ad. III. Miareczkowanie białka CsPinA modelowym peptydem wybranym za pomocą fagowej ekspresji peptydów (phage display) potwierdziło znaczenie reszt helisy3 oraz sąsiedniej pętli w procesie wiązania ligandu (Rys. 5), który moduluje ruchliwość helixy3 oraz reszt centrum katalitycznego po związaniu w 10°C. Glicyna oraz dwa dodatnio naładowane aminokwasy obszaru 310-helisy3 i pętli są konserwowane we wszystkich parwulinach, co dodatkowo potwierdza ich znaczenie funkcjonalne dla tej rodziny białek. Wybrane za pomocą fagowej ekspresji peptydów dwa modelowe peptydy HQSPWNN oraz HKRPRNN zostały użyte do miareczkowania podwójnie znakowanego 13C,15N białka CsPinA w proporcjach 1:0, 1:1, 1:4 oraz 1:8 (stosunek molowy białka do peptydu). Peptyd był dodawany do białka w postaci stężonego roztworu o znanym stężeniu w takim samym jak białko buforze w temperaturze fizjologicznej 10oC. Dla wszystkich kroków miareczkowania białka peptydami wykonywane były widma 1H-15N HSQC, a dla dwóch skrajnych punktów wykonane zostały dodatkowo widma 1H-13C HSQC dla obszaru aromatycznego. Analiza uzyskanych profili zaburzeń przesunięć chemicznych grup H/N (chemical shifts perturbation plots) wykazała, że peptyd HQSPWNN oddziałuje specyficznie z białkiem CsPinA. Reszty wykazujące największe zmiany przesunięć chemicznych w wyniku związania peptydu to: V65, G60, K63, G37, G89, F36, G47, F88, G89, S49, G58, G62 (Rys. 5 – na rysunku numeracja jest przesunieta o -5 reszt) znajdujące się wokół miejsca wiązania substratu jak i samym centrum katalitycznym, jak H14 oraz H91 (Rys. 3). Rysunek 5 | Reszty wykazujące zmiany przesunięć chemicznych po dodaniu HQSPWHH. Ad. IV Rysunek 6 | Efektywny czas korelacji białka CsPinA w funkcji temperatury. Zbadano różnice w dynamice funkcjonalnej i strukturze przestrzennej izomerazy CsPinA w różnych temperaturach: w temperaturze optymalnej dla pracy tego enzymu 10°C oraz wysokiej i niższej. Dynamika łańcucha głównego białka CsPinA w polu 9.4 T w temperaturach 1°C, 10°C, 19°C oraz 28°C zmienia się znacząco z temperaturą przy zachowaniu ogólnego upakowania oraz zwinięcia białka (Rys. 6). Badania zmian przesunięć chemicznych protonów amidowych łańcucha głównego w funkcji temperatury pozwoliło zauważyć zmiany na poziomie lokalnym (Rys. 7). Pomiary widm 2D 1H-15N HSQC w funkcji temperatury w przedziale od -1°C do 36°C, wykazały, że powyżej 15°C kilka reszt z centralnej części białka (C12, I15, V26), a powyżej 25°C wiele dodatkowych reszt aminokwasowych, kluczowych dla procesu izomeryzacji w temperaturze 10°C, charakteryzuje się nieliniowym przebiegiem amidowych przesunięć chemicznych w funkcji temperatury. Wskazuje to na zmiany struktury oraz zachodzący proces wymiany chemicznej. Wyniki te zostały dodatkowo potwierdzone przez wartości lokalnych parametrów MFA w 28oC, w szczególności wartości charakteryzujące wymianę chemiczną, Rex. Rysunek 7 | A – najstabilniejsze reszty białka CsPinA (kolor niebieski); B - zmiany przesunięć chemicznych amidów łańcucha głównego wybranych reszt aminokwasowych w funkcji temperatury; C – nałożenie sekwencji z zaznaczeniem reszt, których przesunięcia chemiczne protonów amidowych nie są liniowe ze zmianami temperatury. Ad. V Rysunek 8 | Wpływ eliminowania danych {1H}-15N NOE na parametry lokalne. Ponieważ pomiary magnetycznej relaksacji jądrowej w białkach są czasochłonne, szczególnie z powodu mało czułego pomiaru {1H}-15N NOE, który jak pokazały wcześniejsze i nasze badania wymaga stosowania długich przerw relaksacyjnych, ważne stają się nowe metody skrócenia czasu pomiarowego. Wykonanie pomiarów relaksacyjnych dla białka CsPinA w temperaturze 10oC w czterech polach magnetycznych umożliwiło całkowite wyeliminowanie pomiarów {1H}-15N NOE z analizy EMFA (Rys. 8). Zarówno globalne jak i lokalne parametry relaksacyjne otrzymane wyłącznie z zestawu czterech par R1 oraz R2 jak i dodatkowo z pomiarami {1H}-15N NOE okazały się być w granicach błędów identyczne (Rys. 9). Potwierdza to zasadność rezygnacji z danych {1H}-15N NOE i zastąpienia tego eksperymentu pomiarem R1 i/albo R2 w dodatkowym polu. Rysunek 9 | Zgodność między parametrami lokalnymi otrzymanymi z i bez {1H}-15N NOE. Ad.VI Od dawna znany jest fakt, iż parametry strukturalne białek, takie jak długości wiązań N-H oraz anizotropia przesunięcia chemicznego jąder 15N, wykazują zmienność w obrębie spotykanych wartości. Często w analizie danych relaksacyjnych stosuje się uproszczenie polegające na przyjęciu stałych wartości rN-H oraz Δσ15N) dla wszystkich reszt aminokwasowych. Dane relaksacyjne dla CsPinA z czterech pól okazały się wystarczające do wyznaczenia wartości rN-H oraz Δσ15N) dla poszczególnych reszt (Rys. 10). Wyniki te zgadzają się z wynikami otrzymanymi przez innych badaczy odmiennymi metodami. Obserwowana jest ciekawa zależność między wydłużeniem się wiązania N-H a zmniejszeniem wartości Δσ15N). Rysunek 10 | Wyznaczone wartości 15N CSA [Δσ15N)] oraz rN-H białka CsPinA.

The structural dynamics of proteins and other macromolecules typically serve crucial roles for their respective biological function. While rigid protein structures are used in classic “lock and key” descriptions of enzymology and receptor-ligand interactions, more and more evidence suggest that the majority of molecular interactions occur on the spectrum between induced-fit binding and conformational selection binding. This model of biomolecular interaction requires, to differing degrees, conformation plasticity and dynamics of the protein itself. To characterize the determinants and implications of protein dynamics, there exists no more suited biophysical technique than nuclear magnetic resonance (NMR) spectroscopy. This method is capable of probing the individual atomic nuclei of proteins in a site-specific manner. Furthermore, NMR spectroscopy is unique in being able to access timescales from picoseconds to seconds, providing information on events from bond vibration and libration to protein folding and ligand binding. The breadth of biophysical information accessible by NMR spectroscopy has led to its widespread use in the study of protein dynamics. The work presented herein involves i) the use of NMR for investigation of structure and dynamics in two separate biological systems that demonstrate a high degree of flexibility for folded proteins and ii) the improvement of pulse sequences and methodology for better characterizing picosecond to nanosecond backbone and side-chain dynamics. The organizing principle of this work, which is best exemplified in the structural studies of the piRNA-pathway protein Gametocyte-specific factor 1, is the unmatched capability of NMR spectroscopy to decipher molecular details within dynamic protein systems. First, the molecular structure and RNA-binding properties of gametocyte-specific factor 1 (GTSF1) of the piRNA effector pathway were investigated. A partially disordered protein with two Zn finger domains, the work presented here describes the isolation of a GTSF1 protein construct amendable to study by NMR spectroscopy. Chemical shift assignment of GTSF1 allowed site-specific observation of amide correlations, which established the basis for NMR structure calculation of GTSF1 and the evaluation of binding to candidate RNA sequences, with goal of the identification of an in vivo RNA binding partner for GTSF1. The work presents compelling data that indicate GTSF1 Zn finger 1 specifically binds a motif GGUUC(G/A) RNA, which in this study was found in the T-arm loop of transfer RNA. Zn finger 2 is affected by the interaction with RNA, but the available structural and binding data indicate that the second Zn finger is a more dynamic, breathable entity, supported by cysteine chemical shift and structural differences between the two GTSF1 Zn fingers. Although it’s currently speculative, the function of GTSF1 might first require binding of RNA to the more stable Zn finger 1, which then leaves Zn finger 2 poised for binding to another molecular species. tRNA-derived fragments that include the T-arm TC loop have been recently implicated in silencing of transposable elements in mammalian cells. GTSF1, which was identified in a genetic screen for piRNA-pathway proteins as vitally required for gene silencing, might plausibly act as a sensor of transcription of transposable elements and help initiate Piwi-piRISCs-mediated chromatin modification and heterochromatin formation. Next, NMR spectroscopy is used to investigate protein thermostability in psychrophilic (cold-loving) cytochrome c552. Isolated from the bacterium Colwellia psychrerythraea (Cp), previous work has implicated two conserved Cpcyt c552 methionine residues, which are both conserved across psychrophilic and psychrotolerant cytochromes, as acting in dynamical ligand substitution with a third methionine that is the axial heme ligand. It is proposed that elevated backbone dynamics in these methionine residues and the ability for them swap into the axial ligand position accounts for an uncharacteristically high melting temperature (Tm) compared to meso- and thermophile c-type cytochromes. Progress was made in NMR sample preparation and backbone chemical shift assignment of both redox states of Cpcyt c552, and insight from 1D 1H NMR experiments focused on the heme group bound to Cp cytochrome c552 is discussed. Additionally, chemical shifts are used to predict protein dynamics as a first test of a multiple methionine axial ligand hypothesis. Initial data analysis predicts relatively large measures of Random Coil Index for residues surrounding the native axial heme ligand, and shows the hyperfine shifts localized to the residues surrounding the heme. Future experiments will selectively record methyl group dynamics of methionine residues for elucidation of rate constants of methionine substitution and to determine the structural properties of this minor conformation. Finally, two NMR methodology studies are presented in this thesis: a novel simultaneous-acquisition TROSY pulse sequence for measurement of backbone spin relaxation rates (R1 and {1H}-15N heteronuclear NOE) and a side-chain 2H spin relaxation method for using multifield experimental datasets for better sampling of the spectral density function. Together, these pulse sequences represent significant advancements in NMR measurement of microscopic rate constants and more nuanced detail of protein dynamics.

碩士
國立臺灣大學
物理學研究所
90
The E2 component of human mitochondrial branched-chain α-ketoacid dehydrogenase (BCKD) complex is a homo-24-meric dihydrolipoyl transacylase. Mutations in BCKD complex are related to the maple syrup urine disease (MSUD). The structure of lipoic acid-bearing domain (LBD) of the E2 component was solved by NMR techniques comprises two four-stranded β sheets forming a flattened β-barrel. We have measured the spin—lattice relaxation rate (R1), spin-spin relaxation rate (R2), and 15N{H} nuclear Overhauser enhancement (NOE) of LBD using 2-D heteronuclear NMR pulse sequences at 11.74T and 14.09T. We used quadric diffusion analysis to obtain the diffusion tensor and model-free formalism to determine the order parameter (S2), the correlation time of local motion (τe), and the chemical exchange contribution, Rex. The spectral density functions were further extracted from reduced spectral density mapping. TROSY-CPMG relaxation experiment was also applied to measure the transverse relaxation rates with different τcp at 14.07 T, which permits the characterization of chemical exchange processes in micro- to milli-second timescale. When the motional behavior is mapped to the structure, β—barrel fold is rigid in general and the lipoylation site, Lys44, exposed to the solvent and located in a turn connecting two β-strands has high amplitude of motion. The loop forming V-shaped groove region which is close to Lys44 in space also appears to display conformational fluctuation in micro- to milli-second timescale.

This thesis presents an activity mode intent recognition approach for safe, robust and reliable control of powered backbone exoskeleton. The thesis presents the background and a concept for a powered backbone exoskeleton that would work in parallel with a user. The necessary prerequisites for the thesis are presented, including the collection and processing of surface electromyography signals and inertial sensor data to recognize the user’s activity. The development of activity mode intent recognizer was described based on decision tree classification in order to leverage its computational efficiency. The intent recognizer is a high-level supervisory controller that belongs to a three-level control structure for a powered backbone exoskeleton. The recognizer uses surface electromyography and inertial signals as the input and CART (classification and regression tree) as the classifier. The experimental results indicate that the recognizer can extract the user’s intent with minimal delay. The approach achieves a low recognition error rate and a user-unperceived latency by using sliding overlapped analysis window. The approach shows great potential for future implementation on a prototype backbone exoskeleton.

In recent years, several interesting biological roles played by RNA have come to light. Apart from their known role in translation of genetic information from DNA to protein, they have been shown to act as enzymes as well as regulators of gene expression. Protein-RNA complexes are involved in regulating cellular processes like cell division, differentiation, growth, cell aging and death. A number of clinically important viruses have RNA as their genetic material. Defective RNA molecules have been linked to a number of human diseases. The ability of RNA to adopt stunningly complex three-dimensional structures aids in diverse functions like catalysis, metabolite sensing and transcriptional control. Several secondary structure motifs are observed in RNA, of which the double-helical RNA motif is ubiquitous and well characterized. Though DNA duplexes have been shown to be present in many polymorphic states, RNA duplexes are believed to exhibit conservatism. Early fibre diffraction analysis on molecular structures of natural and synthetically available oligo- and polynucleotides suggested that the double-helical structures of RNA might exist in two forms: A-form and A′-form. New improved crystallographic methods have contributed to the increased availability of atomic resolution structures of many biologically significant RNA molecules. With the available structural information, it is feasible to try and understand the contribution of the variations at the base pair, base-pair step and backbone torsion angle level to the overall structure of the RNA duplex. Further, the effect of protein binding on RNA structure has not been extensively analysed. These studies have not been investigated in greater detail due to the focus of the research community on understanding conformational changes in proteins when bound to RNA, and due to the lack of a significant number of solved RNA structures in both free and protein-bound state. While studies on the conformation of the DNA double-helical stem have moved beyond the dinucleotide step into tri-, tetra-, hexa- and octanucleotide levels, similar knowledge for RNA even at the dinucleotide step level is lacking. In this thesis, the results of detailed analyses to understand the contribution of the base sequence towards RNA conformational variability as well as the structural changes incurred upon protein binding are reported. Objectives The primary objective of this thesis is to understand the following through detailed analyses of all available high-resolution crystal structures of RNA. 1 Exploring sequence-dependent variations exhibited by dinucleotide steps formed by Watson-Crick (WC) base pairs in RNA duplexes. 2 Identifying sequence-dependent variations exhibited by dinucleotide steps containing non-Watson-Crick (NWC) base pairs in RNA duplexes. 3 Developing a web application for the generation of sequence-dependent non-uniform nucleic acid structures. 4 Investigating the relationship between base sequence and backbone torsion-angle preferences in RNA double helices followed by molecular dynamics simulation using various force fields, to check their ability to reproduce the above experimental findings. Chapter 1 gives an overview of the structural features and polymorphic states of RNA duplexes and the present understanding of the structural architecture of RNA, thereby laying the background to the studies carried out subsequently. The chapter also gives a brief description on the methodologies applied. Relevant methodologies and protocols are dealt with in detail in the respective chapters. Sequence-dependent base-pair step geometries in RNA duplexes A complete understanding of the conformational variability seen in duplex RNA molecules at the dinucleotide step level can aid in the understanding of their function. This work was carried out to derive geometric information using a non-redundant RNA crystal structure dataset and to understand the conformational features (base pair and base-pair step parameters) involving all Watson-Crick (WC) (Chapter 2) and non-Watson-Crick (NWC) base pairs (Chapter 3). The sequence-dependent variations exhibited by the base-pair steps in RNA duplexes are elaborated. Further, potential non-canonical hydrogen bond interactions in the steps are identified and their relationship with dinucleotide step geometry is discussed. Comparison of the features of dinucleotide steps between free and protein-bound RNA datasets suggest variations at the base-pair step level on protein binding, which are more pronounced in non-Watson-Crick base pair containing steps. Chapter 4 describes a web-server NUCGEN-Plus, developed for building and regeneration of curved and non-uniform DNA and RNA duplexes. The main algorithm is a modification of our earlier program NUCGEN that worked mainly for DNA. The WC step parameters and intra-base parameters for RNA were obtained from the work detailed in Chapter 2. The FORTRAN code and input sequence file format was modified. The program has two modules: a) Using the model-building module, the program can build duplex structures for a given input DNA/RNA sequence. Options are available for selecting various derived or user specified base-pair step parameters, and fibre diffraction parameters that can be used in the building process. The program can generate double-helical structures up to 2000 nucleotides in length. In addition, the program can calculate the curvature of the generated duplex at defined length scale. b) Using the regeneration module, double-helical structures of nucleic acids can be rebuilt from the existing solved structures. Further, variants of an existing structure can be generated by varying the input geometric parameters. The web-server has a user-friendly interface and is freely available in the public domain at: http://nucleix.mbu.iisc.ernet.in/nucgenplus/index.html Sequence dependence of backbone torsion angle conformers in RNA duplexes RNA molecules consist of covalently linked nucleotide units. Each of these units has six rigid torsional degrees of freedom (α, β, γ, δ, ε, and ζ) for the backbone and one (χ) around the glycosidic bond connecting the base to the ribose, thereby providing conformational flexibility. An understanding of the relationship between base sequence and structural variations along the backbone can help deduce the rationale for sequence conservation and also their functional importance. Chapter 5 describes in detail the torsion angle-dependent variations seen in dinucleotide steps of RNA duplex. A non-redundant, high resolution (≤2.5Å) crystal structure dataset was created. Base-specific preferences for the backbone and glycosidic torsion angles were observed. Non-A-form torsion angle conformers were found to have a greater prevalence in protein-bound duplexes. Further validation of the above observation was performed by analysing the RNA backbone conformers and the effect of protein binding, in the crystal structure of E. coli 70S ribosome. Chapter 5 further describes the molecular dynamics simulation studies carried out to understand the effect of force fields on the RNA backbone conformer preferences. A 33mer long duplex was simulated using seven different force fields available in AMBER and CHARMM program, each for 100 ns. Trajectory analyses suggest the presence of sequence-dependent torsion angle preferences. Torsion angle conformer distribution closer to that of crystal structures was observed in the system simulated using parmbsc0 force field. Molecular dynamics simulation studies of AU/AU base-pair step A unique geometric feature, unlike that in other purine-pyrimidine (RY) steps in the crystal dataset analysis, was reported for AU/AU step (see Chapter 2). Appendix 1 describes the work carried out to validate these features observed in the crystal structures using simulation studies. Additionally, the effect of nearest-neighbor base pairs on the AU/AU step geometry were examined. General Conclusion Overall, the findings of this thesis work suggest that RNA duplexes exhibit sequence-dependent structural variations and sample a large volume of the double-helical conformational space. Further, protein binding affects the local base-pair step geometry and backbone conformation.