Human immunodeficiency virus reverse transcriptase (HIV RT) is a virally encoded polymerase responsible for replicating the HIV genome. Most HIV treatments include nucleotide RT inhibitors (NRTIs) which inhibit HIV RT replication by serving as a substrate for the polymerase reaction but then blocks subsequent polymerization after incorporation. However, resistance to these NRTIs may occur through specific mutations in HIV RT that increase the discrimination of HIV RT for natural nucleotides over NRTIs. The role of enzyme conformational dynamics in specificity and substrate selection was studied using transient kinetic methods on HIV RT enzymes that have been site-specifically labeled with a conformationally sensitive fluorophore, to measure the rates of binding and catalysis. First, HIV RT with the mutation of lysine to arginine at the residue position 65 (K65R) was examined for its resistance against the NRTI tenofovir diphosphate (TFV), an acyclic deoxyadenosine triphosphate (dATP) analog. It was found that HIV RT K65R resistance to TFV was achieved through decreased rates of catalysis and increased rates of dissociation for TFV over dATP when compared with the kinetics of wild-type HIV RT. Moreover, global fitting analysis confirmed a mechanism where a large conformational change, after initial ground state binding of the substrate, contributed significantly to enzyme specificity. This led to our investigation of the molecular basis for enzyme specificity using HIV RT as a model system. Again, transient kinetic methods were applied with the addition of molecular dynamics simulations. The simulated results were substantiated by the corroborating experimental results. It was found that a substrate-induced conformational change in the transition of HIV RT from an open nucleotide-bound state to a closed nucleotide-bound state was the major determinant in enzyme specificity. The molecular basis for substrate selection resulted from the molecular alignments of the substrate in the active-site, which induced the conformational change. When the correct nucleotide was bound, optimal molecular interactions in the active-site yielded a stably closed complex, which promoted nucleotide incorporation. In contrast, when an incorrect nucleotide was bound, the molecular interactions at the active-site were not ideal, which yielded an unstable closed complex, which promoted substrate dissociation rather than incorporation. / text

Polymers possess bulk elastic properties due to entanglement of the polymer chains, not due to an inherit elasticity found within the monomers. An appropriately disubstituted bicyclo[8.8.8]hexacosane monomer should impart inherit elasticity when utilized in a polymer. A stochastic search of disubstituted bicyclo[m.m.m]alkanes demonstrated that these systems will adopt an out,out configuration and bicycles with medium and large values of m possess variable bridgehead-bridgehead distances. A stochastic search of disubstituted bicyclo[8.8.n]alkanes demonstrated an even-odd effect within the bite-angle of the bicycle. Two model compounds with methyl and hydroxyl groups at the bridgehead carbons were synthesized that demonstrated solid-state structures that correlated extremely well with the computational search. The solid-state structures were observed with both an out,out configuration and variable bridgehead-bridgehead distances. To investigate this hypothesis, polyurethanes will be made from the following diol monomers: 1,10-decanediol, a monocyclic diol, and a bicyclo[8.8.8]hexacosane diol.

Dendritic encapsulation of poly(phenyleneethynylene)s or PPEs has been shown to enhance photoluminescent quantum efficiency and facilitate energy transfer by funneling photonic energy absorbed on the dendron periphery efficiently to the conjugated polymeric core. The research presented herein focuses on incorporating degradable dendron onto PPEs, examining whether or not similar benefits were conferred upon the proposed system and controlling polymer luminescence through the elimination of the insulating macromolecules. PPEs appended with disassembling dendrons of various generation sizes were synthesized and their optical properties studied. Polymer luminescence was then quenched via chemical degradation of the disassembling dendrons. Furthermore, the macromolecules resulting from disassembly exhibited tunable luminescence properties upon manipulation of pH. Consequently, it was determined that polymer luminescence could be controlled upon forming phenolic moieties along the PPE backbone. Tunable emission was later realized in the thin film as well through the integration of crosslinkable dendrons onto the polymer core.Recently, helical synthetic linear polymers have demonstrated the ability to facilitate stereoselective processes such as catalysis, recognition and separation. Consequently, it has become increasingly desirable to develop new platforms capable of imparting asymmetry. The work presented herein describes the synthesis of a series of polymers based upon chiral hydrobenzoin and the subsequent conformational analysis performed on these materials. It was envisioned that these polymeric materials might inherently possess conformational asymmetry and as result could be able to impart configurationally chirality by introducing a diastereomeric bias for the formation of one enantiomer over the other during the course of the reaction.

<p>The structural plasticity conferred by conformational flexibility has increasingly been recognized as a likely determinant of function. For example, multiscale heterogeneity in the calmodulin central helix most likely helps it in binding over 100 protein targets, and a concerted motion seen in both nuclear magnetic resonance (NMR) and crystal structures of ubiquitin is proposed to underlie its functional plasticity of promiscuous binding to many different proteins with high affinity. However, flexibility is manifested in a variety of ways, depending both on the protein itself and on how it is observed. Conformational heterogeneity (the term we use for flexibility when studied by X-ray crystallography) is evident in electron density, either as fully separated peaks or as anisotropic density shapes showing fluctuation of atom groupings. Many phenomena contribute to conformational heterogeneity in crystal structures, from diverse crystal contacts to functionally relevant conformational fluctuations on a wide range of time and size scales.</p><p>In addition to ubiquitin and calmodulin, the Staphylococcus aureus virulence factor staphylococcal protein A (SpA) is an example of a highly heterogeneous protein. SpA is a major contributor to bacterial evasion of the host immune system, through high-affinity binding to host proteins such as antibodies, von Willebrand factor, and tumor necrosis factor receptor 1 (TNFR1). The protein includes five small three-helix-bundle domains (E-D-A-B-C) separated by conserved flexible linkers. Prior attempts to crystallize individual domains in the absence of a binding partner were apparently unsuccessful. There are also no previous structures of tandem domains. In this thesis, I report the high-resolution crystal structures of a single C domain (collected at both cryogenic and room temperatures), a single A domain, and two B domains connected by the conserved linker. All four apo structures exhibit extensive multiscale conformational heterogeneity, which required novel modeling protocols. Comparison of domain structures shows that helix1 orientation is especially heterogeneous, coordinated with changes in sidechain conformational networks and contacting protein interfaces.</p><p>The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure wasn't properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. In this thesis, I report the 2.3-A structure of a fully folded SpA domain in complex with Fc. My structure indicates that there are extensive structural rearrangements necessary for binding Fc, including concerted rotamer changes and coupled backbone rearrangements that lead to a difference in helix1 angle. The conformational heterogeneity of the helix1/2 interface is also eliminated in the complex, with previously poly-rotameric interfacial residues locking into single rotamer conformations. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur in SpA and in its multiple binding partners and may be an important structural paradigm in other functionally plastic proteins.</p> / Dissertation

Proteins function as worker molecules in the cell and their natural environment is crowded. How they fold in a cell-like environment and how they recognize their interacting partners in such conditions, are questions that underlie the work of this thesis. Two distinct subjects were investigated using a combination of biochemical- and biophysical methods. First, the unfolding/dissociation of a heptameric protein (cpn10) in the presence of the crowding agent Ficoll 70. Ficoll 70 was used to mimic the crowded environment in the cell and it has been used previously to study macromolecular crowding effects, or excluded volume effects, in protein folding studies. Second, the conformational changes upon interaction between the Mediator subunit Med25 and the transcription factor Dreb2a from Arabidopsis thaliana. Mediator is a transcriptional co-regulator complex which is conserved from yeast to humans. The molecular mechanisms of its action are however not entirely understood. It has been proposed that the Mediator complex conveys regulatory signals from promoter-bound transcription factors (activators/repressors) to the RNA polymerase II machinery through conformational rearrangements. The results from the folding study showed that cpn10 was stabilized in the presence of Ficoll 70 during thermal- and chemical induced unfolding (GuHCl). The thermal transition midpoint increased by 4°C, and the chemical midpoint by 0.5 M GuHCl as compared to buffer conditions. Also the heptamer-monomer dissociation was affected in the presence of Ficoll 70, the transition midpoint was lower in Ficoll 70 (3.1 μM) compared to in buffer (8.1 μM) thus indicating tighter binding in crowded conditions. The coupled unfolding/dissociation free energy for the heptamer increased by about 36 kJ/mol in Ficoll. Altogether, the results revealed that the stability effect on cpn10 due to macromolecular crowding was larger in the individual monomers (33%) than at the monomer-monomer interfaces (8%). The results from the interaction study indicated conformational changes upon interaction between the A. thaliana Med25 ACtivator Interaction Domain (ACID) and Dreb2a. Structural changes were probed to originate from unstructured Dreb2a and not from the Med25-ACID. Human Med25-ACID was also found to interact with the plant-specific Dreb2a, even though the ACIDs from human and A. thaliana share low sequence homology. Moreover, the human Med25-interacting transcription factor VP16 was found to interact with A. thaliana Med25. Finally, NMR, ITC and pull-down experiments showed that the unrelated transcription factors Dreb2a and VP16 interact with overlapping regions in the ACIDs of A. thaliana and human Med25. The results presented in this thesis contribute to previous reports in two different aspects. Firstly, they lend support to the findings that the intracellular environment affects the biophysical properties of proteins. It will therefore be important to continue comparing results between in vitro and cell-like conditions to measure the magnitude of such effects and to improve the understanding of protein folding and thereby misfolding of proteins in cells. Better knowledge of protein misfolding mechanisms is critical since they are associated to several neurodegenerative diseases such as Alzheimer’s and Parkinson's. Secondly, our results substantiate the notion that transcription factors are able to bind multiple targets and that they gain structure upon binding. They also show that subunits of the conserved Mediator complex, despite low sequence homologies, retain a conserved structure and function when comparing evolutionary diverged species.

No description available.

In der vorliegenden Arbeit wird die Aufklärung der Konformation von Peptiden mit speziell modifizierten Aminosäuren beschrieben. Die Methoden der theoretischen Chemie (Quantenchemie, Molekülmechanik, Moleküldynamik) bilden dabei die Grundlage der Konformationsanalysen. Durch systematische Anwendung dieser Methoden werden im ersten Teil der Arbeit die konformativen Eigenschaften verschiedener [beta]-Aminosäuren und ihrer Oligomere ([beta]-Peptide) untersucht. Aus diesen Ergebnissen werden anschließend Regeln für das Sekundärstrukturdesign von ß-Peptiden abgeleitet. Der zweite Teil beschäftigt sich mit der theoretischen Konformationsanalyse von [alpha]- Hydrazinosäuren und ihrer Oligomere (Hydrazinopeptide). Aus den gewonnenen Erkenntnissen über die Ausbildung charakteristischer Sekundärstrukturelemente in diesen Verbindungen wird ebenfalls ein Regelwerk für das Design von Sekundärstrukturen aufgestellt. / The present work describes the conformational characteristics of pepttides with specifically modified amino acid constituents. For this purpose, the methods of theoretical chemistry (quantum chemistry, molecular mechanics, molecular dynamics) are utilisied for the conformational analyses. The conformation of various [beta]-amino acids and their oligomers ([beta]-peptides) are inverstigated in the first part of this work applying these methods. Rules for the design of definite secondary structures in [beta]-peptides are then derived from the obtained results. In the second part, systematic theoretical conformational analyses on [alpha]-hydrazino acids and their oligomers (hydrazino peptides) are described. The results are then used to compile a set of rules for the formation of characteriasitc secondary structures in this class of compounds.

Thesis (M.Sc. (Tropical Medicine))--Mahidol University, 2003.

Thesis (M. S.)--Mechanical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Zhu, Cheng; Committee Member: Harvey, Stephen; Committee Member: Hud, Nicholas; Committee Member: Zamir, Evan; Committee Member: Zhu, Ting.

The main focus of this thesis is on photochemical modulation of molecular recognition in various host-guest systems. This involves the design, synthesis and integration of light-triggered conformational switches into peptidomimetic guests and molecular tweezer hosts. The impact of the switches on guest and host structures has been assessed by spectroscopic and computational conformational analysis. Effects of photochemical structure modulation on molecular recognition in protein-ligand and supramolecular host-guest systems are discussed. Phototriggerable peptidomimetic inhibitors of the enzyme M. tuberculosis ribonucleotide reductase (RNR) were obtained by incorporation of a stilbene based amino acid moiety into oligopeptides between 3-9 residues long (Paper I). Interstrand hydrogen bond probability in the E and Z forms of the peptidomimetics was used as a tool for predicting conformational preferences. Considerable differences in inhibitory potency for the E and Z photoisomers were demonstrated in a binding assay. In order to advance the concept of photomodulable inhibitors, synthetic routes towards amino acid derivatives based on the more rigid stiff-stilbene chromophore were developed (Paper II).  The effect of E-Z isomerization on the conformational properties of peptidomimetic inhibitors incorporating the stiff-stilbene chromophore was also assessed computationally (Paper III). It was indicated that inhibitors with the more rigid amino acid derivative should display larger conformational divergence between photoisomers than corresponding stilbene derivatives. Bisporphyrin tweezers with enediyne and stiff-stilbene spacers have been synthesized, and the conformational characteristics imposed by the spacers have been studied and compared to a glycoluril linked tweezer. The effects of spacers on tweezer binding of diamine guests and helicity induction by chiral guests have been investigated (Paper IV). Connections between spacer flexibility and host-guest binding strength have been established. The structural properties of the stiff-stilbene spaced tweezer made it particularly susceptible to helicity induction by both monotopic and bitopic chiral guests. Finally, the possibility of photochemical bite-size variation of tweezers with photoswitchable spacers has been assessed. Initial studies have shown that photoisomerization of the tweezers is possible without photochemical decomposition. Conformational analyses indicate that isomerization should impact binding characteristics of the tweezers to a significant extent (Paper V).