The kinetic mechanism of an α/β hydrolase fold epoxide hydrolase from potato, StEH1, has been studied with the aims of explaining the underlying causes for enantio- and regioselectivity, both being important for product purity. Further effort has been laid upon understanding the causes of a hysteretic behavior discovered in the measurements leading to Paper I. The enantioselectivity was investigated with substrates differing only in substituent size at one carbon of the oxirane ring structure. In catalysis with trans-stilbene oxide and styrene oxide, enantioselectivity is the result of differences in alkylation rates. In pre-steady state measurement with trans-2-methylstyrene oxide (2-MeSO), a rate-limiting step involving slow transitions, referred to as hysteresis, was discovered. With this substrate enantioselectivity is proposed to be a consequence of the catalytic rate of (1R,2R)-enantiomer being more influenced by the hysteretic behavior than was the rate of the other enantiomer. In steady-state measurements with (1R,2R)-2-MeSO, at different temperatures and pH, hysteretic cooperativity was displayed. It can be concluded that this behavior is dependent on the relationship between kcat and the rate of transition between two Michaelis complexes. From the differences in pH dependence of kcat/KM in formation of the two diols resulting from low regioselectivity in catalysis of (1R,2R)-2-MeSO, it is suggested that hysteresis is a result of the substrates placed in different conformational modes within the active site cavity. Regioselectivity is proposed to be the result of specific interactions between the catalytically important Tyr and the substrate, with a link between KM-values and degree of regioselectivity. Furthermore, the hysteretic kinetic model proposed can explain hysteresis, cooperativity and regioselectivity resulting from StEH1 catalyzed hydrolysis of (1R,2R)-2-MeSO.

G protein-coupled receptors (GPCRs) are known to exist as oligomers, but there is much uncertainty over the oligomeric size, the number of interacting G proteins and the stability of that interaction. The present approach to these questions has been threefold. Monomers of the M2 muscarinic receptor were purified from Spodoptera frugiperda (Sf9) cells and reconstituted in phospholipid vesicles, where they spontaneously formed tetramers. The size of the reconstituted complex was determined from its electrophoretic mobility after cross-linking and inferred from a quantitative, model-based assessment of cooperative effects in the binding of two muscarinic antagonists: N-methylscopolamine and quinuclidinylbenzilate. Binding of the agonist oxotremorine-M to receptor reconstituted with purified G proteins revealed at least three classes of sites that interconverted from higher to lower affinity upon the addition of guanylylimidotriphosphate (GMP-PNP). The binding properties resemble those of muscarinic receptors in myocardial preparations, thereby implying the existence of tetramers in native tissues. G proteins that copurify with the M2 receptor from cardiac membranes also were found to exist as oligomers, some of which contain both alpha(o) and alpha(i2), and the purified complexes contained receptor and G protein in near-equal amounts. A tetrameric receptor implies a tetramer of G proteins, a conclusion that is supported by the distribution of sites between different states identified in the binding of [35S]GTPgammaS to the purified complex. Covalent adducts of a GPCR fused to a Galpha-subunit provide a model system in which the relationship between receptor and G protein complex is defined with respect to stability and composition. Such a fusion of the M2 receptor and Galpha(i1) underwent a cleavage near the amino terminus of the alpha-subunit, however, flagging the likelihood of similar effects in other such adducts. Truncation of the amino terminus prior to fusion generated a stable product that revealed GMP-PNP-sensitive, biphasic binding of oxotremorine-M and noncompetitive interactions between N-methylscopolamine and quinuclidinylbenzilate. A covalent RG complex therefore exhibits the functional properties of M2 receptors in native systems. These observations are consistent with the notion that signaling through the M2 receptor occurs via cooperative interactions within a stable complex that comprises four receptors and four G proteins.

Motivated by the need to design minimally-invasive treatments for wide-necked cerebral aneurysms, we used computational modeling to assess aneurysm hemodynamics, examined in vitro cellular responses arising from mechanical and chemical stresses, and designed novel materials that cooperatively adhere to the endothelium. We first hypothesized that because aneurysm geometry plays an important role in hemodynamics, changes in flow patterns may affect the shear stress experienced on the aneurysm wall. We defined flow regimes based on aneurysm hemodynamic and geometric parameters, which may correlate with aneurysm rupture. Because of the direct contact between endothelial cells (ECs) and blood flow, we then evaluated how changes in hemodynamics and inflammatory cytokines affect the expression of cell adhesion molecules (CAMs) and matrix remodeling factors on ECs. We subsequently designed biomaterials that complement the dynamic EC surface and have the ability to conform to any geometry through in situ crosslinking. Antibody-conjugated hydrogels facilitated synergistic EC adhesion using cooperativity as an adhesion strategy. We optimized the presentation of antibodies to inflammatory CAMs on polyvinyl alcohol (PVA) and alginate hydrogels to achieve strong adhesion to inflamed ECs. We synthesized photocrosslinkable, aminated PVA hydrogels and determined the effect of substrate stiffness on cell adhesion. We also evaluated the effects of antibody presentation on cell adhesion strength and dynamics using alginate hydrogels. Taken together, the results of this work may be used to design hydrogels for vascular remodeling applications under shear stress, including embolic agents for cerebral aneurysms. / Engineering and Applied Sciences

Regulation of gene expression is essential for many biological processes. Binding of transcription factors to DNA is a key regulatory step in the control of gene expression. It is commonly observed that DNA sequences with high affinity for transcription factors occur more frequently in the genome than the instances of genes bound or regulated by these factors. However, the mechanism by which transcription factors selectively identify and regulate these genes was unclear. I utilized the transcriptional control of the phosphate-responsive signaling pathway (PHO) in Saccharomyces cerevisiae as a model system to address this problem.

Alpha-helical coiled-coils are common protein structural motifs with varied mechanical roles, such as, tropomyosin in muscle contraction or neck-stalks of kinesins and myosins, in motor proteins. Using computer simulations, we characterized elastic properties of coiled-coils both, globally and locally. Normal mode analysis for global elastic properties revealed a buckling instability due to inherently present weak non-bonded forces. We characterized this using a critical buckling length (lc). For coiled-coils, lc was significantly less than their persistence length thereby governing the filament conformation. We also found that mutations to the hydrophobic residues at the knob-into-hole interface affect elasticity of coiled-coils significantly. We built a flexibility map of tropomyosin using a local fluctuation analysis and found regional variations in flexibilities due to such breaks in the knob-into-hole packing. Overall, flexibility varies by more than twofold and increases towards the C-terminal region of the molecule. Actin binding sites in zones and broken core regions due to acidic residues at the hydrophobic face such as, the Asp137 and the Glu218, are found to be the most labile with moduli for splay and broad face bending as 70 nm and 116 nm, respectively. Such variations in flexibility could be relevant to the tropomyosin function, especially for moving across the non-uniform surface of F-actin to regulate myosin binding. Non-claret disjunction (Ncd), is a Kinesin-14 family protein that walks to the microtubule's minus end. Although available structures show its alpha-helical coiled-coil neck in either pre- or post-stroke orientations, little is known about the transition between these two states. Using a combination of molecular dynamics simulations and structural analyses, we find that the neck travel is a guided diffusion involving sequential intermediate contacts with the motor head. The post-stroke is at a higher free-energy minimum than the pre-stroke. The importance of intermediate contacts correlates with the existing motility data including those of mutant Ncds and other members of the kinesin-14 family. While the forward motion has a ~4.5 kBT (kB: Boltzmann constant, T = 300 K) free energy barrier, recovery stroke goes nearly downhill in free energy. The hysteresis in forward and reverse neck motion energetics arises from the mechanical compliance of the protein, and together with guided diffusion, it may be key for the directed motility of Ncd. Although it is known that neighboring Ncds on a microtubule (MT) have an attractive interaction and a group of Ncds act cooperatively, the physical basis of neither this attraction nor the cooperativity is known. From structural analysis of Ncd neighbors on an MT lattice we find that steric hindrances between the coiled-coil neck-stalks of longitudinal neighbors drive synchrony among a group of Ncds on a single protofilament. Across lateral dimers, surface loop L2 of the motor-head (MH) that is not bound to the MT (unbound-MH) in a pre-stroke dimer, is seen to have strong attraction to the nucleotide pocket in the MH that is bound to MT (bound-MH) of its off-axis neighbor. Such an attraction will however impede the motility in both the dimers. We hence propose rules that drive motor binding to an MT site in the presence of immediate neighbors such that motility of the group is not compromised. The unbound-MH, whose role in the walking step of an Ncd was unclear, is thus seen to regulate MT decoration.

G protein-coupled receptors (GPCRs) are known to exist as oligomers, but there is much uncertainty over the oligomeric size, the number of interacting G proteins and the stability of that interaction. The present approach to these questions has been threefold. Monomers of the M2 muscarinic receptor were purified from Spodoptera frugiperda (Sf9) cells and reconstituted in phospholipid vesicles, where they spontaneously formed tetramers. The size of the reconstituted complex was determined from its electrophoretic mobility after cross-linking and inferred from a quantitative, model-based assessment of cooperative effects in the binding of two muscarinic antagonists: N-methylscopolamine and quinuclidinylbenzilate. Binding of the agonist oxotremorine-M to receptor reconstituted with purified G proteins revealed at least three classes of sites that interconverted from higher to lower affinity upon the addition of guanylylimidotriphosphate (GMP-PNP). The binding properties resemble those of muscarinic receptors in myocardial preparations, thereby implying the existence of tetramers in native tissues. G proteins that copurify with the M2 receptor from cardiac membranes also were found to exist as oligomers, some of which contain both alpha(o) and alpha(i2), and the purified complexes contained receptor and G protein in near-equal amounts. A tetrameric receptor implies a tetramer of G proteins, a conclusion that is supported by the distribution of sites between different states identified in the binding of [35S]GTPgammaS to the purified complex. Covalent adducts of a GPCR fused to a Galpha-subunit provide a model system in which the relationship between receptor and G protein complex is defined with respect to stability and composition. Such a fusion of the M2 receptor and Galpha(i1) underwent a cleavage near the amino terminus of the alpha-subunit, however, flagging the likelihood of similar effects in other such adducts. Truncation of the amino terminus prior to fusion generated a stable product that revealed GMP-PNP-sensitive, biphasic binding of oxotremorine-M and noncompetitive interactions between N-methylscopolamine and quinuclidinylbenzilate. A covalent RG complex therefore exhibits the functional properties of M2 receptors in native systems. These observations are consistent with the notion that signaling through the M2 receptor occurs via cooperative interactions within a stable complex that comprises four receptors and four G proteins.

Ce manuscrit présente la synthèse et la caractérisation de deux nouvelles familles de complexes mononucléaires, [Fe(L)2(NCS)2] et [Fe(L)(H2B(pz)2)2], montrant des propriétés bistables. L’étude des structures cristallographiques a permis de discuter l’influence de l’élongation des ligands L sur le réseau cristallin et sur les contacts intermoléculaires ainsi que l’existence de corrélations structures / propriétés. Enfin, la dernière partie de ce travail est consacrée à l’élaboration de façon contrôlée d’objets à transition de spin à l’échelle nanométrique, sous la forme de films déposés par sublimation et de nanoclusters synthétisés en milieu confiné. / This thesis presents the synthesis and characterization of two new families of mononuclear complexes, [Fe(L)2(NCS)2] and [Fe(L)(H2B(pz)2)2] showing bistable properties. The crystallographic studies allowed us to discuss the influence of the elongation of the ligands L on the crystal lattice, intermolecular contacts and the existence of structure / properties correlations. The final part of this work shows the controlled synthesis of spin crossover objects at the nanometric scale as thin films prepared by sublimation and nanoclusters synthesized in confined media.

Uma das várias formas da hemoglobina do peixe Leporinus Frederici (piava) não apresenta efeito Borh (variação da afinidade ao O2 com o pH). Purificação, caracterização e experimentos de cristalização foram conduzidos visando a determinação da estrutura através de difração de raios X, embora sem resultados positivos. O espectro óptico desta forma particular de hemoglobina foi medido no intervalo de 300-700 nm e subseqüentemente simulado, interpretado e comparado com o espectro da humana. Em outra área do trabalho experimental, três pequenas estruturas moleculares foram determinadas: uma é um intermediário na síntese de alcalóides, com um esqueleto sarpagina; outra é um dipeptídeo complexado com Cu2+; e a terceira é um complexo de picrato com Ce3+. As intensidades das reflexões foram medidas com um difratômetro automático de quatro ciclos CAD-4. As estruturas foram resolvidas por Patterson ou Métodos Diretos e foram refinadas por método de mínimo quadrado. Cetona, C19H21N3O, é um intermediário chave no caminho de reação para síntese de indoloquinolisidinas, pertence ao sistema, P21/C, a=12.200(7), b=16,795(2), c=16,655(l)&#197, &#946=104,18(3)&#176, Z=8, Dc= 1,234 gcm-3, V=3308(3)&#1973. As duas moléculas independentes são aproximadamente relacionadas por um centro de inversão, a principal diferença sendo relativa às configurações dos grupos nitril e metil. As moléculas enantioméricas estão mantidas por ligação de hidrogênio através do N(3)-O(1\') e N(3\')-O(1). A junção N(1)-C(6) é trans e o grupo CH2CN é axial. L-(triptofil)-L-glicinato-cobre(II). C13H13CuN3O3, um composto modelo para conseguir informações para interpretar os dados disponíveis para proteínas azuis, é ortorrômbico, P212121, a=8,284(6), b=9,345(2). c=16.503(2)&#197, Z=4, Dc=1.678 gcm-3, V=1277(2)&#1973. O íon Cu2+ é coordenado por um oxigênio e dois nitrogênios de um dipeptídeo e com um oxigênio de um ligante simetricamente relacionado. A estrutura polimérica resultante está alinhada com o eixo b e tem uma estabilidade maior devido a uma ligação de hidrogênio entre o oxigênio carbonil de um dipeptídeo e o nitrogênio do triptofano do ligante vizinho. A coordenação é essencialmente quadrado planar. O complexo de picrato com Ce3+, CeO33N9C18H30, foi analisado dentro de um grande programa de pesquisa para estudar a química de coordenação dos lantanóides. Duas formas cristalinas são estudadas: uma é monoclínica, P21/n, a=7,799(2), b=26,925(2), c=17,465(2)&#197, &#946=98,93(3)&#176 , Z=4, Dc= 1,908 gcm-3, V=3623(2)޵ e a outra é monoc1ínica, C2/c, a=40,225(5), b=8,08(4), c=24,35l(9), &#946=111,46(2), Z=8, Dc= 1,893 gcm-3, V=7300(8)&#1973. A primeira é relativamente instável sobre a incidência de raios-X e embora a medida das intensidades apresentasse erros sistemáticos significantes, a estrutura pode ser resolvida. O número de coordenação dos dois complexos é 9 e os poliedros de coordenação são intermediários entre antiprisma quadrado monoencapuzado e prisma trigonal triencapuzado / One of the various forms of hemoglobin of the fish Leporinus Frederici (piava) does not present any Bohr effect (variation of the affinity to O2 with pH). Purification, characterization and crystallization experiments were conducted, aimed at the structure determination through X-ray diffraction, although with no positive results. The optical spectrum of this particular hemoglobin form was measured in the range 300-700 nm and subsequently simulated, interpreted and compared with the human hemoglobin spectra. In another area of experimental work, three small molecules structures were determinate: one is an intermediate in the synthesis of alkaloids, with a sarpagine backbone; another is a dipeptide complexes with Cu2+; and a third one is a complex of picrate with Ce3+. The intensities of the reflections were measured with an automatic four-circle difractometer CAD-4. The structures were solved by Patterson or Directs Methods, and were refined by the least squares methods. Ketone, C19H21N3O, is a key intermediate in the reaction pathway for synthesis of indoloquisidines, belongs to the monoclinic system, P21/c, a=12.200(7), b=16,795(2), c=16,655(l)&#197, &#946=104,18(3)&#176, Z=8, Dc= 1,234 gcm-3, V=3308(3)&#1973. The two independent molecule are approximately related by an inversion center, the main difference being the relative configurations of the nitril and methyl groups. The enantiomeric molecules are hydrogen bonded through N(3)-O(1\') and N(3\')-O(1). The junction N(l)-C(6) is trans and the group CH2CN is axial. L-(tryptophyl)-L-glycinate-copper(II), C13H13CuN3O3, a model compound to get information to interpret spectroscopic data available for blues proteins, is orthorhombic, P212121, a=8,284(6), b=9,345(2). c=16.503(2)&#197, Z=4, Dc=1.678 gcm-3, V=1277(2)&#1973. The Cu-ion is coordinated by one oxygen and two nitrogen atoms of the one dípeptide and with an oxygen of a symmetrically related ligand. The resulting polymeric structure is aligned with the b-axis and is further stabilized by an H-bond between the carbonyl-oxygen of the one dipeptide and the tryptophan side-chain nitrogen of the neighboring ligand. The coordination is essentially square-planar. The complex of picrate with Ce3+, CeO33N9C18H30, was analyzed within a broader research program to study the chemistry of coordination of the lantanoids. Two crystalline forms are studied: one is monoclinic, P21/n, a=7,799(2), b=26,925(2), c=17,465(2)&#197, &#946=98,93(3)&#176 , Z=4, Dc= 1,908 gcm-3, V=3623(2)޵ and the other is monoclinic, C2/c, a=40,225(5), b=8,08(4), c=24,35l(9), &#946=111,46(2), Z=8, Dc= 1,893 gcm-3, V=7300(8)&#1973. The former is relatively unstable under the X-rays and although the measured intensities presented significant systematic errors, the structure could be solved. The coordination number of the two complexes is 9 and the coordination polyedra are intermediate between mono-coupled square antiprism and tri-coupled trigonal prism

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