A Series of fluorophores of the general formular P(CF2)nP and P(CF2)n-1CF3 has been synthesized. Copper catalyzed coupling of 1-bromopyrene and the corresponding mono and di-iodoperfluoroalkanes were used in most cases. For the n=3 dimer, a novel 1,w-perfluoroalkylation of pyrene via bis-decarboxylation of hexafluorogultaric acid was utilized. These compounds, along with suitable hydrocarbon analogs, are being used to study the flexibility of fluorocarbon chains using emission. We have found that the excimer formation for the fluorinated pyrene monomers is highly dependent on concentration and is less efficient than for pyene. Excimer formation for the fluorinated pyrene dimers is much more efficient than for the fluorocarbon monomers and is only slightly concentraion dependent. Steady-state emission spectra indicate hydrocarbon dimers-models form excimers more efficiently than the fluorinated dimers suggesting the fluorinated chains are stiffer than the hydrocarbons. We conducted the temperature-dependent studies and quantified the conformational difference.

Orientador: Munir Salomão Skaf / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-25T12:12:30Z (GMT). No. of bitstreams: 1 Stankovic_Ivana_D.pdf: 27307159 bytes, checksum: 42c2cb1272b20efd40baf8597dd8f156 (MD5) Previous issue date: 2014 / Resumo: A degradação enzimática da celulose proveniente de biomassa para a produção de bioetanol é realizada por um conjunto de proteínas denominadas celulases, as quais são produzidas por vários fungos e bactérias. O mecanismo molecular das interações físicas entre as celulases e a celulose é pouco conhecido. Para investigar estas interações, bem como as propriedades conformacionais e dinâmicas, nesta Tese usamos simulações por dinâmica molecular (MD). Abordamos duas celulases: a Endoglucanase 3 de Trichoderma harzianum (ThEG3) e a Endoglucanase de Xanthomas campestris pv. campestris (XccEG). O estudo dos mecanismos de reconhecimento de substrato por ThEG3 que falta o módulo de ligação à celulose (CBM), revela que a ausência de um CBM nesta estrutura é compensada pela presença de resíduos similares aos resíduos típicos de um CBM. O segundo estudo, sobre seletividade da XccEG, explica por que esta enzima catalisa a hidrólise somente de oligossacarídeos de cadeia igual ou maior que quatro unidades. Nossas simulações indicam que os quatro subsítios característicos da fenda de ligação ao substrato da XccEG precisam ser simultaneamente estabilizados pelas interações com substrato para promover uma ligação efetiva substrato-enzima. No terceiro estudo, investigamos as propriedades mecânicas do linker entre o domínio catalítico (CCD) e o CBM desta mesma enzima, composto por blocos de Thr e Pro, utilizando a técnica de replica exchange MD. Nossos resultados sugerem as bases moleculares da maior rigidez deste linker em comparação ao linker de celobiohidrolases II de Trichoderma reesei. Por fim, adaptamos o método de MDFF (flexible fitting MD) para gerar modelos de estrutura terciária a partir de dados de SAXS e o aplicamos para criar um modelo da estrutura intacta CCD-linker-CBM de XccEG / Abstract: The enzymatic degradation of the cellulose for the production of bioethanol is performed by a group of proteins called cellulases which are produced by various fungi and bacteria. The molecular mechanism of the physical interactions between the cellulases and a cellulose is little-known. In this work we use molecular dynamics simulations (MD) to investigate these interactions as well as conformational and dynamic properties. We have studied two cellulases: the endoglucanase 3 from Trichoderma harzianum (ThEG3) and the endoglucanase from Xanthomas campestris pv. campestris (XccEG). The study of the mechanisms of substrate recognition by ThEG3 which lacks the cellulose binding module (CBM) shows that the absence of a CBM in this structure is compensated by the presence of residues similar to typical CBM residues. The second study, on the selectivity of XccEG, explains why this enzyme catalyses only the hydrolysis of four or more units long oligosaccharides. Our simulations indicate that the four characteristic subsites of the substrate binding cleft of XccEG need to be simultaneously stabilized by the interactions with the substrate to provide an effective enzyme-substrate binding. In the third study, we investigated the mechanical properties of the linker between the catalytic domain (CCD) and CBM of the same enzyme, composed of Thr-Pro blocks, using the replica exchange molecular dynamics (REMD). Our results suggest the molecular basis of greater rigidity of this linker in comparison to the linker of cellobiohydrolase 2 from Trichoderma reesei. Finally, we have adapted the method of Molecular Dynamics Flexible Fitting (MDFF) to generate tertiary structure models from SAXS data and applied it to create a model of the intact structure CCD-linker-CBM of the XccEG / Doutorado / Físico-Química / Doutora em Ciências

This thesis set out to explore the conformational properties of short designed peptide sequences, in which transitions between structural states may be anticipated. The use of conformationally constrained residues like α-aminoisobutyric acid (Aib) and D-proline (DPro) permits the design of model sequences for structural studies. The principle of imposing conformational constraints by multiple substitutions at backbone atoms in aminoacid residues may also be extended to the higher homologs of α-amino acids, namely β and residues. The experimental results presented in this thesis also examine the potential of using cross-strand interactions between aromatic residues as a probe of structure in designed peptide β-hairpins. Chapter 1 provides a very brief introduction to the necessary background on which the experimental studies in this thesis are based. Chapter 2 describes studies aimed at establishing chain length effects on helix-hairpin conformational distributions in short synthetic sequences, containing centrally positioned Aib-DAla and Aib-Aib segments.The Aib-DAla dipeptide segment has a tendency to form both type-I'/III' and type-I/III β-turns. The occurrence of prime turns facilitates the formation of β-hairpin conformations, while type-I/III turns can nucleate helix formation. The octapeptide Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (1) has been previously shown to form a β-hairpin in the crystalline state and in solution. The effects of sequence truncation have been examined using the model peptides Boc-Phe-Val-Aib-Xxx-Leu-Phe-NHMe (2, 6), Boc-Val-Aib-Xxx-Leu-NHMe (3, 7) and Boc-Aib-Xxx-NHMe (4, 8), where Xxx = DAla, Aib. For peptides with central Aib-Aib segments, Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6), Boc-Val-Aib-Aib-Leu-NHMe (7) and Boc-Aib-Aib-NHMe (8) local helical conformations have been established by NMR studies in both hydrogen bonding (CD3OH) and non-hydrogen bonding (CDCl3) solvents. In contrast, the corresponding hexapeptide Boc-Phe-Val-Aib-DAla-Leu-Phe-Val-NHMe (2) favors helical conformations in CDCl3 and β-hairpin conformations in CD3OH. β-Turn conformations (type-I /III) stabilized by intramolecular 4 1 hydrogen bonds are observed for the peptide Boc-Aib-DAla-NHMe (4) and Boc-Aib-Aib-NHMe (8) in crystals. The tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts an incipient 310-helical conformation stabilized by three 4 1 hydrogen bonds. The peptide Boc-Val-Aib-DAla- Leu-NHMe (3) adopts a novel -turn conformation, stabilized by three intramolecular hydrogen bonds (two 4 1 and one 5 1). The Aib-DAla segment adopts a type-I' β-turn conformation. The observation of the NOE Val(1) NH HNCH3 (5), in CD3OH, suggests that the solid state conformation of peptide 3 is maintained in methanol solutions. Peptide hairpins provide an ideal scaffold for exploring cross-strand interactions between residues on facing antiparallel strands. Chapter 3 reports studies directed towards probing, aromatic interactions between facing Phe residues, positioned at the non-hydrogen bonding positions in designed octapeptide β-hairpins. The studies described in this Chapter employ ring current shifted aromatic proton resonances as a means of probing aromatic ring orientations. Crystal structures of eight peptide -hairpins with the sequence Boc-Leu-Phe-Val-Xxx-Yyy-Leu-Phe-Val-OMe revealed that the Phe(2) and Phe(7) aromatic rings are in close spatial proximity, with a centroid-centroid distance (Rcen) of 4.4Å to 5.4Å between the two phenyl rings. Proton NMR spectra in chloroform and methanol solutions reveal a significant upfield shift of the Phe(7) C , ′ H2 protons (6.65 ppm to 7.04 ppm). Specific assignments of the aromatic protons have been carried out in the peptide Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe (6). The anticipated ring current shifts have been estimated from the aromatic ring geometries observed in crystals for all eight peptides. Only one of the C , ′ H proton lies in the shielding zone, with rapid ring flipping, resulting in averaging between the two extreme chemical shifts. An approximate estimate of the population of conformations which resemble crystal state orientations may be obtained. Key nuclear Overhauser effects (NOEs) between facing Phe sidechains provide support for close similarity between the solid state and solution conformations. Temperature dependence of aromatic ring proton chemical shifts and line widths for peptide 6 (Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe) and the control peptide Boc-Leu-Val-Val-DPro-Gly-Leu-Phe-Val-OMe establish an enhanced barrier to ring flipping, when the two Phe rings are in proximity. Modeling studies suggest that small, conformational adjustments about the C -C ( 1), and C -C ( 2) bonds of the Phe residues may be required in order to permit unhindered, uncorrelated flipping of both the Phe rings. The maintenance of specific aromatic ring orientations in organic solvents provides evidence for significant stabilizing interactions. Earlier studies from this laboratory established that a centrally positioned DPro-LPro-DAla segment could induce hairpin formation in nonapeptide sequences, facilitated by a three residue loop segment. The DAla residue at position 6 in the nonapeptide Boc-Leu-Phe-Val-DPro-LPro-DAla-Leu-Phe-Val-OMe has been shown to adopt a left handed helical (αL) conformation. The studies described in Chapter 4, examine the effects of aminoacid replacements at positions 5 and 6. NMR studies on eight nonapeptides, with the general sequence Boc-Leu-Phe-Val-DPro-Xxx-Yyy-Leu-Phe-Val-OMe are described. In the case of peptides with a central DPro-LPro-Yyy sequence, two kinds of hairpin conformations are formed in solution. These are; i) β-hairpin structures with a central three residue loop, resulting in registered antiparallel tripeptide strands, and ii) a slipped hairpin structure, nucleated by a central DPro-LPro type-II β-turn, with residue 6 being incorporated into the C-terminal strand. The three residue loop β-hairpins are favored for DAla(6) and Aib(6), while the LAla(6) peptide favors a “slipped” hairpin structure. Replacement of the Pro(5) residue by LAla results in a reduced population of three residue hairpins in the nonapeptide with the DPro-LAla-DAla segment. Replacement of Pro(5) by Aib, abolished hairpin formation. Aromatic proton chemical shifts provide a convenient diagnostic for the presence of three residue loop hairpin conformations in these nonapeptides. A great deal of current interest has focused on the conformations of peptides incorporating β and γ aminoacid residues. Earlier studies from this laboratory have focused on the conformational properties of the β,β -disubstituted γ residue gabapentin (1-aminomethylcyclohexane acetic acid). Subsequent work with the related β aminoacid β3,3Ac6c (1-aminocyclohexaneacetic acid) revealed that intramolecularly hydrogen bonded conformations are infrequently observed in short peptides. The studies described in Chapter 5, examine the conformational properties for model peptides containing the isomeric β-aminoacid, β2,2Ac6c (1-aminomethylcyclohexane-1-carboxylic acid). The effect of gem dialkyl substituents on the backbone conformations of amino acid residues in peptides, has been investigated using four model peptides, Boc-Xxx-2,2Ac6c-NHMe [Xxx = Leu (1), Phe(2)] and Boc-Xxx- 3,3Ac6c-NHMe [Xxx = Leu (3), Phe(4)]. Tetrasubstituted carbon atoms restrict the ranges of stereochemically allowed a C11 helical turn, which is a backbone expanded analog of the type III -turn in sequences. The crystal structure of the peptide Boc-Phe- 3,3Ac6c-NHMe (4) establishes a the asymmetric unit adopt backbone torsion angles of opposite signs. In one of the molecules, the Phe residue adopts an unfavourable backbone conformation, with the energetic penalty being offset by favourable aromatic interactions between proximal molecules in the crystal. NMR studies provide evidence for the maintenance of folded structures in solution, in these hybrid sequences. The result presented in this thesis suggests that it should be possible to construct designed synthetic peptides, which can undergo transitions between two distinct and energetically favourable conformational states. The ability to design peptide sequences that can undergo switching between helical and β-hairpin states, or between hairpin structures with variations in connecting loop length may prove valuable in providing further insights into the factors influencing conformational dynamics.