Estudos anteriores mostram que o polímero Nafion® pode causar deslocamento do equilíbrio conformacional de proteínas em valores de pH que não o fisiológico. Nesse sentido, o Nafion® não só pode ser utilizado como uma sonda interessante para estudos estruturais de proteínas, mas, também, é importante entender seu papel na conformação da proteína. Portanto, a Albunina do Soro Bovino (BSA) foi escolhida como modelo para o estudo dos efeitos do Nafion® na conformação helicoidal de proteínas. A finalidade deste trabalho é entender as alterações na conformação e vizinhanças aromáticas da BSA, na faixa de pH de 2 a 12, na presença e ausência de Nafion®, que pode também revelar o papel do polímero na exposição dos aromáticos e nos processos de transferência de energia. As alterações da estrutura secundária foram medidas por Dicroísmo Circular e os espectros de fluorescência no estado estacionário foram usados para analisar as mudanças nas vizinhanças dos aromáticos. Os resultados mostraram a diminuição discreta do conteúdo helicoidal da conformação da BSA na região extremamente básica, pH 11 em relação à conformação em pH 7. Já na região ácida, pH 2, embora haja considerável diminuição do conteúdo helicoidal, a BSA ainda mantém quase 50% de sua conformação secundária regular. Em relação aos ambientes dos aromáticos triptofano e tirosina, a eficiência quântica da emissão de fluorescência diminui em regiões ácidas e básicas, indicando que, nessas estruturas, os aromáticos encontram-se em restrição conformacional em relação ao observado na proteína nativa. Estes resultados apontam para a mudanças na conformação da BSA em ambas as regiões: ácidas e básicas, incluindo mudanças das estruturas secundárias e nas vizinhanças dos aromáticos. A adição do Nafion®, por outro lado, acentua o deslocamento para o azul e diminuição da exposição dos aminoácidos, tanto em solução quanto em estado sólido. A estrutura secundária da proteína é completamente modificada pelo polímero na região ácida, e esta conformação é mantida nas regiões neutra e básica, sugerindo que o Nafion® não estabiliza estruturas helicoidais / Previous studies have shown that Nafion® can disturb the conformational equilibrium of some proteins when at pH other than physiological ones. In this sense, Nafion® can used to study protein conformation, but is also important to understand its interaction with the proteins. In this work, Bovine Serum Albimun (BSA) was chosen as a model to understand the modifications caused by Nafion® at helicoidal proteins conformation. More specifically, the aim encloses the understanding of changes in BSA secondary conformation and aromatic vicinities, at pH range from 2 to 12, in the Nafion®s presence and absence. Secondary changes were measured by Circular Dichroism and steady-state fluorescence was used to study the aromatic vicinities. Results have shown small differences at helix content in the extremely basic pH (pH 11) when compared to BSA conformation at pH 7 (native one). At pH 2, on the other hand, although a decreasing in helical content was observed, BSA was able to keep almost 50% of secondary regular conformation. Regarding the aromatic vicinities (tryptophans and tyrosines) the fluorescence emission quantum eficience decreased in both regions (acid and basic), suggesting that the aromatics in these conformations are found in a more restrict environment. Nafion®, when added, promoted a decreasing in aromatic exposition, both in solution and solid state, while the secondary structure is completelu modified by its presence in all pH range, suggesting that helical conformations are not stabilized by Nafion®

The sensitivity of biological studies performed between 190 and 650 nm is greatly reduced due to the autofluorescence of biomolecules and impurities in this region. Therefore, the enhanced signal-to-noise ratios encountered at longer wavelengths makes biological analysis within the near infrared (NIR) region from 650 nm to 1100 nm far more advantageous. This dissertation describes the noncovalent binding interactions of near-infrared (NIR) carbocyanine dyes with human serum albumin (HSA) and poly-L-lysine (PLL) using UV-Vis/NIR absorption spectroscopy, emission spectroscopy, circular dichroism (CD), and fluorescence detected circular dichroism (FDCD). The optical spectroscopy methods used in this work are described in detail in Chapter 1. The various applications of NIR dyes in protein analysis are introduced in Chapter 2. In general, the sensitivity of cyanines to the polarity of their local environment makes them quite suitable for protein labeling schemes. In aqueous media, cyanines have a high propensity for self-association. Yet in the hydrophobic binding sites of globular proteins, these aggregates often dissipate. Absorption and emission spectroscopy can be utilized to observe the differential spectral properties of monomer, intra-molecular and intermolecular aggregates. In Chapter 3, the photophysical properties of bis(cyanine) NIR dyes containing di-, tri-, and tetraethylene glycol linkers were each examined in the presence of HSA are discussed. Variations in chain length as well as probe flexibility were demonstrated through distinct differences in absorption and emission spectra. The observed changes in the spectral properties of the NIR dyes in the presence and absence of HSA were correlated to the physical parameters of the probes' local environment (i.e., protein binding sites and self-association). All three bis-cyanines examined exhibited enhanced fluorescence in the presence of HSA. The bis-cyanine dye containing the tri(ethylene glycol) spacer allowed for a complete overlap of the benzene rings, to form π-π interactions which were observed as intra-molecular H-aggregate bands. The dye exhibited no fluorescence in buffer, owing to the H-aggregation observed in the absorption data. In the presence of HSA, the intra-molecular dimers were disrupted and fluorescence was then detected. The "cut-on" fluorescence displayed by the dye in the presence of HSA made it ideal for noncovalent labeling applications. The utility of several NIR dyes for use as secondary structural probes was investigated in Chapter 4. NIR dyes were screened thoroughly using UV-Vis/NIR absorption spectroscopy dyes with spectral properties which were sensitive to protein secondary structure models of such as PLL in basic solution. Two NIR dyes were found to be quite sensitive to the structural features of uncharged α- and β-PLL. The chiral discrimination of these probes for basic protein secondary structures was also evaluated through CD measurements within the NIR probes' absorption bands.

No description available.

No description available.

S100A12 porcina é um membro da família das proteínas S100, um grupo de pequenas proteínas ligantes de cálcio caracterizado pela presença de dois motivos “EF-hand". Estas proteínas estão envolvidas em diversos eventos celulares, como a regulação da fosforilação protéica, atividade enzimática, tamponamento de Ca+2, processos inflamatórios e a polimerização de filamentos intermediários. Adicionalmente, algumas dessas proteínas podem ligar Zn+2, o qual pode afetar a ligação do íon Ca+2, particularmente para as proteínas S100. Neste trabalho, a seqüência gênica que codifica a proteína S100A12 porcina foi obtida por meio da construção de um gene sintético usando códons preferenciais para E.coli, permitindo a produção recombinante de grandes quantidades da proteína. Um estudo termodinâmico da estabilidade estrutural foi realizado, assim como a interação da proteína recombinante com íons divalentes usando técnicas de dicroísmo circular (CD) e fluorescência extrínseca. A desnaturação e renaturação induzidas por uréia ou temperatura indicam que se trata de um processo reversível e que a ligação dos íons Zn+2 e ou Ca+2 à rS100A12 aumenta sua estabilidade. A interação da sonda ANS com a proteína na presença de seus ligantes expõe superfícies hidrofóbicas podendo assim facilitar sua interação com macromoléculas alvo. Analisados em conjunto, os resultados obtidos indicam que S100A12 porcina é capaz de assumir diferentes conformações as quais podem estar correlacionadas com sua função fisiológica. / Porcine S100A12 is a member of S100 family, a small acidic calcium-binding proteins group characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events as the regulation of protein phosphorylation, enzymatic activity, Ca+2 homeostasis, inflammatory processes and intermediate filament polymerization. In addition, some of these proteins can bind Zn+2, which can affect the binding of Ca+2 particularly to S100 proteins. In this study, the gene sequence encoding S100A12 was obtained by the synthetic gene approach using E. coli codon bias allowing the recombinant production of large amounts of the protein. We report here a thermodynamic study on the structural stability of this recombinant protein and its interaction with divalent ions using circular dichroism and extrinsic fluorescence. The folding/unfolding induced by urea or temperature indicated a reversible process and the binding of Zn+2 or Zn+2 and Ca+2 to S100A12 increasing its stability. The interaction of the ANS probe with the protein in the ligant presence can lead to exposition of hydrofobic regions allowing its interaction with target macromolecules. Taken together, the results indicated that porcine S100A12 may assume different conformations that could be correlated to its physiological function.

S100A12 porcina é um membro da família das proteínas S100, um grupo de pequenas proteínas ligantes de cálcio caracterizado pela presença de dois motivos “EF-hand”. Estas proteínas estão envolvidas em diversos eventos celulares, como a regulação da fosforilação protéica, atividade enzimática, tamponamento de Ca+2, processos inflamatórios e a polimerização de filamentos intermediários. Adicionalmente, algumas dessas proteínas podem ligar Zn+2, o qual pode afetar a ligação do íon Ca+2, particularmente para as proteínas S100. Neste trabalho, a seqüência gênica que codifica a proteína S100A12 porcina foi obtida por meio da construção de um gene sintético usando códons preferenciais para E.coli, permitindo a produção recombinante de grandes quantidades da proteína. Um estudo termodinâmico da estabilidade estrutural foi realizado, assim como a interação da proteína recombinante com íons divalentes usando técnicas de dicroísmo circular (CD) e fluorescência extrínseca. A desnaturação e renaturação induzidas por uréia ou temperatura indicam que se trata de um processo reversível e que a ligação dos íons Zn+2 e ou Ca+2 à rS100A12 aumenta sua estabilidade. A interação da sonda ANS com a proteína na presença de seus ligantes expõe superfícies hidrofóbicas podendo assim facilitar sua interação com macromoléculas alvo. Analisados em conjunto, os resultados obtidos indicam que S100A12 porcina é capaz de assumir diferentes conformações as quais podem estar correlacionadas com sua função fisiológica. / Porcine S100A12 is a member of S100 family, a small acidic calcium-binding proteins group characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events as the regulation of protein phosphorylation, enzymatic activity, Ca+2 homeostasis, inflammatory processes and intermediate filament polymerization. In addition, some of these proteins can bind Zn+2, which can affect the binding of Ca+2 particularly to S100 proteins. In this study, the gene sequence encoding S100A12 was obtained by the synthetic gene approach using E. coli codon bias allowing the recombinant production of large amounts of the protein. We report here a thermodynamic study on the structural stability of this recombinant protein and its interaction with divalent ions using circular dichroism and extrinsic fluorescence. The folding/unfolding induced by urea or temperature indicated a reversible process and the binding of Zn+2 or Zn+2 and Ca+2 to S100A12 increasing its stability. The interaction of the ANS probe with the protein in the ligant presence can lead to exposition of hydrofobic regions allowing its interaction with target macromolecules. Taken together, the results indicated that porcine S100A12 may assume different conformations that could be correlated to its physiological function.

No description available.

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such ‘normalization’ to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.

Aus den frühen Bindungsstudien des globalen Regulators AbrB mit der ausgedehnten phyC-Promotorregion von Bacillus amyloliquefaciens FZB45 konnte ein mehrstufiger kooperativer Bindungsprozess abgeleitet werden. Dabei verlangt die AbrB-vermittelte Repression von phyC nach Integrität zweier großer Bindungsstellen, ABS1 und ABS2, die 162 bp voneinander entfernt liegen. In der vorliegenden Arbeit wurden die ersten Echtzeitkinetiken zur DNA-AbrB-Interaktion mittels der Oberflächenplasmonresonanz (SPR) gemessen und analysiert. AbrB zeigte hohe Affinitäten zu den 40 bp langen Oligonukleotiden, die den beiden Bindungsstellen entstammen. Dabei verursachten alle Oligonukleotide der ABS2 und nur eine kurze Region innerhalb der ABS1 bei der Bindung von AbrB Konformationsänderungen im Protein und in der DNA (CD - Zirkulardichroismusspektroskopie) und wiesen eine Kooperativität von 2 / In previous binding studies it could be demonstrated that a global regulator AbrB and the extensive phyC promoter region of Bacillus amyloliquefaciens FZB45 interact in a complex manner. AbrB binding is a multistep cooperative process. The integrity of both binding sites, ABS1 and ABS2, which are separated by 162 bp, is crucial for the AbrB-mediated repression of phyC. This work presents the first real-time binding kinetics of the AbrB-DNA interaction using surface plasmon resonance (SPR). AbrB exhibited high affinities to all analyzed 40-bp oligonucleotides that were derived from the ABSs of phyC. All parts of the ABS2, but only a small region within ABS1, were bound cooperatively to AbrB with a stoichiometry of 2 DNA to 1 AbrB tetramer and with 2