Estudo das bases moleculares da especificidade pelo substrato de uma beta-glicosidase (AF052729) de Spodoptera frugiperda

Rozenfeld, Julio H.K.

doi:10.11606/d.46.2006.tde-19092006-163823

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“…Uma segunda região altamente central é a região logo após a fita beta 3, que contém dois resíduos frequentemente centrais, os resíduos Y141 e H142. Estes dois resíduos são bastante conservados em beta-glicosidases, sendo que o resíduo H142 faz parte do sítio ativo e atua na ligação do glicone do substrato (Rozenfeld, 2006;Tamaki et al, 2016).…”

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Papel das redes estruturais proteicas nas propriedades de uma beta-glicosidase

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Analysis of protein structures as networks has been shown a powerful tool to understand their properties and to identify important residues. In the network analysis, residues that interact with each other are called connected. Some residues are essential to shorten the connection pathways between distant residues in the protein structure, being called central. Central residues have been proposed to have important roles in catalysis, thermal stability and allostery. In order to experimentally assess the correlation between the residue centrality and its importance in the protein properties, we use two approaches (chapter 1): The first one is to make single mutations at the central residues of a betaglucosidase Sfβgly, changing those residues to alanine. The second one is to perturb a central residue (F251) by changing its environment through single mutations that introduces voids or additional volume. Next, we evaluate how those mutations affect the protein thermostability and function. In general, we have observed (chapter 2) that mutations at central residues reduce the Tm in 2-15°C and increase the unfolding rate up to 20 times, suggesting that damages in the central residues make the protein more unstable. Moreover, we have observed (chapter 3) that the perturbation of the central residues reduces Sfβgly catalysis, which seems to arise from the same cause that lead to the loss of thermal stability. Besides that, in chapter 4, the investigation of oligomeric state of Sfβgli using SAXS indicated that this protein is mainly a dimer in 100 mM citrate-phosphate pH 6,0, whereas it forms large oligomers, possibly dodecamers, in 10 mM phosphate pH 6,0. In parallel it was shown that Sfβgly undergoes an activation process in 10 mM phosphate and its kinetic parameters converge to those observed for Sfβgly in 100 mM citrate-phosphate. Protein Structural Networks were built considering also that there are links between the polypeptidic chains of the Sfβgly oligomers. We observed 5 residues that are central in all kind of oligomeric structures here analyzed. Three of these residues, E187, P188 and N329, play important roles in the catalysis of this enzyme, and two of them (S247 and N249 are described in this thesis. Lastly, in the chapter 5, we observed that central residues by closeness, betweeness and C ΔLp are concentrated at the top of the beta-barrel (C-terminal end of the beta-strands and subsequent loops), suggesting that this region, where the active site is placed, is close, in terms of contacts, to the whole Sfβgly structure. Moreover, we have built the Protein Structural Network of 21 beta-glucosidases of the Glucoside Hydrolases family 1, revealing that the closeness central residues are highly conserved, being located in the active site of these enzymes. On the other hand, betweeness central residues are located in the same sites in the structure of different beta-glucosidases, but they are not always conserved. Shortly, these data experimentally support the hypothesis that the residue centrality in Protein Str...

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Papel das redes estruturais proteicas nas propriedades de uma beta-glicosidase

Souza¹

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Estudo das bases moleculares da especificidade pelo substrato de uma beta-glicosidase (AF052729) de Spodoptera frugiperda

Cited by 1 publication

References 0 publications

Papel das redes estruturais proteicas nas propriedades de uma beta-glicosidase

Papel das redes estruturais proteicas nas propriedades de uma beta-glicosidase

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