Estudo de interações biomoleculares de nanopartículas de ouro ultrapequenas com α- trombina
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Data
2018-02-22
Autores
Silva, Andre Luis Lira da [UNIFESP]
Orientadores
Sousa, Alioscka Augusto Caldeira Araujo [UNIFESP]
Tipo
Dissertação de mestrado
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Resumo
Nanopartículas de ouro (NPs) possuem diversas aplicações na área biomédica, podendo ser utilizadas como biosensores para diagnósticos de doenças in vitro, ou ainda como plataformas para novas terapias e diagnósticos por imagem in vivo. Quando imersas em fluido biológico, as NPs podem ser recobertas por proteínas e outras biomoléculas presentes no meio, e essa adsorção não-específicas pode acarretar em alterações indesejadas na estrutura e atividade biológica. Portanto, a compreensão dos mecanismos de interação das NPs com proteínas serve como ponto de partida para entender, ou até mesmo prever, seus efeitos biológicos. Dessa forma, este trabalho teve como objetivo caracterizar as interações moleculares entre NPs ultrapequenas de carga negativa e a α-trombina humana (utilizada como enzima modelo), e estudar as consequências dessas interações na estrutura e atividade enzimática da proteína. Os estudos utilizaram uma combinação de ferramentas biofísicas e bioquímicas, tais como fluorescência, ressonância plasmônica de superfície, e diversos ensaios enzimáticos. Os resultados demonstraram que as NPs interagem preferencialmente em regiões de carga positiva na trombina, denominadas exosítios 1 e 2. A ligação é de caráter eletrostático e possui constate de afinidade KD ≅ 44 nM. As NPs induzem modificações na estrutura secundária da trombina e também alterações estruturais na região do sítio ativo. A ligação das NPs foi capaz de inibir parcialmente a atividade enzimática da trombina (atividade residual de ~ 40%), apresentando características de inibição parcial mista. Esta resposta de inibição parcial por mecanismo alostérico não havia sido descrita anteriormente nas interações de NPs sintéticas com enzimas.
Gold nanoparticles (NPs) can be utilized in several different applications in the biomedical area, including as biosensors for in vitro diagnosis of diseases, or still as platforms for diagnostic and therapeutic interventions in vivo. However, when immersed in a biological fluid, the NPs can be coated with proteins and other biomolecules, and this nonspecific adsorption can lead to undesired changes in protein structure and function. Therefore, characterizing the mechanisms of interaction between NPs and proteins is important towards trying to understand, or even predict, the biological effects of NPs. In this work we characterized the molecular interactions of ultrasmall gold NPs of negative charge with human -thrombin (used as a model enzyme), and the effect of these interactions on thrombin’s structure and activity. We employed in these studies a combination of biophysical and biochemical tools, such as fluorescence, surface plasmon resonance and various enzymatic assays. The results showed that the negatively charged NPs interacted preferentially with exosites 1 and 2 of thrombin, which are regions of high positive charge that flank the active site. The interactions were mostly electrostatically driven, and had an associated KD ≅ 44 nM. NP binding induced secondary structural changes on thrombin, in addition to local changes around the active site. Enzyme activity was allosterically inhibited by NP interactions, with a mechanism characterized as partial mixed inhibition (residual activity ~40%). This partial inhibition response has not been described before in the interactions of synthetic NPs with enzymes.
Gold nanoparticles (NPs) can be utilized in several different applications in the biomedical area, including as biosensors for in vitro diagnosis of diseases, or still as platforms for diagnostic and therapeutic interventions in vivo. However, when immersed in a biological fluid, the NPs can be coated with proteins and other biomolecules, and this nonspecific adsorption can lead to undesired changes in protein structure and function. Therefore, characterizing the mechanisms of interaction between NPs and proteins is important towards trying to understand, or even predict, the biological effects of NPs. In this work we characterized the molecular interactions of ultrasmall gold NPs of negative charge with human -thrombin (used as a model enzyme), and the effect of these interactions on thrombin’s structure and activity. We employed in these studies a combination of biophysical and biochemical tools, such as fluorescence, surface plasmon resonance and various enzymatic assays. The results showed that the negatively charged NPs interacted preferentially with exosites 1 and 2 of thrombin, which are regions of high positive charge that flank the active site. The interactions were mostly electrostatically driven, and had an associated KD ≅ 44 nM. NP binding induced secondary structural changes on thrombin, in addition to local changes around the active site. Enzyme activity was allosterically inhibited by NP interactions, with a mechanism characterized as partial mixed inhibition (residual activity ~40%). This partial inhibition response has not been described before in the interactions of synthetic NPs with enzymes.
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Citação
SILVA, André Luís Lira da. Estudo de interações biomoleculares de nanopartículas de ouro ultrapequenas com alfa-trombina. São Paulo, 2018. Dissertação (Mestrado em Ciências biológicas: biologia molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2018.