A rational protocol for the successful crystallization of l-amino-acid oxidase from Bothrops atrox

Autor(es)

Alves, Raquel Melo ; Feliciano, Patricia Rosa ; Sampaio, Suely Vilela ; Nonato, Maria Cristina

• Tipo de documento: Artigo Científico
• Fonte: ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS; v. 67, p. 4-pg., 2011-04-01.

Resumo

Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme l-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 A, beta = 96.03 degrees. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V (M) of 2.12 A3 Da-1, corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques. (AU)

• Processo FAPESP: 05/54855-0 - Toxinas animais: estrutura, função e aplicações biotecnológicas
• Beneficiário: Suely Vilela
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 09/10454-3 - Estudos da correlação entre estrutura e função da enzima fumarato hidratase em Leishmania major
• Beneficiário: Patrícia Rosa Feliciano
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 07/51700-1 - Análise da estrutura tridimensional da L-aminoácido oxidase de Bothops atrox e suas interações in vitro e in vivo
• Beneficiário: Raquel de Melo Alves Paiva
• Modalidade de apoio: Bolsas no Brasil - Doutorado