RESUMO
Chicken avidin is a key component used in a wide variety of biotechnological applications. Here we present a circularly permuted avidin (cpAvd4-->3) that lacks the loop between beta-strands 3 and 4. Importantly, the deletion of the loop has a positive effect on the binding of 4'-hydroxyazobenzene-2-carboxylic acid (HABA) to avidin. To increase the HABA affinity of cpAvd4-->3 even further, we mutated asparagine 118 on the bottom of the ligand-binding pocket to methionine, which simultaneously caused a significant drop in biotin-binding affinity. The X-ray structure of cpAvd4--> 3(N118M) allows an understanding of the effect of mutation to biotin-binding, whereas isothermal titration calorimetry revealed that the relative binding affinity of biotin and HABA had changed by over one billion-fold between wild-type avidin and cpAvd4-->3(N118M). To demonstrate the versatility of the cpAvd4-->3 construct, we have shown that it is possible to link cpAvd4-->3 and cpAvd5-->4 to form the dual-chain avidin called dcAvd2. These novel avidins might serve as a basis for the further development of self-organising nanoscale avidin building blocks.
Assuntos
Avidina/genética , Avidina/metabolismo , Mutagênese/genética , Deleção de Sequência/genética , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Avidina/química , Compostos Azo/metabolismo , Sítios de Ligação , Biotina/metabolismo , Domínio Catalítico , Galinhas , Cristalografia por Raios X , Cinética , Ligantes , Espectrometria de Massas , Modelos Moleculares , Desnaturação Proteica , Especificidade por Substrato , Termodinâmica , Temperatura de TransiçãoRESUMO
The chicken genome encodes several biotin-binding proteins, including avidin and avidin-related protein 4 (AVR4). In addition to D-biotin, avidin binds an azo dye compound, 4-hydroxyazobenzene-2-carboxylic acid (HABA), but the HABA-binding properties of AVR4 are not yet known. Differential scanning calorimetry, UV/visible spectroscopy, and molecular modeling were used to analyze the binding of 15 azo molecules to avidin and AVR4. Significant differences are seen in azo compound preferences for the two proteins, emphasizing the importance of the loop between strands beta3 and beta4 for azo ligand recognition; information on these loops is provided by the high-resolution (1.5 A) X-ray structure for avidin reported here. These results may be valuable in designing improved tools for avidin-based life science and nanobiotechnology applications.
