RESUMO
One of the most remarkable characteristics of Brucella lumazine synthase (BLS) is its versatility to undergo reversible dissociation and reassociation as a polymeric scaffold. We have proposed a mechanism of dissociation and unfolding of BLS. Using static light scattering (SLS) analysis, we were able to demonstrate that the decameric assembly dissociates into two different conditions [pH 5 or 2M guanidinium chloride (GdnHCl) pH 7] forming stable folded pentamers. The transition from folded pentamers to unfolded monomers by GdnHCl denaturation is highly cooperative and can be measured by different spectroscopic techniques. In this work, we show the successful insertion of an intrinsic probe to study in more detail the equilibria described in previous publications. For that purpose, we performed single-point mutations of Phe residues 121 and 127, located at the pentamer-pentamer and monomer-monomer interface, respectively, to Trp residues. These mutations produced only a marginal perturbation of the BLS structure. We analyzed the unfolding and stability of the mutants through different techniques: far-and near-UV CD, SLS, dynamic light scattering, and fluorescence spectroscopy. The introduced intrinsic probe could be used to gain insights into the detailed folding and assembly mechanism of this protein.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação Puntual , Algoritmos , Proteínas de Bactérias/genética , Brucella/enzimologia , Guanidina , Concentração de Íons de Hidrogênio , Luz , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Análise Espectral , Termodinâmica , TriptofanoRESUMO
The multiple display of protein domains on polymeric scaffolds is an emerging technology for many applications. BLS is a highly immunogenic protein that has an oligomeric structure formed by a 17.2 kDa subunit arranged as a dimer of pentamers. Here we describe the production as well as the structural, functional, and immunological properties of a 9 kDa double-stranded RNA-binding domain (RBD3) fused to the structure of BLS. We demonstrate that the BLS and RBD3 modules are stably and independently folded in the structure of the chimera and form a decameric structure of 255 kDa as the native BLS oligomers. The polymeric display of RBD3 in the structure of BLS increases the dsRNA binding strength of this domain both in vitro and in vivo, and also enhances its immunogenicity to the point that it breaks the tolerance of mice to the RBD3 self-antigen. Our results underscore the BLS display strategy as a powerful tool for biotechnological and therapeutic applications.
Assuntos
Complexos Multienzimáticos/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sítios de Ligação , Brucella/enzimologia , Dicroísmo Circular , Clonagem Molecular , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Desnaturação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , TermodinâmicaRESUMO
Protein assemblies with a high degree of repetitiveness and organization are known to induce strong immune responses. For that reason they have been postulated for the design of subunit vaccines by means of protein engineering. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably owing to its homodecameric arrangement and remarkable thermodynamic stability. Structural analysis has shown that it is possible to insert foreign peptides at the ten amino terminus of BLS without disrupting its general folding. These peptides would be displayed to the immune system in a highly symmetric three-dimensional array. In the present work, BLS has been used as a protein carrier of foreign peptides. We have established a modular system to produce chimeric proteins decorated with ten copies of a desired peptide as long as 27 residues and have shown that their folding and stability is similar to that of the wild-type protein. The knowledge about the mechanisms of dissociation and unfolding of BLS allowed the engineering of polyvalent chimeras displaying different predefined peptides on the same molecular scaffold. Moreover, the reassembly of mixtures of chimeras at different steps of the unfolding process was used to control the stoichiometry and spatial arrangement for the simultaneous display of different peptides on BLS. This strategy would be useful for vaccine development and other biomedical applications.