RESUMO
Light activated photosensitizers generate reactive oxygen species (ROS) that interfere with cellular components and can induce cell death, e.g., in photodynamic therapy (PDT). The effect of cellular components and especially proteins on the photochemistry and photophysics of the sensitizers is a key aspect in drug design and the correlating cellular response with the generation of specific ROS species. Here, we show the complex range of effects of binding of photosensitizer to a multidrug resistance protein, produced by bacteria, on the formers reactivity. We show that recruitment of drug like molecules by LmrR (Lactococcal multidrug resistance Regulator) modifies their photophysical properties and their capacity to induce oxidative stress especially in 1O2 generation, including rose bengal (RB), protoporphyrin IX (PpIX), bodipy, eosin Y (EY), riboflavin (RBF), and rhodamine 6G (Rh6G). The range of neutral and charged dyes with different exited redox potentials, are broadly representative of the dyes used in PDT.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Fármacos Fotossensibilizantes/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Corantes/metabolismo , Corantes/efeitos da radiação , Lactococcus/química , Luz , Mutação , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/efeitos da radiação , Ligação Proteica , Oxigênio Singlete/químicaRESUMO
Proteins have been used as templates to stabilize fluorescent metal nanoclusters thus obtaining stable fluorescent structures, and their fluorescent properties being modulated by the type of protein employed. Designed consensus tetratricopeptide repeat (CTPR) proteins are suited candidates as templates for the stabilization of metal nanoclusters due to their modular structural and functional properties. Here, we have studied the ability of CTPR proteins to stabilize fluorescent gold nanoclusters giving rise to designed functional hybrid nanostructures. First, we have investigated the influence of the number of CTPR units, as well as the presence of cysteine residues in the CTPR protein, on the fluorescent properties of the protein-stabilized gold nanoclusters. Synthetic protocols to retain the protein structure and function have been developed, since the structural and functional integrity of the protein template is critical for further applications. Finally, as a proof-of-concept, a CTPR module with specific binding capabilities has been used to stabilize gold nanoclusters with positive results. Remarkably, the protein-stabilized gold nanocluster obtained combines both the fluorescence properties of the nanoclusters and the functional properties of the protein. The fluorescence changes in nanoclusters fluorescence have been successfully used as a sensor to detect when the specific ligand was recognized by the CTPR module.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Ácido Ascórbico/química , Ouro/química , Proteínas de Choque Térmico HSP90/química , Proteínas Heterotriméricas de Ligação ao GTP/química , Nanopartículas Metálicas/química , Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Proteínas Adaptadoras de Transporte Vesicular/genética , Sequência de Aminoácidos , Sítios de Ligação , Desenho de Fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes , Expressão Gênica , Proteínas de Choque Térmico HSP90/síntese química , Proteínas Heterotriméricas de Ligação ao GTP/biossíntese , Proteínas Heterotriméricas de Ligação ao GTP/genética , Ligantes , Nanopartículas Metálicas/ultraestrutura , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Soroalbumina Bovina/química , Espectrometria de FluorescênciaRESUMO
The use of biological molecules as platforms for templating and nanofabrication is an emerging field. Here, we use designed protein building blocks based on small repetitive units (consensus tetratricopeptide repeat - CTPR) to generate fibrillar linear nanostructures by controlling the self-assembly properties of the units. We fully characterize the kinetics and thermodynamics of the assembly and describe the polymerization process by a simple model that captures the features of the structures formed under defined conditions. This work, together with previously established functionalization potential, sets up the basis for the application of these blocks in the fabrication and templating of complex hybrid nanostructures.