RESUMO
High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (~650-900 nm) open a possibility for in vivo imaging of biological processes at the micro- and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes - natural NIR light-absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded fluorescent probes, similarly to GFP-like fluorescent proteins. In this review, we discuss photophysical and biochemical properties of NIR fluorescent proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR fluorescent probes are discussed. Applications of NIR fluorescent proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole-body imaging in vivo, are described. We specifically focus on the use of NIR fluorescent probes in advanced imaging technologies that combine fluorescence and bioluminescence methods with photoacoustic tomography.
Assuntos
Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Proteínas Luminescentes/química , Fitocromo/química , Engenharia de Proteínas/métodos , FluorescênciaRESUMO
The influence of various factors on the physico-chemical characteristics and complexation of glucose with a mutant form of D-glucose/D-galactose-binding protein which can be regarded as a sensor of the glucometer, namely the protein GGBP/H152C with solvatochromic dye BADAN attached to the cysteine residue Cys 152, has been investigated. The point mutation His 152Cys and attaching BADAN reduced the affinity of the mutant form GGBP/H152C to glucose more than 8-fold compared to the wild type protein. This allows using this mutant for the determination of sugar content in biological fluids extracted by transdermal technologies. Sufficiently rapid complexation of GGBP/H152C with glucose (the time of protein-glucose complex formation is not more than three seconds even in solutions with a viscosity of 4 cP) provides timely monitoring changes in the concentration of sugar. The changes of ionic strength and pH within the physiological range of values of these variables do not have significant influence on fluorescent characteristics of GGBP/H152C-BADAN. At acidic pH, (see symbol) some of the molecules GGBP/H152C is in the unfolded state. It has been shown that mutant form GGBP/H152C has relatively low resistance to guanidine hydrochloride denaturing effects. This result indicates the need for more stable proteins to create a sensor for glucose biosensor system.
Assuntos
2-Naftilamina/análogos & derivados , Técnicas Biossensoriais , Proteínas de Escherichia coli/química , Glucose/isolamento & purificação , Proteínas de Transporte de Monossacarídeos/química , 2-Naftilamina/química , Glicemia/isolamento & purificação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Guanidina/química , Humanos , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Conformação ProteicaRESUMO
Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. At present novel naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by stability, value of quantum yield, absorption and fluorescence spectra position and photochemical properties. GFP-like proteins are the fast growing family. This review is an attempt to characterize the main groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.
Assuntos
Proteínas Luminescentes/química , Proteínas Luminescentes/fisiologia , Sequência de Aminoácidos , Animais , Técnicas Biossensoriais/métodos , Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/fisiologia , Indicadores e Reagentes , Cinética , Microscopia de Fluorescência , Modelos Moleculares , Biologia Molecular/métodos , Dados de Sequência Molecular , Mutação , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
A system for actin expression in cells of yeast Pichia pastoris was constructed. Drosophila actin 5C, by 90% homologous to beta-actin of higher eukaryotes, was used as a target protein. To improve the procedures of target protein biosynthesis in yeast cells and of extraction and purification of recombinant actin the fusion protein GFP-actin 5C, having fluorescence protein GFP as a reporter part, was expressed and purified. The dimensions and resistance of yeast cells producing recombinant actin were characterized. It was shown that the size and form of cells depended on the accumulation of recombinant protein. The purified fusion protein was used for obtaining polyclonal antibody for testing recombinant actin.