RESUMO
Visualizing and manipulating the behavior of proteins is crucial to understanding the physiology of the cell. Methods of biorthogonal protein labeling are important tools to attain this goal. In this review, we discuss advances in probe technology specific for self-labeling protein tags, focusing mainly on the application of HaloTag and SNAP-tag systems. We describe the latest developments in small-molecule probes that enable fluorogenic (no wash) imaging and super-resolution fluorescence microscopy. In addition, we cover several methodologies that enable the perturbation or manipulation of protein behavior and function towards the control of biological pathways. Thus, current technical advances in the HaloTag and SNAP-tag systems means that they are becoming powerful tools to enable the visualization and manipulation of biological processes, providing invaluable scientific insights that are difficult to obtain by traditional methodologies. As the multiplex of self-labeling protein tag systems continues to be developed and expanded, the utility of these protein tags will allow researchers to address previously inaccessible questions at the forefront of biology.
Assuntos
Corantes Fluorescentes/química , Proteínas/análise , Fenômenos Biológicos , Humanos , Microscopia de Fluorescência , Proteínas/metabolismoRESUMO
While organic donor-acceptor (D-A) molecules are widely employed in multiple areas, the application of more D-A molecules could be limited because of an inherent polarity sensitivity that inhibits photochemical processes. Presented here is a facile chemical modification to attenuate solvent-dependent mechanisms of excited-state quenching through addition of a ß-carbonyl-based polar substituent. The results reveal a mechanism wherein the ß-carbonyl substituent creates a structural buffer between the donor and the surrounding solvent. Through computational and experimental analyses, it is demonstrated that the ß-carbonyl simultaneously attenuates two distinct solvent-dependent quenching mechanisms. Using the ß-carbonyl substituent, improvements in the photophysical properties of commonly used D-A fluorophores and their enhanced performance in biological imaging are shown.
Assuntos
Corantes Fluorescentes/química , Compostos Orgânicos/química , Teoria Quântica , Estrutura Molecular , Processos Fotoquímicos , Solventes/químicaRESUMO
We present a fluorogenic method to visualize misfolding and aggregation of a specific protein-of-interest in live cells using structurally modulated fluorescent protein chromophores. Combining photophysical analysis, X-ray crystallography, and theoretical calculation, we show that fluorescence is triggered by inhibition of twisted-intramolecular charge transfer of these fluorophores in the rigid microenvironment of viscous solvent or protein aggregates. Bioorthogonal conjugation of the fluorophore to Halo-tag fused protein-of-interests allows for fluorogenic detection of both misfolded and aggregated species in live cells. Unlike other methods, our method is capable of detecting previously invisible misfolded soluble proteins. This work provides the first application of fluorescent protein chromophores to detect protein conformational collapse in live cells.