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Development of specific l-methionine sensors by FRET-based protein engineering.

ABSTRACT: Amino acids are essential nutrients that are not only used as protein building blocks but are also involved in various biochemical processes and in the development of human diseases. Quantitative analysis of amino acids in complex biological samples is an important analytical process used for understanding amino acid biochemistry and diagnosis of human diseases. In this study, a protein sensor based on fluorescence resonance energy transfer (FRET) was designed for the quantitative analysis of l-Met, in which a fluorescent unnatural amino acid (CouA) and YFP were used as a FRET pair. A natural Met-binding protein (MetQ) was chosen as a sensor protein, and CouA and YFP were incorporated into the protein by genetic code expansion technology and genetic fusion. Among the four sites screened for CouA incorporation into MetQ, R189 was selected as the best site for l-Met sensing. The sensor protein (YFP-MetQ-R189CouA) showed a large FRET signal change (2.7-fold increase) upon l-Met binding. To improve amino acid specificity of the sensor protein, the ligand-binding site was engineered, and the mutant sensor (YFP-MetQ-R189CouA-H88F) with the H88F mutation was identified, which showed no FRET signal change with d-Met and l-Gln at 50 μM concentration and retained the maximum FRET signal change with l-Met. The optimized sensor protein was evaluated for biochemical applications. l-Met concentration in FBS and optical purity in a mixture of d- and l-Met were successfully determined. Because l-Met is biochemically important owing to its involvement in cancer cell growth and autophagy, the sensor protein would be useful for quantitative analysis of l-Met in a complex biological sample. In addition, the design strategy used in this study can be applied to other small molecule-binding proteins for the development of protein sensors for important biomolecules.

SUBMITTER: Ko W

PROVIDER: S-EPMC9064335 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cyclic AMP is a ubiquitous second messenger that orchestrates a variety of cellular functions over different timescales. The mechanisms underlying specificity within this signaling pathway are still not well understood. Several lines of evidence suggest the existence of spatial cAMP gradients within cells, and that compartmentalization underlies specificity within the cAMP signaling pathway. However, to date, no studies have visualized cAMP gradients in three spatial dimensions (3D: x, y, z).This is in part due to the limitations of FRET-based cAMP sensors, specifically the low signal-to-noise ratio intrinsic to all intracellular FRET probes. Here, we overcome this limitation, at least in part, by implementing spectral imaging approaches to estimate FRET efficiency when multiple fluorescent labels are used and when signals are measured from weakly expressed fluorescent proteins in the presence of background autofluorescence and stray light. Analysis of spectral image stacks in two spatial dimensions (2D) from single confocal slices indicates little or no cAMP gradients formed within pulmonary microvascular endothelial cells (PMVECs) under baseline conditions or following 10 min treatment with the adenylyl cyclase activator forskolin. However, analysis of spectral image stacks in 3D demonstrates marked cAMP gradients from the apical to basolateral face of PMVECs. Results demonstrate that spectral imaging approaches can be used to assess cAMP gradients-and in general gradients in fluorescence and FRET-within intact cells. Results also demonstrate that 2D imaging studies of localized fluorescence signals and, in particular, cAMP signals, whether using epifluorescence or confocal microscopy, may lead to erroneous conclusions about the existence and/or magnitude of gradients in either FRET or the underlying cAMP signals. Thus, with the exception of cellular structures that can be considered in one spatial dimension, such as neuronal processes, 3D measurements are required to assess mechanisms underlying compartmentalization and specificity within intracellular signaling pathways.

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Development of specific l-methionine sensors by FRET-based protein engineering.

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