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Fluorescent protein biosensors

Genetically encoded (protein-based) fluorescent biosensors have been developed to enable imaging and monitoring of a variety of metabolites and cellular events, as highlighted in this collection of recent papers from Nature Portfolio. Engineering these biosensors is often non-trivial, requiring careful mutagenesis and insertion of active domains, but insights gained from past studies can be used to inform more rational design, and thus more rapid access to useful variants, in the future.

Fluorescent protein engineering:

In this Perspective the authors discuss strategies for the development of improved fluorescent proteins, with a focus on probes at the red end of the spectrum. They synthesize the literature on chromophore photochemistry and protein structure to identify residues for targeted mutagenesis, and consider improvements in molecular evolution methodologies to enable improved screening for desired probes.

Perspective | | Nature Methods

Fluorescent proteins are widely used to image cellular structures. Here, Shemiakina and colleagues develop an enhanced version of a red fluorescent protein that is monomeric and less cytotoxic, thereby improving the quality of images that can be obtained in the red part of the visible spectrum.

Article | | Nature Communications

Improved photostability of fluorescent proteins would benefit many applications but is usually an afterthought in selection screens. Setting photostability as the primary selection criterion in screens for improved fluorescent proteins yielded highly photostable variants of existing orange and red fluorescent proteins without compromising other beneficial characteristics.

Article | | Nature Methods

Roger Tsien left us on August 24. His untimely passing has saddened and shocked the scientific community. Roger literally and figuratively brightened our world, illuminated the dark matter of biology, and forever changed our view of the interface of chemistry and biology.

Obituary | | Nature Chemical Biology

Calcium biosensors

Current calcium-sensitive probes based on red fluorescent proteins are unsuitable for two-photon excitation at the near-infrared wavelengths commonly used for green fluorescent probes. Wu et al. use a structure-guided approach to engineer a red fluorescent probe with optimal two-photon excitation at these wavelengths.

Article | | Nature Communications

Metabolite biosensors

In the construction of single fluorescent protein biosensors, selection of the insertion point of a fluorescent protein into a ligand-binding domain is a rate-limiting step. Here, the authors develop an unbiased, high-throughput approach, called domain insertion profiling with DNA sequencing (DIP-seq), to generate a novel trehalose biosensor.

Article | Open Access | | Nature Communications

A ratiometric fluorescent sensor that reports the ATP/ADP concentration ratio in living cells was created by fusing the bacterial regulatory protein GlnK1 to a circularly permuted fluorescent protein. The sensor detected inhibition of cellular metabolism caused by transient removal of glucose from the cellular medium or administration of a glycolytic inhibitor.

Article | | Nature Methods

Neuronal biosensors

Genetically encoded calcium sensors have brought neuronal recording to the tiny brains of invertebrates, but the methodology has lagged behind classical electrophysiology in vertebrates. Now Douglas Kim and colleagues have used selective mutagenesis to engineer a new ultrasensitive probe, GCaMP6, demonstrating improved spatial and temporal resolution in vivo, from flies to zebrafish. In addition, in mouse visual cortex GCaMP6 can reliably detect single action potentials and single-spine orientation tuning. GCaMP6 sensors can be used to image large groups of neurons as well as tiny synaptic compartments over multiple imaging sessions separated by months, offering a flexible new tool for brain research and calcium signalling studies.

Article | | Nature

An improved version of the GCaMP genetically encoded calcium indicator, called GCaMP3, has higher calcium affinity and increased baseline fluorescence, dynamic range and stability. GCaMP3 performs better than existing genetically encoded calcium indicators in several assays and organisms, including in vivo imaging of neuronal signaling in worms, flies and mice.

Article | | Nature Methods