Dependence of fluorescent protein brightness on protein concentration in solution and enhancement of it

Morikawa, Takamitsu; Fujita, Hideaki; Kitamura, Akira; Horio, Takashi; Yamamoto, Johtaro; Sasaki, Akira; Machiyama, Hiroaki; Yoshizawa, Keiko; Ichimura, Taro; Imada, Katsumi; Nagai, Takeharu; Watanabe, Tomonobu M.

doi:10.1038/srep22342

Cited by 47 publications

(63 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Other sensors have been developed, including a synthetic sensor based on polyethylene glycol that is compressed by macromolecular crowding (15), and a genetically encoded sensor that is based on protein-induced destabilization of an impaired YFP (16). The polyethylene glycol 10,000 (PEG)-based sensor may function via a similar mechanism to our sensor, whereas the mechanism behind the destabilization of the YFP sensor is not yet clear.…”

Section: Introductionmentioning

confidence: 99%

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

Zhang

Åberg

Eerden

et al. 2017

Biophysical Journal

100

View full text Add to dashboard Cite

Cells are highly crowded with proteins and polynucleotides. Any reaction that depends on the available volume can be affected by macromolecular crowding, but the effects of crowding in cells are complex and difficult to track. Here, we present a set of Fö rster resonance energy transfer (FRET)-based crowding-sensitive probes and investigate the role of the linker design. We investigate the sensors in vitro and in vivo and by molecular dynamics simulations. We find that in vitro all the probes can be compressed by crowding, with a magnitude that increases with the probe size, the crowder concentration, and the crowder size. We capture the role of the linker in a heuristic scaling model, and we find that compression is a function of size of the probe and volume fraction of the crowder. The FRET changes observed in Escherichia coli are more complicated, where FRETincreases and scaling behavior are observed solely with probes that contain the helices in the linker. The probe with the highest sensitivity to crowding in vivo yields the same macromolecular volume fractions as previously obtained from cell dry weight. The collection of new probes provides more detailed readouts on the macromolecular crowding than a single sensor.

show abstract

Section: Introductionmentioning

confidence: 99%

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

Zhang

Åberg

Eerden

et al. 2017

Biophysical Journal

100

View full text Add to dashboard Cite

show abstract

“…However, in vitro calibrations may not fully recapitulate the sensor behavior in cells, considering that its signal can be affected by the cellular environment (Dittmer, Miranda, Gorski, & Palmer, 2009). For example, genetically encoded, engineered protein sensors can be sensitive to molecular crowding (Dittmer et al, 2009;Morikawa et al, 2016), which is a major distinction between the purified protein and the in-cell calibrations.…”

Section: (B) (A)mentioning

confidence: 99%

Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor

Díaz‐García

Lahmann

Martínez-François

et al. 2019

J of Neuroscience Research

View full text Add to dashboard Cite

Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR‐TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose‐binding protein with a circularly permuted variant of the monomeric fluorescent protein T‐Sapphire. Here, we provide more details on iGlucoSnFR‐TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2–10 mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20% of the nominal external glucose concentration (~0.4–2 mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7–2.5 mM in cortical neurons from awake mice.

show abstract

“…4A). Recently, Morikawa et al found that the fluorophore of YFP is sensitive to protein crowding (Morikawa et al, 2016). They introduced a mutation to increase the sensitivity to the protein crowding and generated a proteincrowding sensor (Fig.…”

Section: Fret Biosensors Based On the Intrinsic Properties Of Fluoresmentioning

confidence: 99%

Two Decades of Genetically Encoded Biosensors Based on Förster Resonance Energy Transfer

Terai

Imanishi

2019

Cell Struct. Funct.

View full text Add to dashboard Cite

Two decades have passed since the development of the first calcium indicator based on the green fluorescent protein (GFP) and the principle of Förster resonance energy transfer (FRET). During this period, researchers have advanced many novel ideas for the improvement of such genetically encoded FRET biosensors, which have allowed them to expand their targets from small molecules to signaling proteins and physicochemical properties. Although the merits of "genetically encoded" FRET biosensors became clear once various cell lines were established and several transgenic organisms were generated, the road to these developments was not necessarily a smooth one. Moreover, even today the development of new FRET biosensors remains a very laborintensive, trial-and-error process. Therefore, at this junction, it may be worthwhile to summarize the progress of the FRET biosensor and discuss the future direction of its development and application.

show abstract

Dependence of fluorescent protein brightness on protein concentration in solution and enhancement of it

Cited by 47 publications

References 62 publications

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor

Two Decades of Genetically Encoded Biosensors Based on Förster Resonance Energy Transfer

Contact Info

Product

Resources

About