Fluorescent Protein Sensor Research Articles

Ratiometric Fluorescent Sensor Proteins with Subnanomolar Affinity for Zn(II) Based on Copper Chaperone Domains

The ability to image the concentration of transition metals in living cells in real time is important for further understanding of transition metal homeostasis and its involvement in diseases. The goal of this study was to develop a genetically encoded FRET-based sensor for copper(I) based on the copper-induced dimerization of two copper binding domains involved in human copper homeostasis, Atox1 and the fourth domain of ATP7B (WD4). A sensor has been constructed by linking these copper binding domains to donor and acceptor fluorescent protein domains. Energy transfer is observed in the presence of Cu(I), but the Cu(I)-bridged complex is easily disrupted by low molecular weight thiols such as DTT and glutathione. To our surprise, energy transfer is also observed in the presence of very low concentrations of Zn(II) (10(-)(10) M), even in the presence of DTT. Zn(II) is able to form a stable complex by binding to the cysteines present in the conserved MXCXXC motif of the two copper binding domains. Co(II), Cd(II), and Pb(II) also induce an increase in FRET, but other, physiologically relevant metals are not able to mediate an interaction. The Zn(II) binding properties have been tuned by mutation of the copper-binding motif to the zinc-binding consensus sequence MDCXXC found in the zinc transporter ZntA. The present system allows the molecular mechanism of copper and zinc homeostasis to be studied under carefully controlled conditions in solution. It also provides an attractive platform for the further development of genetically encoded FRET-based sensors for Zn(II) and other transition metal ions.

Imaging brain activity with voltage- and calcium-sensitive dyes.

This paper presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes and then discusses the methodological aspects of the measurements that are needed to achieve an optimal signal-to-noise ratio. Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations. Both invertebrate and vertebrate ganglia can be bathed in voltage-sensitive dyes to stain all of the cell bodies in the preparation. These dyes can then be used to follow the spike activity of many neurons simultaneously while the preparations are generating behaviors. Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb. There they can be used to measure the input from the nose to the bulb. Three kinds of noise are discussed. a. Shot noise from the random emission of photons from the preparation. b. Vibrational noise from external sources. c. Noise that occurs in the absence of light, the dark noise. Three different parts of the light measuring apparatus are discussed: the light sources, the optics, and the cameras. The major effort presently underway to improve the usefulness of optical recordings of brain activity are to find methods for staining individual cell types in the brain. Most of these efforts center around fluorescent protein sensors of activity.

All solid-state GFP sensor.

An all-solid-state green fluorescent protein (GFP) sensor for GFP measurement was developed. It is immune to interference from ambient light and works with standard flow-through cuvettes. The sensor is practically insensitive to the scattered excitation light encountered in microbial suspensions. It has a range of 0.0002-1 g/L (7.4 x 10(-9) - 3.7 x 10(-5) M) with limit of detection 0.00019 g/L (7.0 x 10(-9) M). The sensor could be used with a UV or blue light emitting diode (LED) as a light source, depending on required sensitivity, selectivity, and background levels. Its very low cost makes it useful in a variety of applications. This article describes the construction and validation of the sensor both off- and on-line in fermentation processes.

Fluorescent protein sensors for detection of anaIytes (R Tsien and A Miyawaki, CA, USA)

Fluorescent protein sensors for detection of anaIytes (R Tsien and A Miyawaki, CA, USA)

On-line green fluorescent protein sensor with LED excitation

We present an intensity based sensor designed for on-line monitoring of green fluorescent protein, a revolutionary marker of protein expression. The device consisted of a blue light emitting diode as the excitation source. A band pass excitation filter cut off light longer than 490 nm. The light was directed into a bifurcated optical fiber bundle with the common end inserted into a stainless steel housing equipped with a quartz window. The fiber bundle and stainless steel housing are steam sterilizable. The emission radiation was collected through a long wave pass filter to reject the excitation light shorter than 505 nm and was detected by a photomultiplier tube. The signal was amplified and sent to a computer for recording time course data. The sensor was tested in an Escherichia coli fermentation of JM105 transformed with pBAD-GFP. The on-line signal was compared to off-line fluorescence spectrophotometer measurements. The on-line profile closely followed the off-line. Western blot data showed that with a time shift, the sensor was able to both continuously and quantitatively monitor expression of green fluorescent protein on-line in real time.