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Analytical Chemistry

A New Fluorescent Protein For Studying Living Organisms

Pacifichem News: Engineered near-IR fluorescent protein could aid imaging of multiple biological processes in whole animals

by Stu Borman
December 18, 2015

Engineered Protein
[+]Enlarge
Credit: RCSB PDB
Shown is a crystal structure of BphP1-FP. The ball-and-stick structure is the bound biliverdin chromophore, green spheres are water molecules, and the gap between the main protein and segment at the top is a part of the protein disordered in the crystal structure.
Credit: RCSB PDB
Shown is a crystal structure of BphP1-FP. The ball-and-stick structure is the bound biliverdin chromophore, green spheres are water molecules, and the gap between the main protein and segment at the top is a part of the protein disordered in the crystal structure.

Green fluorescent proteins (GFPs) revolutionized scientists’ ability to monitor happenings inside cells. But the glowing proteins have a serious limitation that prevents them from helping scientists watch biological phenomena in whole, living animals. The wavelengths of light used to excite GFPs are readily absorbed by animal tissue, making it difficult for researchers to peer past the surface of an organism. To overcome that limitation, some scientists have engineered fluorescent proteins that absorb and emit near-infrared (near-IR) light, which can penetrate through tissue.

On Tuesday at the 7th International Chemical Congress of Pacific Basin Societies, or Pacifichem, in Honolulu, scientists reported a newly engineered near-IR fluorescent protein that could improve whole-organism imaging of biological processes.

Two Colors
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Credit: Courtesy of Vladislav Verkhusha
In this image, green corresponds to the nucleus of a human cell labeled by BphP1-FP, and red denotes the cell’s lysosomes labeled by a red-shifted near-IR fluorescent protein.
Credit: Courtesy of Vladislav Verkhusha
In this image, green corresponds to the nucleus of a human cell labeled by BphP1-FP, and red denotes the cell’s lysosomes labeled by a red-shifted near-IR fluorescent protein.

The new protein is the brightest in a bacterial class of phytochrome fluorescent proteins, and it emits close to the edge of the near-IR spectra. These features could enhance the sensitivity and ease of imaging experiments that monitor multiple biological processes at once.

Phytochrome fluorescent proteins bind near-IR absorbing tetrapyrroles called bilins. But the native proteins “are not meant to fluoresce,” explains biophysicist John T. M. Kennis of VU University Amsterdam, who was not involved in the new work. “They are signaling proteins that have evolved to detect near-IR light and give off a signal to the organism to act in certain ways. So these proteins have to be engineered to become fluorescent.”

Vladislav Verkhusha and Daria Shcherbakova of Albert Einstein College of Medicine have been working on engineering the proteins to absorb and emit light from different stretches of the near-IR spectrum. Proteins with different spectral properties allow scientists to tag more than one type of cell, tissue, or organ so they can image them simultaneously.

Recently, Verkhusha, Shcherbakova, and coworkers found that they could tune the proteins’ spectral properties by changing the amino acid residue to which a bilin called biliverdin attaches to the protein. Their best success to date is a protein they call BphP1-FP (Chem. Biol. 2015, DOI: 10.1016/j.chembiol.2015.10.007). The emission of the new protein is blue-shifted by about 40 nm compared to native and previously engineered phytochrome proteins—from over 700 nm to 669 nm, near the edge of the near-IR region.

BphP1-FP also has a fluorescence quantum yield, a measure that contributes to the protein’s brightness, of 13%, “which is the highest ever reported for bacterial phytochromes,” Kennis says. He notes that the first reported near-IR bacterial phytochrome fluorescent protein, developed in 2009, had a quantum yield of about 7% and that there have been few improvements on that in the past six years.

Verkhusha and Shcherbakova’s team used BphP1-FP and a spectrally distinct phytochrome protein for multicolor imaging of different compartments in human cells. The group’s work suggests that “the ideal of the transparent mouse comes within reach,” Kennis says.

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Comments
Emma (December 20, 2015 8:03 AM)
Can someone explain what they mean by "part of the protein disordered in the crystal structure" for the model of the protein?
Stu Borman (December 21, 2015 9:43 AM)
Emma -- It's a part of the protein that didn't crystallize and therefore couldn't be visualized crystallographically. The main body of the protein crystallized OK, and the part at top left crystallized as well, but the part represented by the gap did not crystallize and is therefore missing from the crystal structure. -- Stu Borman
Ho Leung Ng (December 25, 2015 4:12 AM)
It means that part of the protein does not adopt defined, discrete conformations that could be observed in the crystals. It is present in the crystal but it was not experimentally observed in the crystal structure.

Nice work by Drs. Verkhusha and Shcherbakova and their team!

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