Special luciferase tracks cellular processes when activated with light

May 6, 2013 § Leave a comment

The letters were produced by covering HEK293T cells expressing photoactivatable luciferase with aluminum foils in the shapes of the letters before photolysis. Image provided by Jingyi Zhao.

The letters were produced by covering HEK293T cells expressing photoactivatable luciferase with aluminum foils in the shapes of the letters before photolysis. Image provided by Jingyi Zhao.

Being able to control protein activity with respect to time and location with pulses of laser light has become an increasingly important scientific pursuit to understand how proteins work together. In a paper just out in the Journal of the American Chemical Society, researchers demonstrated a new fluorescent marker based on the well-known luciferase method. This new marker allowed the investigators to track dynamic ATP fluctuation within living cells, something that couldn’t be done with other fluorescent markers.

A new class of fluorescent markers called photocaged unnatural amino acids have proved to be useful as a research tool to activate proteins at specific places and times. “The repertoire of photocaged unnatural amino acids has expanded lately, permitting the utilization of light to directly manipulate a specific amino acid residue, such as an essential lysine residue, on a given protein in diverse living species,” explains Peng Chen of Peking University in China. Chen led the team of investigators, along with Jing Zhao , also at Peking University.

But Chen says,  “It remains a challenge to assess the photolysis efficiency of photocaged lysine residues in the context of their embedded proteins in living cells.”

The luciferase-based bioluminescent imaging and tracking approach is used routinely in biomedical research. The system has good sensitivity and allows for quantitative measurements. “Intracellular activation of luciferase in a spatial and temporal fashion would allow more precise tracking of the cellular events or gene expression within intact cells or animals,” says Chen.

The investigators set out to make a luciferase whose activity could be controlled by light. Luciferase converts its substrate, luciferin, into the highly luminescent oxyluciferin in a two-step process. The investigators noted that lysine 529 on the enzyme was critical for the reaction. They envisioned that replacing this important lysine residue with a genetically encoded photocaged lysine analog would block the substrate binding in the enzyme’s active site. This photocaged lysine analog then could be zapped with an ultraviolet laser to remove the cage, produce a free lysine and restore luciferase’s activity.

The investigators demonstrated just that with their modified luciferase in live cultured cells. They were careful to use low doses of UV light to avoid photodamage. They were able to use their special enzyme to track ATP changes in the cells over time. Chen says the photocaged lysine residue can be dropped into another kinds of enzyme to study “cellular processes such as epigenetic histone regulations, p53 homeostasis and protein ubiquitination, all of which heavily depend on posttranslational lysine modifications.”

Chen does say that the photoactivatable luciferase has only been used so far in cultured cell lines, not in primary cells, tissues or multicellular organisms. “Indeed, one of the major advantages of luciferase over fluorescent proteins is its applicability in animals, which requires deep [light] penetration.” Chen says the investigators are currently looking into generating the photoactivable luciferase in multicellular organisms.

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