New tools catch and release molecules at the flip of a light switch


Date:
August 13, 2020
Source:
Princeton University, Engineering School
Summary:
A team has developed a class of light-switchable, highly adaptable
molecular tools with new capabilities to control cellular
activities. The antibody-like proteins, called OptoBinders, have
potential applications including protein purification, the improved
production of biofuels, and new types of targeted cancer therapies.



FULL STORY ==========================================================================
A Princeton team has developed a class of light-switchable, highly
adaptable molecular tools with new capabilities to control cellular
activities. The antibody-like proteins, called OptoBinders, allow
researchers to rapidly control processes inside and outside of cells
by directing their localization, with potential applications including
protein purification, the improved production of biofuels, and new types
of targeted cancer therapies.


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In a pair of papers published Aug. 13 in Nature Communications,
the researchers describe the creation of OptoBinders that can
specifically latch onto a variety of proteins both inside and outside
of cells. OptoBinders can bind or release their targets in response to
blue light. The team reported that one type of OptoBinder changed its
affinity for its target molecules up to 330-fold when shifted from dark
to blue light conditions, while others showed a five-fold difference in
binding affinity -- all of which could be useful to researchers seeking
to understand and engineer the behaviors of cells.

Crucially, OptoBinders can target proteins that are naturally present in
cells, and their binding is easily reversible by changing light conditions
-- "a new capability that is not available to normal antibodies," said co-author Jose' Avalos, an assistant professor of chemical and biological engineering and the Andlinger Center for Energy and the Environment. "The ability to let go [of a target protein] is actually very valuable for
many applications," said Avalos, including engineering cells' metabolisms, purifying proteins or potentially making biotherapeutics.

The new technique is the latest in a collaboration between Avalos and
Jared Toettcher, an assistant professor of molecular biology. Both joined
the Princeton faculty in 2015, and soon began working together on new
ways to apply optogenetics -- a set of techniques that introduce genes
encoding light- responsive proteins to control cells' behaviors.

"We hope that this is going to be the beginning of the next era of optogenetics, opening the door to light-sensitive proteins that can
interface with virtually any protein in biology, either inside or outside
of cells," said Toettcher, the James A. Elkins, Jr. '41 Preceptor in
Molecular Biology.

Avalos and his team hope to use OptoBinders to control the metabolisms
of yeast and bacteria to improve the production of biofuels and other
renewable chemicals, while Toettcher's lab is interested in the molecules' potential to control signaling pathways involved in cancer.



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The two papers describe different types of light-switchable binders:
opto- nanobodies and opto-monobodies. Nanobodies are derived from the antibodies of camelids, the family of animals that includes camels,
llamas and alpacas, which produce some antibodies that are smaller
(hence the name nanobody) and simpler in structure than those of humans
or other animals.

Nanobodies' small size makes them more adaptable and easier to work
with than traditional antibodies; they recently received attention for
their potential as a COVID-19 therapy. Monobodies, on the other hand,
are engineered pieces of human fibronectin, a large protein that forms
part of the matrix between cells.

"These papers go hand in hand," said Avalos. "The opto-nanobodies take advantage of the immune systems of these animals, and the monobodies have
the advantage of being synthetic, which gives us opportunities to further engineer them in different ways." The two types of OptoBinders both incorporate a light-sensitive domain from a protein found in oat plants.

"When you turn the light on and off, these tools bind and release their
target almost immediately, so that brings another level of control" that
was not previously possible, said co-author Ce'sar Carrasco-Lo'pez, an associate research scholar in Avalos' lab. "Whenever you are analyzing
things as complex as metabolism, you need tools that allow you to
control these processes in a complex way in order to understand what
is happening." In principle, OptoBinders could be engineered to target
any protein found in a cell. With most existing optogenetic systems,
"you always had to genetically manipulate your target protein in a
cell for each particular application," said co-author Agnieszka Gil, a postdoctoral research fellow in Toettcher's lab. "We wanted to develop an optogenetic binder that did not depend on additional genetic manipulation
of the target protein."


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In a proof of principle, the researchers created an opto-nanobody that
binds to actin, a major component of the cytoskeleton that allows cells to move, divide and respond to their environment. The opto-nanobody strongly
bound to actin in the dark, but released its hold within two minutes
in the presence of blue light. Actin proteins normally join together
to form filaments just inside the cell membrane and networks of stress
fibers that traverse the cell. In the dark, the opto-nanobody against
actin binds to these fibers; in the light, these binding interactions are disrupted, causing the opto-nanobody to scatter throughout the cell. The researchers could even manipulate binding interactions on just one side
of a cell -- a level of localized control that opens new possibilities
for cell biology research.

OptoBinders stand to unlock scores of innovative, previously inaccessible
uses in cell biology and biotechnology, said Andreas Mo"glich, a professor
of biochemistry at the University of Bayreuth in Germany who was not
involved in the studies. But, Mo"glich said, "there is much more to the research" because the design strategy can be readily translated to other molecules, paving the way to an even wider repertoire of customized, light-sensitive binders.

"The impressive results mark a significant advance," he said.

"Future applications will depend on being able to generate
more OptoBinders" against a variety of target proteins, said
Carrasco-Lo'pez. "We are going to try to generate a platform so we can
select OptoBinders against different targets" using a standardized, high-throughput protocol, he said, adding that this is among the first priorities for the team as they resume their experiments after lab
research was halted this spring due to COVID-19.

Beyond applications that involve manipulating cell metabolism for
microbial chemical production, Avalos said, OptoBinders could someday
be used to design biomaterials whose properties can be changed by light.

The technology also holds promise as way to reduce side effects of drugs
by focusing their action to a specific site in the body or adjusting
dosages in real time, said Toettcher, who noted that applying light inside
the body would require a device such as an implant. "There aren't many
ways to do spatial targeting with normal pharmacology or other techniques,
so having that kind of capability for antibodies and therapeutic binders
would be a really cool thing," he said. "We think of this as a sea change
in what sorts of processes can be placed under optogenetic control."

========================================================================== Story Source: Materials provided by
Princeton_University,_Engineering_School. Note: Content may be edited
for style and length.


========================================================================== Journal Reference:
1. Ce'sar Carrasco-Lo'pez, Evan M. Zhao, Agnieszka A. Gil, Nathan Alam,
Jared E. Toettcher, Jose' L. Avalos. Development of light-responsive
protein binding in the monobody non-immunoglobulin scaffold. Nature
Communications, 2020; 11 (1) DOI: 10.1038/s41467-020-17837-7 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/08/200813142400.htm

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