A new tool that enables thousands of tiny experiments to run simultaneously on a single polymer chip will let scientists study enzymes faster and more comprehensively than ever before.
By Ker Than, Stanford University
July 22, 2021 -- For much of human
history, animals and plants were perceived to follow a different set of rules
than rest of the universe. In the 18th and 19th centuries, this culminated in a
belief that living organisms were infused by a non-physical energy or “life
force” that allowed them to perform remarkable transformations that couldn’t be
explained by conventional chemistry or physics alone.
Scientists now understand that these
transformations are powered by enzymes – protein molecules comprised of chains
of amino acids that act to speed up, or catalyze, the conversion of one kind of
molecule (substrates) into another (products). In so doing, they enable
reactions such as digestion and fermentation – and all of the chemical events
that happen in every one of our cells – that, left alone, would happen
extraordinarily slowly.
“A chemical reaction that would take
longer than the lifetime of the universe to happen on its own can occur in
seconds with the aid of enzymes,” said Polly Fordyce, an
assistant professor of bioengineering and of genetics at Stanford University.
While much is now known about enzymes,
including their structures and the chemical groups they use to facilitate
reactions, the details surrounding how their forms connect to their functions,
and how they pull off their biochemical wizardry with such extraordinary speed
and specificity are still not well understood.
A new technique, developed by Fordyce
and her colleagues at Stanford and detailed this week in the journal Science,
could help change that. Dubbed HT-MEK — short for High-Throughput Microfluidic
Enzyme Kinetics — the technique can compress years of work into just a few
weeks by enabling thousands of enzyme experiments to be performed
simultaneously. “Limits in our ability to do enough experiments have
prevented us from truly dissecting and understanding enzymes,” said study
co-leader Dan
Herschlag, a professor of biochemistry at Stanford’s School of
Medicine.
By allowing scientists to deeply probe
beyond the small “active site” of an enzyme where substrate binding occurs,
HT-MEK could reveal clues about how even the most distant parts of enzymes work
together to achieve their remarkable reactivity.
“It’s like we’re now taking a flashlight
and instead of just shining it on the active site we’re shining it over the
entire enzyme,” Fordyce said. “When we did this, we saw a lot of things we
didn’t expect.”
Enzymatic tricks
HT-MEK is designed to replace a
laborious process for purifying enzymes that has traditionally involved
engineering bacteria to produce a particular enzyme, growing them in large
beakers, bursting open the microbes and then isolating the enzyme of interest
from all the other unwanted cellular components. To piece together how an
enzyme works, scientists introduce intentional mistakes into its DNA blueprint
and then analyze how these mutations affect catalysis.
This process is expensive and time
consuming, however, so like an audience raptly focused on the hands of a
magician during a conjuring trick, researchers have mostly limited their
scientific investigations to the active sites of enzymes. “We know a lot about
the part of the enzyme where the chemistry occurs because people have made
mutations there to see what happens. But that’s taken decades,” Fordyce said.
But as any connoisseur of magic tricks
knows, the key to a successful illusion can lie not just in the actions of the
magician’s fingers, but might also involve the deft positioning of an arm or
the torso, a misdirecting patter or discrete actions happening offstage,
invisible to the audience. HT-MEK allows scientists to easily shift their gaze
to parts of the enzyme beyond the active site and to explore how, for example,
changing the shape of an enzyme’s surface might affect the workings of its
interior.
“We ultimately would like to do
enzymatic tricks ourselves,” Fordyce said. “But the first step is figuring out
how it’s done before we can teach ourselves to do it.”
Enzyme experiments on a chip
The technology behind HT-MEK was
developed and refined over six years through a partnership between the labs of
Fordyce and Herschlag. “This is an amazing case of engineering and enzymology
coming together to — we hope — revolutionize a field,” Herschlag said. “This
project went beyond your typical collaboration — it was a group of people
working jointly to solve a very difficult problem — and continues with the
methodologies in place to try to answer difficult questions.”
HT-MEK combines two existing
technologies to rapidly speed up enzyme analysis. The first is microfluidics,
which involves molding polymer chips to create microscopic channels for the
precise manipulation of fluids. “Microfluidics shrinks the physical space to do
these fluidic experiments in the same way that integrated circuits reduced the
real estate needed for computing,” Fordyce said. “In enzymology, we are still
doing things in these giant liter-sized flasks. Everything is a huge volume and
we can’t do many things at once.”
The second is cell-free protein
synthesis, a technology that takes only those crucial pieces of biological
machinery required for protein production and combines them into a soupy
extract that can be used to create enzymes synthetically, without requiring
living cells to serve as incubators.
“We’ve automated it so that we can use
printers to deposit microscopic spots of synthetic DNA coding for the enzyme
that we want onto a slide and then align nanoliter-sized chambers filled with
the protein starter mix over the spots,” Fordyce explained.
Because each tiny chamber contains only
a thousandth of a millionth of a liter of material, the scientists can engineer
thousands of variants of an enzyme in a single device and study them in
parallel. By tweaking the DNA instructions in each chamber, they can modify the
chains of amino acid molecules that comprise the enzyme. In this way, it’s
possible to systematically study how different modifications to an enzyme
affects its folding, catalytic ability and ability to bind small molecules and
other proteins.
When the team applied their technique to
a well-studied enzyme called PafA, they found that mutations well beyond the
active site affected its ability to catalyze chemical reactions — indeed, most
of the amino acids, or “residues,” making up the enzyme had effects.
The scientists also discovered that a
surprising number of mutations caused PafA to misfold into an alternate state
that was unable to perform catalysis. “Biochemists have known for decades that
misfolding can occur but it’s been extremely difficult to identify these cases
and even more difficult to quantitatively estimate the amount of this misfolded
stuff,” said study co-first author Craig Markin, a research scientist with
joint appointments in the Fordyce and Herschlag labs.
“This is one enzyme out of thousands and
thousands,” Herschlag emphasized. “We expect there to be more discoveries and
more surprises.”
Accelerating advances
If widely adopted, HT-MEK could not only
improve our basic understanding of enzyme function, but also catalyze advances
in medicine and industry, the researchers say. “A lot of the industrial
chemicals we use now are bad for the environment and are not sustainable. But
enzymes work most effectively in the most environmentally benign substance we
have — water,” said study co-first author Daniel Mokhtari, a Stanford graduate
student in the Herschlag and Fordyce labs.
HT-MEK could also accelerate an approach
to drug development called allosteric targeting, which aims to increase drug
specificity by targeting beyond an enzyme’s active site. Enzymes are popular
pharmaceutical targets because of the key role they play in biological
processes. But some are considered “undruggable” because they belong to
families of related enzymes that share the same or very similar active sites,
and targeting them can lead to side effects. The idea behind allosteric
targeting is to create drugs that can bind to parts of enzymes that tend to be
more differentiated, like their surfaces, but still control particular aspects
of catalysis. “With PafA, we saw functional connectivity between the surface
and the active site, so that gives us hope that other enzymes will have similar
targets,” Markin said. “If we can identify where allosteric targets are, then
we’ll be able to start on the harder job of actually designing drugs for them.”
The sheer amount of data that HT-MEK is
expected to generate will also be a boon to computational approaches and
machine learning algorithms, like the Google-funded AlphaFold project, designed
to deduce an enzyme’s complicated 3D shape from its amino acid sequence alone.
“If machine learning is to have any chance of accurately predicting enzyme
function, it will need the kind of data HT-MEK can provide to train on,”
Mokhtari said.
Much further down the road, HT-MEK may
even allow scientists to reverse-engineer enzymes and design bespoke varieties
of their own. “Plastics are a great example,” Fordyce said. “We would love to
create enzymes that can degrade plastics into nontoxic and harmless pieces. If
it were really true that the only part of an enzyme that matters is its active
site, then we’d be able to do that and more already. Many people have tried and
failed, and it’s thought that one reason why we can’t is because the rest of
the enzyme is important for getting the active site in just the right shape and
to wiggle in just the right way.”
Herschlag hopes that adoption of HT-MEK
among scientists will be swift. “If you’re an enzymologist trying to learn
about a new enzyme and you have the opportunity to look at 5 or 10 mutations
over six months or 100 or 1,000 mutants of your enzyme over the same period,
which would you choose?” he said. “This is a tool that has the potential to
supplant traditional methods for an entire community.”
https://news.stanford.edu/2021/07/22/new-tool-drastically-speeds-study-enzymes/
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