New method takes snapshots of proteins as they fold

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New method takes snapshots of proteins as they fold

Scientists have invented a way to ‘watch’ proteins fold — in less than

thousandths of a second -- into the elaborate twisted shapes that determine

their function.

January 10, 2011

By Lutz

An unfolded protein is a string made up of amino acids. When the protein folds

it forms structures like the alpha helix (the corkscrew) and the beta sheet (the

flat ribbon) in the image to the right. These secondary structures then double

back on themselves to form the final structure.

People have only 20,000 to 30,000 genes (the number is hotly contested), but

they use those genes to make more than 2 million proteins. It’s the protein

molecules that do most of the work in the human cell. After all, the word

protein comes from the Greek prota, meaning “of primary importance.”

Proteins are created as chains of amino acids, and these chains of usually fold

spontaneously into what is called their “native form” in milliseconds or a few

seconds.

A protein’s function depends sensitively on its shape. For example, enzymes and

the molecules they alter are often described as fitting together like a lock and

key. By the same token, misfolded proteins are behind some of the most dreaded

neurodegenerative diseases, such as Alzheimer’s, Parkinson's and mad cow

disease.

Scientists can't match the speed with which proteins fold. Predicting how chains

of amino acids will fold from scratch requires either powerful supercomputers or

cloud sourcing (harnessing the pattern recognition power of thousands of people

by means of games such as Folding@home).

Either way, prediction is time-consuming and often inaccurate, so much so that

the protein-structure bottleneck is slowing the exploitation of DNA sequence

data in medicine and biotechnology.

A clever way of watching proteins fold and unfold may finally provide the kind

of detail needed to improve protein structure predictions.

In a recent issue of the online version of the Journal of the American Chemical

Society three scientists, led by L. Gross, PhD, professor of chemistry

in Arts & Sciences and of medicine and immunology in the School of Medicine at

Washington University in St. Louis, describe a proof-of-principle study in which

they use the new approach to watch the folding of a small protein called

barstar.

What they do is roughly analogous to filming flying bullets or bursting balloons

with a stroboscope and a fast camera. The " stills " taken by the camera slow

motion to the point that normally imperceptible events are laid open to

scrutiny.

The scientists are using a version of this old trick to “watch” proteins fold.

The “strobe light” is a temperature jump and the “camera” is a fast chemical

reaction whose outcome is measured by a sensitive mass spectrometer.

Why folding is a complex problem

Courtesy of Fabio Pietrucci

Fabio Pietrucci, PhD,a postdoctoral researcher at Centre Européen de Calcul

Atomique et Moléculaire tries to predict protein structures from scratch, that

is, from the electromagnetic interactions between atoms. This illustration shows

the various misfolded states of a protein called GB1 his computer simulation of

protein folding found. The correct native state is at the top left.

One of the dogmas of modern biology is that the sequence of amino acids

determines how a protein will fold. If the amino acid sequence is known, it

should be possible to calculate the protein’s final structure from scratch.

But like many things in life, it’s harder than it looks.

“Think of a protein as thousands of atoms connected together by springs,” says

Gross, who is also director of the National Institutes of Health/ National

Center for Research Resources (NIH/NCRR) Mass Spectrometry Resource “If you were

to suspend this object with a string from the ceiling and let it flop around,

imagine how many shapes it could take.”

“An enormous number, because it is free to move in so many different ways.”

In practice, scientists often predict protein structure not from scratch but by

analogy. They sift through large databases for proteins with similar sequences

of amino acids and assume similar amino-acid chains will fold in similar ways.

“But,” says Gross, “at some point any method for predicting protein structure

has to be checked against experimental evidence that shows how proteins actually

do fold.”

That’s what his research is all about.

A model protein for the experiment

Wikimedia Commons

The function of barstar (right) is to bind tightly to barnase (left), preventing

it from chopping up RNA, a molecule necessary to life, until it is secreted from

the cell.

Barstar is a small protein synthesized by a soil bacterium that is often used in

folding studies.

Importantly, barstar’s “native state” is known, as is its primary structure, the

sequence of the protein subunits called amino acids of which it is made. What

isn’t known is how the amino-acid chain twists and coils to form the final

structure.

Fortunately for the scientists, barstar, unlike most proteins, is unfolded at

zero degrees Celsius and begins to fold as it warms.

The folding takes place in microseconds (millionths of a second).

How the method works

Nature Publications

To analyze a protein as it folds, a cold solution of an unfolded protein (the

thread in the top image), and hydrogen peroxide (paired red dots) is injected

into a capillary. The solution is hit with a laser at an infrared wavelength

(second image), which heats the protein enough to encourage it to start folding

(the blue coil is an alpha helix, a common protein " fold " ). In the third step,

the solution is exposed to an ultraviolet pulse that breaks the hydrogen

peroxide into two parts that attach oxygen atoms (red dots) to portions of the

protein that are still outside the folding structure and exposed to the

solution. The protein is later cut up and the parts " weighed " to see if they are

carrying additional oxygen atoms. The pathway by which the protein folds can be

deduced by repeatedly " painting " it with oxygen in this way.

The scientists begin by injecting a cold solution of barstar and a tiny amount

of hydrogen peroxide into an optical fiber so thin it is difficult to believe it

is actually hollow.

“Plugs” of sample in the fiber are then hit with two laser pulses in quick

succession.

The first pulse, called a T jump, heats the solution just enough to make a

different protein conformation energetically favorable.

The second pulse then breaks some of the hydrogen peroxide (H2O2) molecules into

two haves, each of which is a very reactive hydroxyl (-OH) radical.

The radicals react with those parts of the protein that are exposed to the

solution, “painting” them with oxygen atoms.

“Imagine,” says Gross, “that you suspended a styrofoam model of a partially

folded protein and spray-painted it blue. The outside parts would be painted

blue; those buried within would remain white.”

The radical reactions must be terminated rapidly; otherwise some " painting " may

occur within the structure. Within a microsecond, a scavenger amino acid clears

away any remaining hydroxyl radicals to prevent them from breaking bonds and

altering the protein’s configuration.

The same process is repeated 500 times, taking rapid-fire “snapshots” of the

protein’s quickly changing confirmation.

“The hydroxyl radicals don’t mark everything,” says Gross. “But they mark about

half the amino acids, which is really pretty good. Most other chemical reagents

are too specific and too slow for this experiment. Compared to hydroxyl radicals

they’re just plain ponderous.”

Weighing the painted proteins

“We collect each drop of marked protein as it emerges from the fiber,” says

Gross. “Then we digest the protein very slowly and carefully with an enzyme that

cleaves the amino acid chains at specific locations, creating a known set of

protein fragments, called peptides

These protein fragments are separated according to type by liquid

chromatography, and a mass spectrometer then “weighs” each fragment type to see

whether it has picked up oxygen atoms.

“Detecting an extra oxygen is child’s play for a modern mass spectrometer,” says

Gross. “Most instruments can even detect an extra proton, with is one-sixteenth

the mass of an oxygen atom.”

“In the same instrument, on the fly, we break apart the protein fragments and

again ‘weigh’ the bits to see which one still carries the oxygen atom. This lets

us deduce the oxygen’s location on the original fragment.”

By following barstar to its first intermediate state, or way station enroute to

its native state, the scientists demonstrated that the new technique can follow

folding and unfolding on a submillisecond time scale.

‘Massive amounts of detail’

Gross is the first to say that this proof-of-principle experiment stands at the

end of a long line of elegant experiments of a similar type, called pump-probe

experiments.

Other techniques probe the change in protein structure by monitoring the

absorption or emission of light--or a similar physical effect. They can provide

only global information, such as the rate constant of a folding reaction.

“Because we use a chemical rather than a physical probe, we can see what’s going

on in much greater detail,” says Gross. “We can say which part of the structure

closes first, which second, and so on.”

The new technique caught the attention of protein scientist Gruebele of

the University of Illinois, who spotlighted it in the Dec. 2, 2010, issue of the

journal Nature.

It “could provide truly massive amounts of detail about fast protein folding,”

wrote Gruebele, which might finally allow scientists “to correctly predict the

biologically active structure of a protein starting from the unfolded state.”