CHAMPAIGN, Ill. -- A new measurement technique developed at the University
of Illinois has shed some light on the early stages of protein folding.
In experiments conducted on the small, oxygen-carrying protein apomyoglobin,
U. of I. researchers have found that the initial steps of helix formation
can occur within several hundred nanoseconds. (A nanosecond is one billionth
of a second.) The entire collapse to a compact structure appears nearly
complete after just a few microseconds.
"Unlike slower spectroscopic methods that are limited in time resolution,
our technique can identify events occurring within nanoseconds," said
Martin Gruebele, a professor of chemistry and a researcher at the Beckman
Institute for Advanced Science and Technology. "We are able to witness
the early steps in protein folding that are being missed by other techniques."
Understanding how proteins fold is a necessary first step toward the eventual
goal of tailoring designed proteins. But, to study how proteins fold, researchers
first must unfold them.
Gruebele and his colleagues -- graduate students Richard Ballew and Jobiah
Sabelko -- do this by supercooling the proteins in an aqueous solution.
Then, to initiate the folding sequence, the solution is heated rapidly by
a single pulse from an infrared laser. As the proteins begin twisting into
their characteristic shapes, a series of pulses from an ultraviolet laser
cause some of the amino acids to fluoresce, revealing to the researchers
a time-sequence of folding events.
"We can monitor the laser-induced fluorescence for up to 60,000 pulses,
which corresponds to one millisecond," Gruebele said. "By studying
how the fluorescence decays over time, we can tell how the amino acids are
coming together and how different parts of the protein are moving around."
Based on recent measurements of apomyoglobin, Gruebele has identified two
distinct phases in the folding process. In the first phase, which lasts
several hundred nanoseconds, the amino acids quickly curl into helixes --
the fundamental building blocks of proteins. In the second phase, which
takes three to four microseconds, the helixes "hunt for and hook into"
their appropriate locations in the protein chain, Gruebele said. At that
point, the collapse phase of the folding process is complete.
"Before we conducted our experiments, many researchers thought protein
folding was a slow and inefficient process," Gruebele said. "Now
that we have pinned it down to the range of microseconds, protein folding
seems to be about as efficient as you could imagine."
In the past, researchers and theoreticians also have disagreed over the
exact nature of the folding sequence: whether the process begins with an
initial random collapse of the protein structure or with the forming of
helixes. "The debate has been going on for decades," Gruebele
said. "Now, with our fast detection and measurement scheme, I think
we can finally resolve some of the arguments."
The researchers' findings appear in the November issue of Nature: Structural
Biology.