Folding, Misfolding, and Lessons from a WW Domain
By Jason Socrates
Bardi
Protein misfolding can be such a difficult and abstract subject
that it becomes easy to lose sight of the simple principles
that scientists would like to understand regarding how and
why proteins misfold.
One reason can be illustrated by the discovery a doctor
made almost 100 years ago. When Dr. Alois Alzheimer examined
a post-mortem patient who died with an unusual mental illness
in 1906, he found clumps of "amyloid" protein plaques in her
brain. These plaques are still a clear sign of the disease
that bears his name—a disease now believed to inflict
some 4 million Americans.
Many diseases are caused by the formation of protein plaques
inside the body. Amyloid-forming diseases like Alzheimer's
and Parkinson's are well known, but others include a collection
of over 80 rare amyloid diseases caused by the misfolding
of the protein transthyretin (TTR), which the liver secretes
into the bloodstream to carry thyroid hormone and vitamin
A. In diseases like familial amyloid polyneuropathy (FAP),
hundreds of these proteins misfold into structures leading
to microscopic fibril plaques, which deposit in internal organs
and interfere with normal function, sometimes lethally.
Another reason for scientists' interest in protein misfolding
can be illustrated by a problem recently facing a graduate
student. The student was toiling away at a routine laboratory
procedure of expressing and purifying a small soluble protein
in bacteria, but every time the student induced the expression
of the protein, the protein molecules would clump together
on the nanoscale, form insoluble "inclusion bodies" on the
microscale, and crash out of solution like a fried egg at
the bottom of the test tube on the macroscale.
In fact, understanding protein misfolding would have many
applications in basic biochemistry, where it could be used
to prevent such aggregation and advance basic laboratory methods.
As such, says Jeffery W. Kelly, who is the Lita Annenberg
Hazen Professor of Chemistry at The Scripps Research Institute
(TSRI) and vice president of academic affairs at TSRI, understanding
how and why proteins misfold is a high priority for many scientists.
And, he adds, "In order to completely understand misfolding,
we have to understand how proper protein folding takes place."
A Model For Beta Sheet Formation
Now two studies in an upcoming issue of Proceedings of
the National Academy of Sciences (PNAS) provide insight
into the folding process of what are known as beta sheet structures.
This is a common fold or "motif" wherein fully extended peptide
strands hydrogen bond to each other to form a sheet—the
same way that plastic teeth in a flat comb line up.
In one article, Kelly, his colleagues at The Skaggs Institute
for Chemical Biology at TSRI, and a group at the University
of Illinois examined the kinetics of beta sheet formation
using a protein fragment called the formin-binding protein
(FBP) WW domain. The "WW" refers to the fact that there are
two conserved tryptophan residues in the hundreds of sequences
comprising this domain family (tryptophan is abbreviated "W").
This FBP WW domain is a small protein fragment of a few
dozen residues that folds rapidly into its structure in tens
of microseconds. The structure is very basic—its chain
folds to form three strands of a beta sheet connected by two
loops.
Significantly, this structure is incredibly tolerant to
mutagenesis—two sequence-dissimilar WW domains from divergent
species will nevertheless fold into the same three-dimensional
structure. This tolerance is important to be able to mutate
residues of this protein and gauge which ones may be particularly
important for the folding of these WW domains.
And that's exactly what Kelly and his colleagues did. They
made changes to the protein, including mutating certain residues,
cutting off the end of the FBP WW domains, and observing protein
folding under temperature variations. They observed this by
monitoring a change in fluorescence as the proteins adopts
or changes its structure using a laser heating and rapid fluorescence
measurement technique employed by the University of Illinois
group.
What the researchers found was that temperature, mutation,
and truncation changes all have the ability to alter the kinetics
of the protein folding—changing the pathway by which
these proteins fold, not their final structure.
The folding kinetics or the way that FBP and other WW domain
proteins fold can generally be divided into two separate classes—the
two-state folders and the three-state folders.
Two-state folders form the characteristic beta sheet in
a single transition. That is, they go from one (unfolded)
state to a second (folded) state. Three-state folders, on
the other hand, go through an intermediate state, which means
that they have two transitions along their folding
pathways (unfolded to intermediate and intermediate to folded).
Kelly and his colleagues found that when the FBP WW domains
folded with three-state kinetics, the formation of a particular
loop between two individual beta strands is the rate-limiting
step for the folding of the beta sheet, meaning that these
connections form more slowly. They also found that it is possible
to "tune" the way the proteins folded by changing the sequence
or the temperature of the protein. Specifically, Kelly and
his colleagues were able to switch a slow folding, two-transition
WW domain into a more rapid, single transition WW domain.
Kelly explains that higher temperatures, the removal of
the C-terminus of the protein, or cetain internal mutations
destabilize the intermediate state, so the proteins fold in
a single transition.
A Model of the Folding
A second PNAS paper, also scheduled for publication
soon, by TSRI graduate student John Karanicolas and Charles
L. Brooks III, professor of molecular biology at TSRI, predict
this behavior with a new model.
Karanicolas and Brooks model the folding kinetics of two
different WW domains—which both fold into the classic
WW beta sheet, but via the two different mechanisms. Significantly,
Karanicolas and Brooks found through their simulations that
the origin of the three-state folding is a particular set
of residues that form mismatched contacts with each other
in the intermediate state.
These contacts occur during the formation of a particular
loop that must form before the beta strands can line up to
form a beta sheet. This process has the propensity to form
a misaligned loop in which the incorrect neighbors line up,
which is sort of like mismatching the buttons on your shirt.
This mismatched intermediate, Karanicolas and Brooks found,
is what drives the rate-limiting step. They observed this
misaligned loop and inferred that one particular residue that
is responsible for fixing the alignment is removed, the slow
phase gets slower.
Karanicolas and Brooks shared this result with Kelly, and
Kelly and his colleagues made mutations to the proteins, tested
their folding, and verified the prediction experimentally.
What emerges from both of these studies is a complicated
description best explained by a simple analogy: the folding
of the WW domain is something like buttoning up your shirt
in the morning. If you misalign the buttons, it takes time
to unbutton the shirt and button it up again.
In other words, it's faster to button your shirt right the
first time than to button it incorrectly and have to fix it,
and it's possible to "tune" the process by adjusting surrounding
conditions, like the light in the room. Turn up the dimmer,
and you can see the shirt better. The better you see, the
less likely you are to make a mistake buttoning your shirt,
and the faster you will be able to do it.
"This [research] was an extraordinarily good set of interactions,"
says Brooks. "Jeff [Kelly] and his colleagues were really
listening to our theoretical input, and we were getting all
this wonderful feedback that helped us understand our models
better and led, ultimately, to the correct picture of the
folding for this protein."
To read the article "Tuning the free energy landscape of
a WW domain by temperature, mutation, and truncation" by Houbi
Nguyen, Marcus Jager, Alessandro Moretto, Martin Gruebele,
and Jeffery W. Kelly, please see: http://www.pnas.org/cgi/doi/10.1073/pnas.0538054100.
To read the article "The structural basis for biphasic kinetics
in the folding of the formin-binding protein WW domain: Lessons
for protein design?" by John Karanicolas and Charles L. Brooks
III, please see: http://www.pnas.org/cgi/doi/10.1073/pnas.0731771100.
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