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"The consistency is very viscous," said Wittung-Stafshede. "It's
something like Jell-O or the freeway at rush hour."
The study, which was co-authored by UH physicist Margaret Cheung, is
available online and slated to appear in the Nov. 27 issue of the
Proceedings of the National Academies of Science.
"Our simulations pinpointed specific places in the protein's structure
where compaction was occurring and secondary structures improved,"
Cheung said. "This offers the first observed evidence - both in silico
and in vitro - for structural effects on proteins in the native state."
To find out how crowded environments affect the stability, structure
and folding of proteins, Wittung-Stafshede and Rice graduate student
Loren Stagg set up a series of biophysical experiments involving the
protein apoflavodoxin. This is an excellent model system because it is
well-characterized in dilute conditions and can be made in the lab.
Using sucrose-based polymers (inert synthetic mimics of real
macromolecules), the pair created several test environments designed
to mimic the gooey milieu that proteins experience inside a cell.
Using spectroscopic methods, Stagg and Wittung-Stafshede then probed
how the structural content as well as the thermal stability of
apoflavodoxin changed as a function of added crowding agents.
At UH, Cheung and graduate student Shao-Qing Zhang used sophisticated
computer simulations in a parallel set of tests. In the computer
simulations, crowding was mimicked by solid spheres of the same size
as the inert polymers used in the test tubes. In the end, the results
from the lab and the computer on the same protein matched almost
perfectly, lending weight to the final report.
The researchers found the protein's native state becomes more compact
and more ordered. The secondary structure of the folded protein
increased by as much as 25 percent based on circular dichroism data.
�From the simulations, it is evident that these changes occur in the
ends of the helices and in the core, where the peptide chain packs
better," Cheung said. Also, the unfolded state becomes more compact,
as predicted by excluded volume theory. These effects on the folded
and unfolded states made the native state of the protein 20 degrees
Celsius more resistant to thermal perturbations.
Wittung-Stafshede said the group is following up with similar in vitro
studies of several other proteins. The flavodoxin results and
preliminary evidence from follow-up studies indicate that the native
state of proteins - the form they take when they are carrying out
their normal functions inside living cells - may be markedly different
from the folded state that scientists most often study in the lab.
"Most lab experiments are done with purified proteins in dilute
buffers," Wittung-Stafshede said. "In those conditions, the protein
has more space to move around in than it would in its native
environment. Our findings may have serious implications for the
folding processes of proteins in cells and the structures of enzyme
active sites in vivo. We are now beginning to assess the magnitude of
these issues in the lab."
Proteins are the workhorses of biology, and their form and function
are intertwined. Proteins are chains of amino acids strung end-to-end
like beads on a necklace. The order comes from DNA blueprints, but
proteins fold into a 3-D shape as soon as the chain is complete, and
scientists can determine a protein's function only by studying its
folded shape. It is still an open question how a long floppy chain of
amino acids is programmed to adopt a unique 3-D shape in a timely
manner (often seconds to minutes).
The science of protein folding has grown dramatically in the past
decade, due in part to the discovery that misfolded proteins play key
roles in diseases like Alzheimer's and Parkinson's.
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