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
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