Free-energy Theory Borne Out In Large-scale Protein Folding

The interdisciplinary research appears this week in two back-to-backpapers in the Proceedings of the National Academy of Sciences.

"Researchers have successfully combined computer modeling andexperimental results in folding studies for small proteins, but this isthe first effective combination for a large, multi-domain protein,"said study co-author Kathleen Matthews, Dean of the Wiess School ofNatural Sciences and Stewart Memorial Professor of Biochemistry."Pioneering efforts were required to establish comparable experimentaland theoretical data, and the method worked remarkably well. We expectothers to adopt it in their own studies."

Proteins are the workhorses of biology. At any given time, eachcell in our bodies contains 10,000 or more of them. Each of theseproteins is a chain of amino acids strung end-to-end like beads innecklace. For longer proteins, the chain can contain hundreds of aminoacids.

Thanks to modern genomics, scientists can use DNA to decipherthe amino acid sequence in a protein. But knowing the sequence gives noclue to the protein's function, because function is inextricably tiedto shape, and every protein self-assembles into its characteristicshape within seconds of being created.

"The folded, functional form of the protein is what reallymatters, and our interest is in creating a folding roadmap of sorts, aplot of the thermodynamic route that the protein follows as it movestoward equilibrium," said co-author Cecilia Clementi, the NormanHackerman-Welch Young Investigator Assistant Professor of Chemistry.

The Rice research team included Clementi, Clementi's graduatestudent Payel Das, experimentalist Pernilla Wittung-Stafshede,associate professor of biochemistry and cell biology, Matthews andgraduate student Corey Wilson of biochemistry and cell biology.

"We know that misfolded proteins play a key but mysterious rolein Alzheimer's, Parkinson's, diabetes and a host of other diseases, somapping the normal route a protein takes � and finding the off-rampsthat might lead to misfolding � are vitally important,"Wittung-Stafshede said.

Rice's studies were carried out on monomeric lactose repressorprotein, or MLAc, a variant of the protein used by E. coli to regulateexpression of the proteins that transport and metabolize lactose. MLAccontains about 360 amino acids.

While scientists know proteins containing 100 or fewer aminoacids fold in a very cooperative (all-or-none) fashion, it is believedthat larger proteins fold through the formation of partially foldedintermediate structures before settling into their final state.

Simulating large-scale protein folding is too complex for eventhe most powerful supercomputer. In developing a theoretical approachthat allows studying protein folding on a computer, Clementi and Dasrelied on the techniques of statistical mechanics, building up anoverall picture of MLAc folding based upon statistical approximationsof molecular events.

On the experimental side, Wittung-Stafshede, Matthews andWilson prepared samples of MLAc and added urea to cause them to unfold.The team then injected water into the solution very fast, diluting themixture and causing the proteins to fold. Using spectroscopy, theycaptured fluorescence and ultraviolet polarization patterns given offby the proteins as they folded.

"The novelty of this work is the direct and quantitativecomparison of the time-dependent simulation data with the experimentalmeasurements from circular dichroism and tryptophan fluorescence," Dassaid. "The excellent agreement between experiment and theoryillustrates that the existence of a well-defined "folding route", atleast for large proteins, can be predicted within the framework offree-energy landscape theory. This has been a very controversial issuein the field of protein folding."

Study co-authors also included Giovanni Fossati, assistantprofessor of physics and astronomy, who helped the team analyze andinterpret the simulation data.

The research was funded bythe National Science Foundation, the National Institutes of Health, theTexas Advanced Technology Program and the Welch Foundation.