[excerpt] New method allows study of protein folding under native conditions
A new method to probe the forces that control higher order structure in proteins under native conditions—that is, structure-promoting conditions for the protein—has been developed by chemists at the University of Wisconsin, Madison.
To evaluate such forces, scientists typically use a spectroscopic method to monitor changes in structure when the protein is treated with heat or a chemical agent such as guanidinium chloride. But these procedures can give an incomplete picture because a protein’s folded and unfolded states under native and denaturing conditions may be different.
For examining protein folding under native conditions, the method developed by Matthew G. Woll and Samuel H. Gellman may be particularly useful [J. Am. Chem. Soc., published online Aug. 19, http://dx.doi.org/10.1021/ja046891i].
The method is based on replacing a backbone amide of a protein with a thioester linkage. When this backbone-modified protein is mixed with a thiol-containing small molecule (red), a thiol/thioester exchange (shown) occurs rapidly at room temperature and neutral pH. The process can be monitored by high-performance liquid chromatography. The equilibrium constant for the exchange can be used to estimate the protein’s folding thermodynamics under native conditions, Gellman says.
The researchers demonstrate the method using bovine pancreatic polypeptide, a 36-residue protein with a well-defined tertiary structure. They say the approach provides information that is “complementary to that available from thermal and chemical denaturation studies.”
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