Physical-organic molecular biology: pathway and stability of protein folding

Protein engineering, the design and synthesis of novel proteins by genetic engineering, allows complex problems in molecular biology to be studied by structure-activity relationships in an analogous manner to the application of physical-organic chemistry to simple organic molecules. This approach has been applied to study the folding pathway and stability of barnase, the RNAse from Bacillus amyloliquefaciens. The strategy is two fold: i , Mutations are made which remove defined interactions that stabilise barnase. The changes in stability on mutation are measured from the changes in free energy of unfolding of the protein. In this way a library of interaction energies is achieved. ii, Kinetic measurements are made on the folding and unfolding of wild-type and mutant proteins. Combination of kinetic and thermodynamic data enables the roles of side chains in the stabilization of the final, transition and intermediate states of the protein to be measured. This gives the order of events during protein folding. The protein engineering experiments are corroborated by N M R studies of hydrogen exchange during the folding process. The combined procedures show that the folding is a multiphasic process in which secondary structure is formed very early on. Formation of the hydrophobic core by docking helix and sheet is (partly) rate determining.