| Understanding how the amino acid sequence of a protein determines its three dimensional structure is one of the outstanding questions in biology today. Finding the answer will greatly improve our grasp on the self-assembly of molecular complexes, aid the interpretation of the proliferating genome sequence data, and unveil the mystery of protein misfolding diseases. Although some progress has been made in the ab initio folding of proteins using solely computer programs, experimental studies are crucial for the continuous testing and refinement of computational methods.; This work describes the elucidation of the folding mechanism of the src SH3 domain, a small independently folding domain. Our strategy has been to characterize all the states along the folding reaction coordinate of this protein in order to understand how the polypeptide chain limits the conformational freedom of the denatured ensemble to reach the unique folded state. First, the kinetic and thermodynamic parameters of the native state were determined to reveal its stability and the extent of local fluctuations around the folded conformation. Second, using point mutagenesis and covalent modifications we have examined the extent of structure formation at the folding transition state. We have determined that at the rate-limiting step parts of the src SH3 domain are well ordered, while others are still quite flexible. This suggests the existence of a well defined hierarchy of structure accumulation during folding, rather than a multitude of routes all of which can lead to the folded state.; Although different proteins are likely to fold by different mechanisms, our studies, together with work on other model proteins, have made apparent the general rules governing the folding process. Native state topology seems to be the primary determinant of the folding mechanism of a protein, while the details in the specific interactions are not as important. For example, distant homologs of the src SH3 domain have a very similar transition state despite the low sequence conservation and the only way to change the folding mechanism of the src SH3 domain is by changing its topology, as in circularization. |
