A Tightly Packed Hydrophobic Cluster Directs the Formation of an Off-pathway Sub-millisecond Folding Intermediate in the α Subunit of Tryptophan Synthase, a TIM Barrel Protein

Protein misfolding is now recognized as playing a crucial role in both normal and pathogenic folding reactions. An interesting example of misfolding at the earliest state of a natural folding reaction is provided by the α-subunit of tryptophan synthase, a (β/α)8 TIM barrel protein. The molecular basis for the formation of this off-pathway misfolded intermediate, IBP, and a subsequent on-pathway intermediate, I1, was probed by mutational analysis of 20 branched aliphatic side-chains distributed throughout the sequence. The elimination of IBP and the substantial destabilization of I1 by replacement of a selective set of the isoleucine, leucine or valine residues (ILV) with alanine in a large ILV cluster external-to-the-barrel and spanning the N and C termini (cluster 2) implies tight-packing at most sites in both intermediates. Differential effects on IBP and I1 for replacements in α3, β4 and α8 at the boundaries of cluster 2 suggest that their incorporation into I1 but not IBP reflects non-native folds at the edges of the crucial (β/α)1–2β3 core in IBP. The retention of IBP and the smaller and consistent destabilization of both IBP and I1 by similar replacements in an internal-to-the-barrel ILV cluster (cluster 1) and a second external-to-the-barrel ILV cluster (cluster 3) imply molten globule-like packing. The tight packing inferred, in part, for IBP or for all of I1 in cluster 2, but not in clusters 1 and 3, may reflect the larger size of cluster 2 and/or the enhanced number of isoleucine, leucine and valine self-contacts in and between contiguous elements of secondary structure. Tightly packed ILV-dominated hydrophobic clusters could serve as an important driving force for the earliest events in the folding and misfolding of the TIM barrel and other members of the (β/α)n class of proteins.