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 alpha-subunit of tryptophan synthase, a (beta/alpha)(8) TIM barrel protein. The molecular basis for the formation of this off-pathway misfolded intermediate, I-BP, 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 I-BP 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 externalto-the-barrel and spanning the N and C termini (cluster 2) implies tightpacking at most sites in both intermediates. Differential effects on I-BP and I1 for replacements in alpha 3, beta(4) and alpha 8 at the boundaries of cluster 2 suggest that their incorporation into I1 but not I-BP reflects non-native folds at the edges of the crucial (beta/alpha)(1-2)O-3 core in I-BP. The retention of I-BP and the smaller and consistent destabilization of both I-BP and I1 by similar replacements in an internal-to-the-barrel ILV cluster (cluster 1) and a second external-to-thebarrel ILV cluster (cluster 3) imply molten globule-like packing. The tight packing inferred, in part, for I-BP 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 class of proteins. (c) 2006 Elsevier Ltd. All rights reserved.