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 (β/α) 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 external-to-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 α3, β 4 and α 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 (β/α) 1-2 β 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-the-barrel 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. Tightlypacked 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.

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The role of hither-to-fore unrecognized long-range hydrogen bonds between main-chain amide hydrogens and polar side chains on the stability of a well-studied (ba) 8 , TIM barrel protein, the alpha subunit of tryptophan synthase (aTS), was probed by mutational analysis. The F19-D46 and I97-D124 hydrogen bonds link the N terminus of a b-strand with the C terminus of the succeeding antiparallel ahelix, and the A103-D130 hydrogen bond links the N terminus of an a-helix with the C terminus of the succeeding antiparallel b-strand, forming clamps for the respective ba or ab hairpins. The individual replacement of these aspartic acid side chains with alanine leads to what appear to be closely related partially folded structures with significantly reduced far-UV CD ellipticity and thermodynamic stability. Comparisons with the effects of eliminating another main-chain-side-chain hydrogen bond, G26-S33, and two electrostatic side-chain-side-chain hydrogen bonds, D38-H92 and D112-H146, all in the same N-terminal folding unit of aTS, demonstrated a unique role for the clamp interactions in stabilizing the native barrel conformation. Because neither the asparagine nor glutamic acid variant at position 46 can completely reproduce the spectroscopic, thermodynamic, or kinetic folding properties of aspartic acid, both size and charge are crucial to its unique role in the clamp hydrogen bond. Kinetic studies suggest that the three clamp hydrogen bonds act in concert to stabilize the transition state leading to the fully folded TIM barrel motif.Keywords: circular dichroism; protein folding; site-directed mutagenesis; thermodynamic and kinetic mechanisms; ba and ab hairpins Side chains stabilize the native, functional conformations of proteins by van der Waals interactions between nonpolar partners, hydrogen bonds (H-bonds) between polar donors and acceptors, and electrostatic interactions between opposing charged groups. Mutational analysis of buried nonpolar side chains has shown that replacements with alanine can lead to destabilizations of up to 7 kcal mol À1 (Shortle et al. 1990;Eriksson et al. 1992;Serrano et al. 1992). The magnitudes of the effects correlate with the local packing density (Shortle et al. 1990) and the burial of nonpolar surface area (Jackson et al. 1993

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