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

Abstract

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.

Modular Control of Cross-oligomerization

“…S2). Protein folding studies indicate that clusters of these three aliphatic amino acids form strong associations capable of directing folding pathways (25,26). The aliphatic patch at the C-domain dimer interface is consistent with the critical role of subunit association in the function of the Hsp90 and our observations that subunit dissociation does not appear to be required for essential Hsp90 function.…”

Folding of the RNA Recognition Motif (RRM) Domains of the Amyotrophic Lateral Sclerosis (ALS)-linked Protein TDP-43 Reveals an Intermediate State

“…Hydrogen exchange experiments on the TIM barrel protein family have shown that clusters of isoleucine, leucine, and valine residues can accurately predict cores of stability (67) and early folding events (68,69). Using an in-house algorithm (see the Clusters of Branched Aliphatic Side Chains in Globular Proteins (BASiC) Web site) (70,71)), clusters of the branched aliphatic side chain residues in RRM1 (Fig.…”

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

“…Its conservation might reflect the (βα) n -repeat architecture of these barrels and, thereby, the opportunity for local and rapid folding reactions to forms of sufficient stability to be transiently populated during folding. As has been previously speculated, 51 this structure may not be able to propagate to the remainder of the sequence because it adopts a non-native local fold or because its edges adopt a non-native fold that enhances its interactions with solvent before the propagation reaction can occur.…”

“…In both cases, the protection patterns correlate with the presence of large hydrophobic clusters dominated by the branched aliphatic side chains from isoleucine, leucine and valine residues. 24,51 Thus, as the sequences of these (βα) 8 barrels evolved, the location of the structured regions defining the equilibrium intermediate moved with the location of the ILV-dominated hydrophobic clusters.…”