A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the alpha subunit of tryptophan synthase, a TIM barrel protein
Biochemistry & Molecular Pharmacology
Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology
Medical Subject Headings
Alanine; Amino Acid Substitution; Circular Dichroism; Enzyme Stability; Hydrogen Bonding; Hydrogen-Ion Concentration; Hydrophobicity; Kinetics; Mutagenesis, Site-Directed; Protein Conformation; Protein Denaturation; *Protein Folding; Protein Renaturation; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Salmonella typhimurium; Temperature; Thermodynamics; Tryptophan Synthase; Urea; Variation (Genetics)
Life Sciences | Medicine and Health Sciences
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 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 I(BP) and I1 for replacements in alpha3, beta4 and alpha8 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)beta(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. 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 (beta/alpha)(n) class of proteins.
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Citation: J Mol Biol. 2007 Mar 9;366(5):1624-38. Epub 2006 Dec 15. Link to article on publisher's site
DOI of Published Version
Journal of molecular biology
Wu, Ying; Vadrevu, Ramakrishna; Kathuria, Sagar V.; Yang, Xiaoyan; and Matthews, C. Robert, "A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the alpha subunit of tryptophan synthase, a TIM barrel protein" (2007). GSBS Student Publications. 513.