title: 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.
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 (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.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wu
authors.forename: Ramakrishna
authors.initials: R
authors.lastname: Vadrevu
authors.forename: Sagar
authors.initials: S
authors.lastname: Kathuria
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Yang
authors.forename: C Robert
authors.initials: CR
authors.lastname: Matthews
mesh_terms: D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D004795:Enzyme Stability; D014644:Genetic Variation; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D013696:Temperature; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
chemical_list: D021122:Protein Subunits; D014508:Urea; D014367:Tryptophan Synthase; D000409:Alanine