Structural Selection of a Native Fold by Peptide Recognition. Insights into the Thioredoxin Folding Mechanism

摘要

Thioredoxins (TRXs) are monomeric α/βproteins with a fold characterized by a central twistednu0001-sheet surrounded by α-helical elements. The interaction of the C-terminal -helix 5 of TRX against thenremainder of the protein involves a close packing of hydrophobic surfaces, offering the opportunity ofnstudying a fine-tuned molecular recognition phenomenon with long-range consequences on the acquisitionnof tertiary structure. In this work, we focus on the significance of interactions involving residues L94,nL99, E101, F102, L103 and L107 on the formation of the noncovalent complex between reduced TRX1 93nand TRX94 108. The conformational status of the system was assessed experimentally by circularndichroism, intrinsic fluorescence emission and enzymic activity; and theoretically by molecular dynamicsnsimulations (MDS). Alterations in tertiary structure of the complexes, resulting as a consequence of sitenspecific mutation, were also examined. To distinguish the effect of alanine scanning mutagenesis onnsecondary structure stability, the intrinsic helix-forming ability of the mutant peptides was monitorednexperimentally by far-UV CD spectroscopy upon the addition of 2,2,2-trifluoroethanol, and also theoreticallynby Monte Carlo conformational search and MDS. This evidence suggests a key role of residues L99,nF102 and L103 on the stabilization of the secondary structure of α-helix 5, and on the acquisition ofntertiary structure upon complex formation. We hypothesize that the transition between a partially foldednand a native-like conformation of reduced TRX1 93 would fundamentally depend on the consolidationnof a cooperative tertiary unit based on the interaction between α-helix 3 and α-helix 5.