Design and study of Trp-cage miniproteins.

摘要

TC5b is a 20-residue peptide (NLYIQ WLKDG GPSSG RPPPS) that was the result of a design process involving the truncation and mutation of a 39-residue peptide, exendin-4, from Gila monster saliva. TC5b was >95% folded in water and displayed protein-like two-state folding behavior with a Tm of 42°C. This peptide adopted a novel fold, which was designated as the 'Trp-cage' motif. This work describes the detailed studies carried out to test the various hypotheses regarding the stabilizing features of this Trp-cage fold. This was done by making additional mutations in the TC5b sequence; more stable mutant with Tm's as high as 65°C were obtained. These studies provided information about the relative contributions of the various fold stabilizing interactions. The folding of these Trp-cages is hydrophobically driven, with the Trp6 side chain at the center of the hydrophobic core, surrounded by Tyr3, Leu7, and Pro on one side and by Pro18, 19 on the other side. The interactions of Pro 19, presumably with both Trp6 and Tyr3, and a buried H-bonding network including Ser14, were particularly important for fold stabilization. An additional goal was to test whether shorter Trp-cage sequences would still fold yielding the same hydrophobic core. These studies also provided information regarding the folding pathway and potential folding intermediates of the Trp-cages and the effects of local and secondary structural features on the folding scenario. The folding kinetics of some representative Trp-cage constructs were determined using the NMR lineshape analysis method. These experiments indicated that the Trp-cages fold at near 106 s-1 rates with Arrhenius plots that are in other respects, analogous to globular proteins.