Folding dynamics and pathways of the trp-cage miniproteins.

Using alternate measures of fold stability for a wide variety of Trp-cage mutants has raised the possibility that prior dynamics T-jump measures may not be reporting on complete cage formation for some species. NMR relaxation studies using probes that only achieve large chemical shift difference from unfolded values on complete cage formation indicate slower folding in some but not all cases. Fourteen species have been examined, with cage formation time constants (1/kF) ranging from 0.9-7.5 µs at 300 K. The present study does not change the status of the Trp-cage as a fast folding, essentially two-state system, although it does alter the stage at which this description applies. A diversity of prestructuring events, depending on the specific analogue examined, may appear in the folding scenario, but in all cases, formation of the N-terminal helix is complete either at or before the cage-formation transition state. In contrast, the fold-stabilizing H-bonding interactions of the buried Ser14 side chain and the Arg/Asp salt bridge are post-transition state features on the folding pathway. The study has also found instances in which a [P12W] mutation is fold destabilizing but still serves to accelerate the folding process.

Optimal salt bridge for Trp-cage stabilization.

Gai and co-workers [Bunagan, M. R., et al. (2006) J. Phys. Chem. B 110, 3759-3763] reported computational design studies suggesting that a D9E mutation would stabilize the Trp-cage. Experimental studies for this mutation were reported in 2008 [Hudaky, P., et al. (2008) Biochemistry 47, 1007-1016]; the authors suggested that [D9E]-TC5b presented a more compact and melting resistant structure because of the "optimal distance between the two sides of the molecule". Nonetheless, the authors reported essentially the same circular dichroism (CD) melting temperature, 38 ± 0.3 °C, for TC5b and its [D9E] mutant. In this study, a more stable Trp-cage, DAYAQ WLKDG GPSSG RPPPS, was examined by nuclear magnetic resonance and CD with the following mutations: [D9E], [D9R,R16E], [R16O], [D9E,R16O], [R16K], and [D9E,R16K]. Of these, the [D9E] mutant displayed the smallest acidification-induced change in the apparent T(m). In analogy to the prior study, the CD melts of TC10b and its [D9E] mutant were, however, very similar; all of the other mutations were significantly fold destabilizing by all measures. A detailed analysis indicates that the original D9-R16 salt bridge is optimal with regard to fold cooperativity and fold stabilization. Evidence of salt bridge formation is also provided for a swapped pair, the [D9R,R16E] mutant. Model systems reveal that an ionized aspartate at the C-terminus of a helix significantly decreases intrinsic helicity, a requirement for Trp-cage fold stability. The CD evidence that was cited as supporting increased fold stability for [D9E]-TC5b at higher temperatures appears to be a reflection of increased helix stability in both the folded and unfolded states rather than a more favorable salt bridge. Our study also provides evidence of other Trp-cage stabilizing roles of the R16 side chain.

Hyperstable miniproteins: additive effects of D- and L-Ala mutations.

The folding enantioselectivity for D-Ala versus L-Ala at one glycine site in the Trp-cage is 16 kJ mol(-1); judicious introductions of alanines of the correct chirality raises the melting temperature of this 20-residue fold to 83 degrees C.