Probing the Lower Size Limit for Protein-Like Fold Stability: Ten-Residue Microproteins With Specific, Rigid Structures in Water

Abstract

Mutational optimization of two long-range interactions first observed in Ac-WINGKWT-NH₂, (a) bifurcated H-bonding involving the threonine amide H_N and side chain OH and the N-terminal acetyl carbonyl and (b) an H-bond between the entgegen-H_N of the C-terminal amide and the indole ring of Trp6 that stabilizes a face-to-edge indole/indole interaction between Trp1 and Trp6, has afforded ≤10 residue systems that yield a remarkably stable fold in water. Optimization was achieved by designing a hydrophobic cluster that sequesters these H-bonds from solvent exposure. The structures and extent of amide H/D exchange protection for CH₃CH₂CO-WIpGXWTGPS (p = d-Pro, X = Leu or Ile) were determined. These two systems are greater than 94% folded at 298 K (97.5% at 280 K) with melting temperatures >75 °C. The fold appears to display minimal fluxionality; a well-converged NMR structure rationalizes all of the large structuring shifts observed, and we suggest that these designed constructs can be viewed as microproteins.