Structural analysis of a beta-helical protein motif stabilized by targeted replacements with conformationally constrained amino acids.

Here we study conformational stabilization induced in a beta-helical nanostructure by position-specific mutations. The nanostructure is constructed through the self-assembly of the beta-helical building block excised from E. coli galactoside acetyltransferase (PDB code 1krr , chain A; residues 131-165). The mutations involve substitutions by cyclic, conformationally constrained amino acids. Specifically, a complete structural analysis of the Pro-Xaa-Val sequence [with Xaa being Gly, Ac 3c (1-aminocyclopropane-1-carboxylic acid) and Ac 5c (1-aminocyclopentane-1-carboxylic acid)], corresponding to the 148-150 loop region in the wild-type (Gly) and mutated (Ac 3c and Ac 5c) 1krr , has been performed using Molecular Dynamics simulations and X-ray crystallography. Simulations have been performed for the wild-type and mutants of three different systems, namely the building block, the nanoconstruct and the isolated Pro-Xaa-Val tripeptide. Furthermore, the crystalline structures of five peptides of Pro-Xaa-Val or Xaa-Val sequences have been solved by X-ray diffraction analysis and compared with theoretical predictions. Both the theoretical and crystallographic studies indicate that the Pro-Ac n c-Val sequences exhibit a high propensity to adopt turn-like conformations, and this propensity is little affected by the chemical environment. Overall, the results indicate that replacement of Gly149 by Ac 3c or Ac 5c significantly reduce the conformational flexibility of the target site enhancing the structural specificity of the building block and the nanoconstruct derived from the 1krr beta-helical motif.