RESUMEN
The design of beta-sheet proteins is still a challenge in the field of de novo protein design. Here, we have tested the validity of automatic design methods to create and/or improve beta-sheet peptides and proteins. We chose Betanova, a three-stranded beta-sheet peptide, as target system, and, as an automatic design tool, a protein design algorithm called PERLA (protein engineering rotamer library algorithm). PERLA was used to define both stabilising and destabilising single- and multiple-residue mutations of Betanova. Conformational analysis by NMR spectroscopy and far-UV circular dichroism (CD) allowed us to evaluate population differences among the set of designed peptides. Some of the new mutants are approximately 1 kcal/mol more stable than the wild-type peptide. Comparison of the scale of predicted and observed stabilities demonstrates that they are in good agreement for most peptides studied. Our results show that automatic design algorithms can be successfully applied to the design of beta-sheet peptides.
Asunto(s)
Péptidos/química , Ingeniería de Proteínas/métodos , Proteínas/química , Algoritmos , Secuencia de Aminoácidos , Dicroismo Circular , Diseño de Fármacos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Valor Predictivo de las Pruebas , Conformación Proteica , Proteínas/genéticaRESUMEN
Current knowledge about the determinants of beta-sheet formation has been notably improved by the structural and kinetic analysis of model peptides, by mutagenesis experiments in proteins and by the statistical analysis of the protein structure database (Protein Data Bank; PDB). In the past year, several peptides comprising natural and non-natural amino acids have been designed to fold as monomeric three-stranded beta-sheets. In all these cases, the design strategy has involved both the statistical analysis of the protein structure database and empirical information obtained in model beta-hairpin systems and in proteins. Only in one case was rotamer analysis performed to check for the compatibility of the sidechain packing. It is foreseeable that, in future designs, algorithms exploring the sequence and conformational space will be employed. For the design of small proteins (less than 30 amino acids), questions remain about the demonstration of two-state behavior, the formation of a well-defined network of mainchain hydrogen bonds and the quantification of the structured populations.