Electrostatics significantly affect the stability of designed homeodomain variants.

The role of electrostatic interactions in determining the stability of designed proteins was studied by constructing and analyzing a set of designed variants of the Drosophila engrailed homeodomain. Computational redesign of 29 surface positions results in a 25-fold mutant with moderate stability, similar to the wild-type protein. Incorporating helix dipole and N-capping considerations into the design algorithm by restricting amino acid composition at the helix termini and N-capping positions yields a ninefold mutant of the initial design (a 23-fold mutant of wild-type) that is over 3 kcal mol(-1) more stable than the protein resulting from the unbiased design. Four additional proteins were constructed and analyzed to isolate the effects of helix dipole and N-capping interactions in each helix. Based on the results of urea-denaturation experiments and calculations using the finite difference Poisson-Boltzmann method, both classes of interaction are found to increase the stability of the designed proteins significantly. The simple electrostatic model used in the optimization of rotamers by iterative techniques (ORBIT) force-field, which is similar to the electrostatic models used in other protein design force-fields, is unable to predict the experimentally determined stabilities of the designed variants. The helix dipole and N-capping restrictions provide a simple but effective method to incorporate two types of electrostatic interactions that impact protein stability significantly. Copyright 2001 Elsevier Science Ltd.