Thermodynamic and structural characterization of Asn and Ala residues in the disallowed II ' region of the Ramachandran plot

Residue Asn47 at position L1 of a type II' beta -turn of the alpha -spectri n SH3 domain is located in a disallowed region of the Ramachandran plot (ph i = 56 +/- 12, psi = - 118 +/- 17). Therefore, it is expected that replacem ent of Asn47 by Gly should result in a considerable stabilization of the pr otein. Thermodynamic analysis of the N47G and N47A mutants shows that the c hange in free energy is small (similar to0.7 kcal/mol: similar to3 kJ/mol) and comparable to that found when mutating a Gly to Ala in a alpha -helix o r beta -sheet. X-ray structural analysis of these mutants shows that the co nformation of the beta -turn does not change upon mutation and, therefore, that there is no relaxation of the structure, nor is there any gain or loss of interactions that could explain the small energy change. Our results in dicate that the energetic definition of II' region of the Ramachandran plot (phi = 60 +/- 30, psi = -115 +/- 15) should be revised for at least Ala an d Asn in structure validation and protein design.