Redesign of artificial globins: Effects of residue replacements at hydrophobic sites on the structural properties

Artificial sequences of the 153 amino acids have been designed to fit the m ain-chain framework of the sperm whale myoglobin (Mb) structure based on a knowledge-based 3D-1D compatibility method. The previously designed artific ial globin (DG1) folded into a monomeric, compact, highly helical and globu lar form with overall dimensions similar to those of the target structure, but it lacked structural uniqueness at the side-chain level [Isogai, Y., Ot a, M., Fujisawa, T., Izuno, H., Mukai, hi., Nakamura, H., Iizuka, T., and N ishikawa, K. (1999) Biochemistry, 38, 7431-7443]. In this study, we redesig ned hydrophobic sites of DG1 to improve the structural specificity. Several Leu and Met residues in DG1 were replaced with beta-branched amino acids, lie and Val, referring to the 3D profile of DG1 to produce three redesigned globins, DG2-4. These residue replacements resulted in no significant chan ges of their compactness and alpha-helical contents in the absence of denat urant, whereas they significantly affected the dependence of the secondary structure on the concentration of guanidine hydrochloride. The analyses of the denaturation curves revealed higher global stabilities of the designed globins than that of natural apoMb. Among DG1-4, DG3, in which 11 Leu resid ues of DG1 are replaced with seven lie and four Val residues, and one Met: residue is replaced with Val, displayed the lowest stability but the most c ooperative folding-unfolding transition and the most dispersed NMR spectrum with the smallest line width. The present results indicate that the replac ements of Leu (Met) with the beta-branched amino acids at appropriate sites reduce the freedom of side-chain conformation and improve the structural s pecificity at the expense of stability.