Redesign of Artificial Globins: Effects of Residue Replacements at Hydrophobic Sites on the Structural Properties
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- 作者：Yasuhiro Isogai ; Anna Ishii ; Tetsuro Fujisawa ; Motonori Ota ; and Ken Nishikawa
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：May 16, 2000
- 年：2000
- 卷：39
- 期：19
- 页码：5683 - 5690
- 全文大小：451K
- 年卷期：v.39,no.19(May 16, 2000)
- ISSN：1520-4995
文摘
Artificial sequences of the 153 amino acids have been designed to fit the main-chain frameworkof the sperm whale myoglobin (Mb) structure based on a knowledge-based 3D-1D compatibility method.The previously designed artificial globin (DG1) folded into a monomeric, compact, highly helical andglobular form with overall dimensions similar to those of the target structure, but it lacked structuraluniqueness at the side-chain level [Isogai, Y., Ota, M., Fujisawa, T., Izuno, H., Mukai, M., Nakamura,H., Iizuka, T., and Nishikawa, K. (1999) Biochemistry 38, 7431-7443]. In this study, we redesignedhydrophobic sites of DG1 to improve the structural specificity. Several Leu and Met residues in DG1were replaced with 
-branched amino acids, Ile and Val, referring to the 3D profile of DG1 to producethree redesigned globins, DG2-4. These residue replacements resulted in no significant changes of theircompactness and 
-helical contents in the absence of denaturant, whereas they significantly affected thedependence of the secondary structure on the concentration of guanidine hydrochloride. The analyses ofthe denaturation curves revealed higher global stabilities of the designed globins than that of naturalapoMb. Among DG1-4, DG3, in which 11 Leu residues of DG1 are replaced with seven Ile and fourVal residues, and one Met residue is replaced with Val, displayed the lowest stability but the mostcooperative folding-unfolding transition and the most dispersed NMR spectrum with the smallest linewidth. The present results indicate that the replacements of Leu (Met) with the -branched amino acidsat appropriate sites reduce the freedom of side-chain conformation and improve the structural specificityat the expense of stability.
-branched amino acids, Ile and Val, referring to the 3D profile of DG1 to producethree redesigned globins, DG2-4. These residue replacements resulted in no significant changes of theircompactness and -helical contents in the absence of denaturant, whereas they significantly affected thedependence of the secondary structure on the concentration of guanidine hydrochloride. The analyses ofthe denaturation curves revealed higher global stabilities of the designed globins than that of naturalapoMb. Among DG1-4, DG3, in which 11 Leu residues of DG1 are replaced with seven Ile and fourVal residues, and one Met residue is replaced with Val, displayed the lowest stability but the mostcooperative folding-unfolding transition and the most dispersed NMR spectrum with the smallest linewidth. The present results indicate that the replacements of Leu (Met) with the -branched amino acidsat appropriate sites reduce the freedom of side-chain conformation and improve the structural specificityat the expense of stability.