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Construction of heme-binding two-α-helix peptides and reguration of binding property by peptide conformation
120
Journal of Inorganic Biochemistry
Abstracts
D43
C O N S T R U C T I O N OF HEME-BINDING T W O - n - H E L I X PEPTIDES AND REGURATION OF BINDING PROPERTY BY PEPTIDE C O N F O R M A T I O N
$, Sakamoto, A. Ueno and H. Mihara Department of Bioengineering,Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology Nagatsuta, Midori-ku, Yokohama 226 (Japan) Iron porphyrins occur widely in nature as cofactors of hemeproteins and display diverse functions, such as oxygen carrier/storage, electron transfer, energy transfer, redox catalyst and transmission of information. So far, the study of artificial protein design has focused considerable effort on the construction of polypeptide 3D structures and conjugation of porphyrin molecules by chelation [ 1] or covalent linkage [2-4] with peptides. Here, we report a variety of originally designed 2o~-helix peptides His2ot-16, I-Iis2o~19 and His2o~-23 (FIGURE 1), which could bind FelII-mesoporphyrin (heme) in a regulated manner. Each peptide segment was designed to take an amphiphilic c~-helix structure in a manner similar to that of coiled-coil proteins. To construct a parallel 2or-helix structure, the two segments were dimerized by the disulfide linkage of Cys residues at C termini with a flexible spacer of 13-Ala. As axial ligands of heme, His residues were introduced at the 6, 9 or 13th position instead of Leu to coordinate a heme inside the hydrophobic region of the 2or-helix structure. The peptides showed the heme-binding properties dependent on trifluoroethanol (TFE) contents. The conformations of His20t-16 and His2o~-19 in a buffer (pH 7.4) were almost random-coil and these peptides could not bind the heme. By the addition of 10-25% TFE, in which His2o~-16 and His2ot-19 took 25-60% and 30-60% o~-helix structure, respectively, the peptide could effectively bind the heme. His2ot-16 and His2o~-19 showed the highest binding constant at around 15% TFE (Ka=5.8xl05 M -1 and 1.9x106 M -1, respectively). The ~x-helicities of His2o~-16 and His2o~-19 were increased upon the binding of heme and the heme changed from high-spin to low-spin forms. These TFE effects revealed that the 2or-helix structures were annealed by TFE and the consequent formation of hydrophobic pocket was important for heme binding. On the other hand, I-Iis2c~-23 also could not bind the heme in the buffer, although the peptide took a 55% a-helix structure. By the addition of 10% TFE, His2c~-23 could bind the heme (Ka=4.9x 105 M-I). In the case of His2o~-23, the hydrophobic interaction between two a-helix segments in the buffer may be so strong that the heme can not enter the hydrophobic pocket of 2o~-helix structure. Thus, for the effective heme binding with His2ot-23, it is necessary that the helix-helix packing is loosened by the addition of TFE. These results indicated that the heme binding ability of the peptides was controlled by the peptide conformation. Furthermore, Ndemethylase activity of heme was greatly enhanced by the binding with the peptide His2o~-16. Presumably, the peptide could enhance the activity by isolating the heme in the peptide 3D structure and form the active intermediate much easily. To study the relationship between the function and the conformation of heme-peptides, we examined the heme catalytic activity of other elongated peptides.
~ ~
"~
N.~.~ ~ ~--~--J--~ ~ ~s--~
~
Ac-ALEQKHAALEQKLA-I3AlaC-NH2 Ao-ALEQKHAALEQKLA-J3Ala~NH2 His2~'16 Ao-ALAALEQKHAALEQKLA-1I3Aal O-NHis2c~-19 H2 Ac-ALAALEQKHAALEQKLA-I3AlaC-NH2
Ac-ALEQKLAALEQKHAALEQKLA-13AIaO-NH2 Ac_ALEQKLAALEQKHAALEQKLA_I3Aal ~2_NH His2a.23
FIGURE 1. Structuresof designed heme-bindingpeptides. 1. 2. 3. 4.
F. Rabanal, W. E DeGrado and P. L. Dutton, J. Am. Chem. Soc., 118,473 (1996). T. Sasaki and E. T. Kaiser, J. Am. Chem. Soc., 111,380 (1989). H. Mihara, Y. Haruta, S. Sakamoto, N. Nishino and H. Aoyagi, Chem. Lett., 1 (1996); H. Mihara, K. Tomizaki,T. Fujimoto, S. Sakamoto, H. Aoyagi and N. Nishino, Chem. Lett., 187 (1996). D.R. Benson, B. R. Hart, X. Zhu and M. B. Doughty,J. Am. Chem. Soc., 117, 8502 (1995).
Journal of Inorganic Biochemistry
Abstracts
D43
C O N S T R U C T I O N OF HEME-BINDING T W O - n - H E L I X PEPTIDES AND REGURATION OF BINDING PROPERTY BY PEPTIDE C O N F O R M A T I O N
$, Sakamoto, A. Ueno and H. Mihara Department of Bioengineering,Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology Nagatsuta, Midori-ku, Yokohama 226 (Japan) Iron porphyrins occur widely in nature as cofactors of hemeproteins and display diverse functions, such as oxygen carrier/storage, electron transfer, energy transfer, redox catalyst and transmission of information. So far, the study of artificial protein design has focused considerable effort on the construction of polypeptide 3D structures and conjugation of porphyrin molecules by chelation [ 1] or covalent linkage [2-4] with peptides. Here, we report a variety of originally designed 2o~-helix peptides His2ot-16, I-Iis2o~19 and His2o~-23 (FIGURE 1), which could bind FelII-mesoporphyrin (heme) in a regulated manner. Each peptide segment was designed to take an amphiphilic c~-helix structure in a manner similar to that of coiled-coil proteins. To construct a parallel 2or-helix structure, the two segments were dimerized by the disulfide linkage of Cys residues at C termini with a flexible spacer of 13-Ala. As axial ligands of heme, His residues were introduced at the 6, 9 or 13th position instead of Leu to coordinate a heme inside the hydrophobic region of the 2or-helix structure. The peptides showed the heme-binding properties dependent on trifluoroethanol (TFE) contents. The conformations of His20t-16 and His2o~-19 in a buffer (pH 7.4) were almost random-coil and these peptides could not bind the heme. By the addition of 10-25% TFE, in which His2o~-16 and His2ot-19 took 25-60% and 30-60% o~-helix structure, respectively, the peptide could effectively bind the heme. His2ot-16 and His2o~-19 showed the highest binding constant at around 15% TFE (Ka=5.8xl05 M -1 and 1.9x106 M -1, respectively). The ~x-helicities of His2o~-16 and His2o~-19 were increased upon the binding of heme and the heme changed from high-spin to low-spin forms. These TFE effects revealed that the 2or-helix structures were annealed by TFE and the consequent formation of hydrophobic pocket was important for heme binding. On the other hand, I-Iis2c~-23 also could not bind the heme in the buffer, although the peptide took a 55% a-helix structure. By the addition of 10% TFE, His2c~-23 could bind the heme (Ka=4.9x 105 M-I). In the case of His2o~-23, the hydrophobic interaction between two a-helix segments in the buffer may be so strong that the heme can not enter the hydrophobic pocket of 2o~-helix structure. Thus, for the effective heme binding with His2ot-23, it is necessary that the helix-helix packing is loosened by the addition of TFE. These results indicated that the heme binding ability of the peptides was controlled by the peptide conformation. Furthermore, Ndemethylase activity of heme was greatly enhanced by the binding with the peptide His2o~-16. Presumably, the peptide could enhance the activity by isolating the heme in the peptide 3D structure and form the active intermediate much easily. To study the relationship between the function and the conformation of heme-peptides, we examined the heme catalytic activity of other elongated peptides.
~ ~
"~
N.~.~ ~ ~--~--J--~ ~ ~s--~
~
Ac-ALEQKHAALEQKLA-I3AlaC-NH2 Ao-ALEQKHAALEQKLA-J3Ala~NH2 His2~'16 Ao-ALAALEQKHAALEQKLA-1I3Aal O-NHis2c~-19 H2 Ac-ALAALEQKHAALEQKLA-I3AlaC-NH2
Ac-ALEQKLAALEQKHAALEQKLA-13AIaO-NH2 Ac_ALEQKLAALEQKHAALEQKLA_I3Aal ~2_NH His2a.23
FIGURE 1. Structuresof designed heme-bindingpeptides. 1. 2. 3. 4.
F. Rabanal, W. E DeGrado and P. L. Dutton, J. Am. Chem. Soc., 118,473 (1996). T. Sasaki and E. T. Kaiser, J. Am. Chem. Soc., 111,380 (1989). H. Mihara, Y. Haruta, S. Sakamoto, N. Nishino and H. Aoyagi, Chem. Lett., 1 (1996); H. Mihara, K. Tomizaki,T. Fujimoto, S. Sakamoto, H. Aoyagi and N. Nishino, Chem. Lett., 187 (1996). D.R. Benson, B. R. Hart, X. Zhu and M. B. Doughty,J. Am. Chem. Soc., 117, 8502 (1995).