Enhancing the Activity of a -Helical Antifreeze Protein by the Engineered Addition of Coils
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- 作者：Christopher B. Marshall ; Margaret E. Daley ; Brian D. Sykes ; and Peter L. Davies
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：September 21, 2004
- 年：2004
- 卷：43
- 期：37
- 页码：11637 - 11646
- 全文大小：405K
- 年卷期：v.43,no.37(September 21, 2004)
- ISSN：1520-4995
文摘
The effectiveness of natural antifreeze proteins in inhibiting the growth of a seed ice crystalseems to vary with protein size. Here we have made use of the extreme regularity of the 
-helical antifreezeprotein from the beetle Tenebrio molitor to explore systematically the relationship between antifreezeactivity and the area of the ice-binding site. Each of the 12-amino acid, disulfide-bonded central coils ofthe -helix contains a Thr-Xaa-Thr ice-binding motif. By adding coils to, and deleting coils from, theseven-coil parent antifreeze protein, we have made a series of constructs with 6-11 coils. Misfoldedforms of these antifreezes were removed by ice affinity purification to accurately compare the specificactivity of each construct. There was a 10-100-fold gain in activity upon going from six to nine coils,depending on the concentration that was compared. Activity was maximal for the nine-coil construct,which gave a freezing point depression of 6.5 C
at 0.7 mg/mL, but actually decreased for the 10- and11-coil constructs. This small loss in activity might result from the accumulation of a slight mismatchbetween the spacing of the ice-binding threonine residues and the O atoms of the ice lattice.
-helical antifreezeprotein from the beetle Tenebrio molitor to explore systematically the relationship between antifreezeactivity and the area of the ice-binding site. Each of the 12-amino acid, disulfide-bonded central coils ofthe -helix contains a Thr-Xaa-Thr ice-binding motif. By adding coils to, and deleting coils from, theseven-coil parent antifreeze protein, we have made a series of constructs with 6-11 coils. Misfoldedforms of these antifreezes were removed by ice affinity purification to accurately compare the specificactivity of each construct. There was a 10-100-fold gain in activity upon going from six to nine coils,depending on the concentration that was compared. Activity was maximal for the nine-coil construct,which gave a freezing point depression of 6.5 C at 0.7 mg/mL, but actually decreased for the 10- and11-coil constructs. This small loss in activity might result from the accumulation of a slight mismatchbetween the spacing of the ice-binding threonine residues and the O atoms of the ice lattice.