Enhancing the activity of a beta-helical antifreeze protein by the engineered addition of coils.
The effectiveness of natural antifreeze proteins in inhibiting the growth of a seed ice crystal seems to vary with protein size. Here we have made use of the extreme regularity of the beta-helical antifreeze protein from the beetle Tenebrio molitor to explore systematically the relationship between antifreeze activity and the area of the ice-binding site. Each of the 12-amino acid, disulfide-bonded central coils of the beta-helix contains a Thr-Xaa-Thr ice-binding motif. By adding coils to, and deleting coils from, the seven-coil parent antifreeze protein, we have made a series of constructs with 6-11 coils. Misfolded forms of these antifreezes were removed by ice affinity purification to accurately compare the specific activity 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 degrees at 0.7 mg/mL, but actually decreased for the 10- and 11-coil constructs. This small loss in activity might result from the accumulation of a slight mismatch between the spacing of the ice-binding threonine residues and the O atoms of the ice lattice.
Marshall
Christopher B
CB
Department of Biochemistry and Protein Engineering Network Centres of Excellence, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Daley
Margaret E
ME
Sykes
Brian D
BD
Davies
Peter L
PL
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D007053:Ice; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013696:Temperature; D013712:Tenebrio / Q000737:chemistry*
D021301:Antifreeze Proteins; D007053:Ice; D020033:Protein Isoforms