Compound ice-binding site of an antifreeze protein revealed by mutagenesis and fluorescent tagging.
By binding to the surface of ice crystals, type III antifreeze protein (AFP) can depress the freezing point of fish blood to below that of freezing seawater. This 7-kDa globular protein is encoded by a multigene family that produces two major isoforms, SP and QAE, which are 55% identical. Disruptive mutations on the ice-binding site of type III AFP lower antifreeze activity but can also change ice crystal morphology. By attaching green fluorescent protein to different mutants and isoforms and by examining the binding of these fusion proteins to single-crystal ice hemispheres, we show that type III AFP has a compound ice-binding site. There are two adjacent, flat, ice-binding surfaces at 150° to each other. One binds the primary prism plane of ice; the other, a pyramidal plane. Steric mutations on the latter surface cause elongation of the ice crystal as primary prism plane binding becomes dominant. SP isoforms naturally have a greatly reduced ability to bind the prism planes of ice. Mutations that make the SP isoforms more QAE-like slow down the rate of ice growth. On the basis of these observations we postulate that other types of AFP also have compound ice-binding sites that enable them to bind to multiple planes of ice.
Garnham
Christopher P
CP
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Natarajan
Aditya
A
Middleton
Adam J
AJ
Kuiper
Mike J
MJ
Braslavsky
Ido
I
Davies
Peter L
PL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D021343:Antifreeze Proteins, Type III / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D007053:Ice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010473:Perciformes / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D021343:Antifreeze Proteins, Type III; D029941:Fish Proteins; D005456:Fluorescent Dyes; D007053:Ice; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins