Antifreeze protein from shorthorn sculpin: identification of the ice-binding surface.
Shorthorn sculpins, Myoxocephalus scorpius, are protected from freezing in icy seawater by alanine-rich, alpha-helical antifreeze proteins (AFPs). The major serum isoform (SS-8) has been reisolated and analyzed to establish its correct sequence. Over most of its length, this 42 amino acid protein is predicted to be an amphipathic alpha-helix with one face entirely composed of Ala residues. The other side of the helix, which is more heterogeneous and hydrophilic, contains several Lys. Computer simulations had suggested previously that these Lys residues were involved in binding of the peptide to the [11-20] plane of ice in the <-1102> direction. To test this hypothesis, a series of SS-8 variants were generated with single Ala to Lys substitutions at various points around the helix. All of the peptides retained significant alpha-helicity and remained as monomers in solution. Substitutions on the hydrophilic helix face at position 16, 19, or 22 had no obvious effect, but those on the adjacent Ala-rich surface at positions 17, 21, and 25 abolished antifreeze activity. These results, with support from our own modeling and docking studies, show that the helix interacts with the ice surface via the conserved alanine face, and lend support to the emerging idea that the interaction of fish AFPs with ice involves appreciable hydrophobic interactions. Furthermore, our modeling suggests a new N terminus cap structure, which helps to stabilize the helix, whereas the role of the lysines on the hydrophilic face may be to enhance solubility of the protein.
Baardsnes
J
J
Protein Engineering Network of Centres of Excellence, Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Jelokhani-Niaraki
M
M
Kondejewski
L H
LH
Kuiper
M J
MJ
Kay
C M
CM
Hodges
R S
RS
Davies
P L
PL
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D021301:Antifreeze Proteins / Q000737:chemistry*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D029941:Fish Proteins; D005615:Freezing; D007053:Ice; D008239:Lysine / Q000737:chemistry; D008715:Methionine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014461:Ultracentrifugation
D021301:Antifreeze Proteins; D029941:Fish Proteins; D007053:Ice; D010455:Peptides; D020033:Protein Isoforms; C446459:antifreeze protein, Myoxocephalus scorpius; D008715:Methionine; D008239:Lysine; D000409:Alanine