Alternative roles for putative ice-binding residues in type I antifreeze protein.
Two sets of variants of type I antifreeze protein have been synthesized to investigate the role of Leu and Asn in the activity of this 37-residue alpha-helix. Leu and Asn flank the central two of four regularly spaced ice-binding Thr in the i-1 and i + 3 positions, respectively. All three residues project from the same side of the helix to form the protein's putative ice-adsorption site and are considered in some models to act together as an "ice-binding motif". Replacement of Asn by residues with shorter side chains resulted in either a small loss (Ala) or gain (Thr) of antifreeze activity. However, substitution of Asn by its slightly larger homologue (Gln) abolished thermal hysteresis activity. The Gln-containing peptide was very soluble, largely monomeric, and fully helical. Of the three variants in which Leu was replaced by Ala, two of the three were more active than their Leu-containing counterparts, but all three variants began to precipitate as the peptide concentration increased. None of the seven variants tested showed dramatic differences in ice crystal morphology from that established by the wild type. These results are consistent with a primary role for Leu in preventing peptide aggregation at the antifreeze protein concentrations (10 mg/mL) normally present in fish serum. Similarly the role for Asn may have more to do with enhancing the solubility of these rather hydrophobic peptides than of making a stereospecific hydrogen-bonding match to the ice lattice as traditionally thought. Nevertheless, the dramatic loss of activity in the Asn-to-Gln replacement demonstrates the steric restriction on residues in or near the ice-binding site of the peptide.
Loewen
M C
MC
Protein Engineering Network of Centres of Excellence and Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Chao
H
H
Houston
M E
ME
Baardsnes
J
J
Hodges
R S
RS
Kay
C M
CM
Sykes
B D
BD
Sönnichsen
F D
FD
Davies
P L
PL
D000595:Amino Acid Sequence; D021301:Antifreeze Proteins; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D007053:Ice; D007930:Leucine / Q000378:metabolism; D008969:Molecular Sequence Data
D021301:Antifreeze Proteins; D006023:Glycoproteins; D007053:Ice; D001216:Asparagine; D007930:Leucine