New ice-binding face for type I antifreeze protein.
Type I antifreeze protein (AFP) from winter flounder is an alanine-rich, 37 amino acid, single alpha-helix that contains three 11 amino acid repeats (Thr-X(2)-Asx-X(7)), where X is generally Ala. The regularly spaced Thr, Asx and Leu residues lie on one face of the helix and have traditionally been thought to form hydrogen bonds and van der Waals interactions with the ice surface. Recently, substitution experiments have called into question the importance of Leu and Asn for ice-binding. Sequence alignments of five type I AFP isoforms show that Leu and Asn are not well conserved, whereas Ala residues adjacent to the Thr, at right angles to the Leu/Asn-rich face, are completely conserved. To investigate the role of these Ala residues, a series of Ala to Leu steric mutations was made at various points around the helix. All the substituted peptides were fully alpha-helical and remained as monomers in solution. Wild-type activity was retained in A19L and A20L. A17L, where the substitution lies adjacent to the Thr-rich face, had no detectable antifreeze activity. The nearby A21L substitution had 10% wild-type activity and demonstrated weak interactions with the ice surface. We propose a new ice-binding face for type I AFP that encompasses the conserved Ala-rich surface and adjacent Thr.
Baardsnes
J
J
Department of Biochemistry and the Protein Engineering Network of Centres of Excellence, Queen's University, Kingston, Ont., Canada.
Kondejewski
L H
LH
Hodges
R S
RS
Chao
H
H
Kay
C
C
Davies
P L
PL
D000409:Alanine / Q000737:chemistry; D000818:Animals; D021301:Antifreeze Proteins; D002942:Circular Dichroism; D003461:Crystallography; D004305:Dose-Response Relationship, Drug; D005432:Flounder / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006860:Hydrogen Bonding; D007053:Ice; D007930:Leucine / Q000737:chemistry; D016296:Mutagenesis; D011485:Protein Binding; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013912:Threonine / Q000737:chemistry; D014461:Ultracentrifugation
D021301:Antifreeze Proteins; D006023:Glycoproteins; D007053:Ice; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013912:Threonine; D007930:Leucine; D000409:Alanine