Investigation of the Ice-Binding Site of an Insect Antifreeze Protein Using Sum-Frequency Generation Spectroscopy.
We study the ice-binding site (IBS) of a hyperactive antifreeze protein from the beetle Dendroides canadensis (DAFP-1) using vibrational sum-frequency generation spectroscopy. We find that DAFP-1 accumulates at the air-water interface due to the hydrophobic character of its threonine-rich IBS while retaining its highly regular β-helical fold. We observe a narrow band at 3485 cm(-1) that we assign to the O-H stretch vibration of threonine hydroxyl groups of the IBS. The narrow character of the 3485 cm(-1) band suggests that the hydrogen bonds between the threonine residues at the IBS and adjacent water molecules are quite similar in strength, indicating that the IBS of DAFP-1 is extremely well-ordered, with the threonine side chains showing identical rotameric confirmations. The hydrogen-bonded water molecules do not form an ordered ice-like layer, as was recently observed for the moderate antifreeze protein type III. It thus appears that the antifreeze action of DAFP-1 does not require the presence of ordered water but likely results from the direct binding of its highly ordered array of threonine residues to the ice surface.
†FOM-Institute for Atomic and Molecular Physics AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands.
Konrad
K
Meister
†FOM-Institute for Atomic and Molecular Physics AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands.
Stephan
S
Lotze
‡Laboratory of Macromolecular and Organic Chemistry and Institute for Complex Molecular Systems, Eindhoven University of Technology, Postbus 513, 5600 MB Eindhoven, The Netherlands.
Luuk L C
LL
Olijve
§Department of Animal Biology, University of Illinois at Urbana-Champaign, 515 Morrill Hall, Urbana, Illinois 61801, United States.
Arthur L
AL
DeVries
∥Department of Biological Sciences, University of Notre Dame, 100 Galvin Life Sciences Center, Notre Dame, Indiana 46556, United States.
John G
JG
Duman
‡Laboratory of Macromolecular and Organic Chemistry and Institute for Complex Molecular Systems, Eindhoven University of Technology, Postbus 513, 5600 MB Eindhoven, The Netherlands.
Ilja K
IK
Voets
†FOM-Institute for Atomic and Molecular Physics AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands.
Huib J
HJ
Bakker
D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry*; D001665:Binding Sites; D001517:Coleoptera; D007053:Ice; D019476:Insect Proteins / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods; D013912:Threonine / Q000737:chemistry
D021301:Antifreeze Proteins; D007053:Ice; D019476:Insect Proteins; D013912:Threonine
air−water interface; antifreeze proteins; chiral sum-frequency generation spectroscopy; phase-sensitive sum-frequency generation; protein−water interface