Elevated temperature and tyrosine iodination aid in the crystallization and structure determination of an antifreeze protein.
Production and refolding of recombinant Choristoneura fumiferana antifreeze protein (CfAFP) leads to a disulfide-bonded product containing dynamic conformational microheterogeneity. Difficulties in the crystallization of this protein arising from its microheterogeneity were overcome by screening of crystallization conditions at various temperatures and finally using a temperature of 318 K to obtain diffraction-quality crystals. In addition, heavy-atom derivatization of this protein required the iodination of a specific tyrosine residue, leading to the successful single anomalous scattering (SAS) structure determination. The techniques of higher temperature screening, to reduce dynamic conformational microheterogeneity, and defined tyrosine iodination, for specific heavy-atom incorporation, are methods which can be employed with other proteins to aid in structure determination.
Leinala
Eeva K
EK
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Davies
Peter L
PL
Jia
Zongchao
Z
D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D007455:Iodine / Q000737:chemistry*; D007915:Lepidoptera / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D014443:Tyrosine / Q000737:chemistry
D021301:Antifreeze Proteins; D014443:Tyrosine; D007455:Iodine