X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers.
Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 A. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.
Pentelute
Brad L
BL
Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637, USA.
Gates
Zachary P
ZP
Tereshko
Valentina
V
Dashnau
Jennifer L
JL
Vanderkooi
Jane M
JM
Kossiakoff
Anthony A
AA
Kent
Stephen B H
SB
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D011487:Protein Conformation; D005423:Siphonaptera / Q000737:chemistry*; D013237:Stereoisomerism
D021301:Antifreeze Proteins