Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jul 30;130(30):9695-701.
doi: 10.1021/ja8013538. Epub 2008 Jul 4.

X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers

Brad L Pentelute  1 , Zachary P Gates, Valentina Tereshko, Jennifer L Dashnau, Jane M Vanderkooi, Anthony A Kossiakoff, Stephen B H Kent
Affiliations

X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers

Brad L Pentelute et al. J Am Chem Soc. .

Abstract

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
A direct comparison of the results for attempted crystallization of the racemic protein mixture [d-sfAFP + l-sfAFP] or the single enantiomer l-sfAFP under three sets of conditions. Protein crystals appeared rapidly from a solution containing a racemic mixture of d-sfAFP and l-sfAFP, but not for l-sfAFP alone, when screened under a standard set of crystallization conditions. Representative examples are shown in this Figure. X-ray diffraction was used to verify that the crystals were in fact protein. The crystallization conditions shown are a subset of the main findings reported here and are as follows: (a) 0.1 M bicine pH = 9.0, 2% v/v 1,4-dioxane, 10% w/v polyethylene glycol 20,000; (b) 0.1 M bis-Tris pH = 6.5, 1.15 M ammonium sulfate; (c) 0.1 M Tris pH = 8.5, 25% w/v polyethylene glycol 3350. All crystallization trials were carried out at room temperature (∼23 °C).
Figure 2
Figure 2
Quasi-racemate crystal packing in space group P1, showing the d-sfAFP molecule (purple ribbon) and the l-Se-sfAFP (green ribbon) with the “pseudo-Se-Gln” at position 11 shown as CPK solid spheres colored by atom type (C, green; O, red; N, cyan; Se, yellow). (a) Unit cell with two d-sf AFP/l-Se-sfAFP pairs located at positions 0, 1/2b, 1/2c, and 0,0,0, in the asymmetric unit. The d-sfAFP contains right-handed polyproline type II (PPII) helices; the l-Se-sfAFP contains left-handed PPII helices. (b) View of one d-sfAFP/l-Se-sfAFP pair located at 0, 1/2b, 1/2c. Side chains are included as stick representations.
Figure 3
Figure 3
The X-ray structure of snow flea antifreeze protein (sfAFP). Atomic resolution (1.2 A) X-ray structure of sfAFP, obtained from quasi-racemate crystals of d-sfAFP and l-Se-sf AFP in the P1 space group. (a) SigmaA-weighted 2Fo – Fc electron density map (blue, 1.5 sigma level) of the proposed ice-binding surface encompassing residues 17–21, 47–51, 74–78. Five ordered water molecules are shown (magenta spheres) with electron density shown at 1σ. (b) Cartoon of the backbone fold of sfAFP. Amino acid side chains are shown as sticks. The dashed box indicates the region shown in (a) above. (c) The main structural elements of sfAFP are six antiparallel polyproline type II (PPII) (left-handed) helices, stacked in two groups and joined by five reverse turns and interlocked by a complex hydrogen bond network. The N-terminal half of the protein contains two intramolecular disulfide bonds between residues Cys1-Cys28 and Cys13-Cys43. (d) Surface map highlighting sfAFP’s amphiphilic character. A 180° rotation (along the long axis) shows the contrasting surfaces: hydrophobic/apolar (left); hydrophilic/polar (right). (e) Two views related by a 90° rotation of highly ordered first shell water molecules (shown in magenta) interacting with the sfAFP hydrophobic face backbone (dashed magneta lines). Water molecules (magenta spheres) are within hydrogen-bonding distance (2.7–3.0 Å) of the sfAFP backbone amide carbonyls and nitrogens, and of one another (solid magnenta lines). The spacing between water molecules is 4.5 Å and 9.2 Å along the short and long sfAFP axes, respectively. X-ray statistics are given in Table 1. Backbone rmsd deviations between molecules of l-sfAFP and d-sfAFP are given in Table 2.
Figure 4
Figure 4
SigmaA-weighted 2Fo – Fc electron density map (blue, 1.5σ level) of the proposed ice-binding surface encompassing residues 17–21, 47–51, 74–78 for: (a) P21 l-sfAFP; (b) P 1̄ {d-sfAFP + l-sfAFP}. Ordered water molecules with electron density at 1σ are shown (magenta spheres). Five ordered water molecules are conserved in all structures. An additional water molecule is observed in the P21 structure; this water is absent in the racemic crystals because of packing interactions.
Figure 5
Figure 5
Analytical LC-MS data for synthetic folded l-Se-sfAFP. The chromatographic separations were carried out on a Vydac C4 2.1 mm × 150 mm column using a linear gradient of 1–61% buffer B over 15 min (buffer A = 0.1% TFA in H2O; buffer B = 0.08% TFA in acetonitrile). The inset is the ESMS summed over the entire LC peak. Calculated masses were based on average isotope composition.
Scheme 1
Scheme 1
Predicted 81-Residue Glycine-Rich Amino Acid Sequence of sfAFP
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Davies PL, Baardsnes J, Kuiper MJ, Walker VK. Philos. Trans. R. Soc. London, Ser. B. 2002;357:927–935. - PMC - PubMed
    1. DeVries AL, Wohlschlag DE. Science. 1969;163:1073–1075. - PubMed
    1. DeVries AL, Komatsu SK, Feeney RE. J. Biol. Chem. 1970;245:2901–2908. - PubMed
    1. DeVries AL. Science. 1971;172:1152–1155. - PubMed
    1. Prabhu N, Sharp K. Chem. Rev. 2006;106:1616–1623. - PMC - PubMed

Publication types

Substances