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

Article abstract of DOI:10.1021/ja8013538

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.

Full text of DOI:10.1021/ja8013538

Published on Web 07/04/2008
X-ray Structure of Snow Flea Antifreeze Protein Determined
by Racemic Crystallization of Synthetic Protein Enantiomers
†
,‡
†,‡
†,§
Brad L. Pentelute, Zachary P. Gates, Valentina Tereshko,
⊥
⊥
†,§
Jennifer L. Dashnau, Jane M. Vanderkooi, Anthony A. Kossiakoff, and
,
†,‡,§
Stephen B. H. Kent*
Institute for Biophysical Dynamics, Department of Chemistry, and Department of Biochemistry
and Molecular Biology, UniVersity of Chicago, Chicago, Illinois 60637, and Department of
Biochemistry and Biophysics, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104
Received February 22, 2008; E-mail: skent@uchicago.edu
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 Å. 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.
Introduction
Antifreeze proteins are a structurally diverse class of mac-
protein and the surface of the ice crystal, with both van der
Waals-type forces and H-bonds contributing to keep the
hydrophobic protein face bound tightly to the ice crystal.
1
romolecules that have in common the abilities to effect thermal
hysteresis of water (nonequilibrium freezing point depression)
and to inhibit ice recrystallization (growth of large ice crystals
Two years ago, Davies and co-workers reported the discovery
6
of a novel antifreeze protein from the Canadian snow flea. The
1
snow flea antifreeze protein (sfAFP) studied here is the smaller
of two protein isoforms that they isolated by ice-affinity
purification of snow flea homogenates. The predicted 81-residue
amino acid sequence of sfAFP is glycine rich, and is character-
ized by a repetitive Gly-Xaa-Xaa motif, the presence of several
proline residues, and by two intramolecular disulfides, the
connectivity of which has not been experimentally determined
(Scheme 1). sfAFP has no known homologues, antifreeze protein
or otherwise, and no experimental structure has been reported
for the folded protein molecule. Recently, Davies and co-
at the expense of small ones). These proteins are critical to
2
,3
the existence of life at subzero temperatures and also have
potential practical applications, e.g. for the storage of organs
for use in transplantation. Both antifreeze proteins and antifreeze
peptides function by binding to the surface of ice crystals and
4
preventing further crystal growth. The exact molecular basis
of the binding of antifreeze proteins to ice crystals is still subject
5
to investigation; it was originally thought that antifreeze
peptides bind to the surface of an ice crystal through H-bonding
of polar side chain moieties, for example in Thr or Lys residues,
suitably disposed on the peptide molecule. More recent studies
of antifreeze proteins have pointed toward key roles for shape
complementarity between a flat hydrophobic surface of the
7
workers have proposed a structural model for sfAFP. Experi-
mental structure determination has been precluded in the first
instance by a simple lack of materialsisolation of the natural
product gives only microgram quantities, and recombinant
expression is reportedly difficult and does not yield significant
†
Institute for Biophysical Dynamics, University of Chicago.
Department of Chemistry, University of Chicago.
Department of Biochemistry and Molecular Biology, University of
7
amounts of protein.
‡
§
Chemical protein synthesis, based on modern ligation
8
,9
Chicago.
methods, provides a route to the preparation of protein
⊥
Department of Biochemistry and Biophysics, University of Pennsylvania.
(
1) Davies, P. L.; Baardsnes, J.; Kuiper, M. J.; Walker, V. K. Philos.
Trans. R. Soc. London, Ser. B 2002, 357, 927–935.
(6) Graham, L. A.; Davies, P. L. Science 2005, 310, 461.
(7) Lin, F. H.; Graham, L. A.; Campbell, R. L.; Davies, P. L. Biophys. J.
2007, 92, 1717–1723.
(
(
2) DeVries, A. L.; Wohlschlag, D. E. Science 1969, 163, 1073–1075.
3) DeVries, A. L.; Komatsu, S. K.; Feeney, R. E. J. Biol. Chem. 1970,
2
45, 2901–2908.
(8) Schnolzer, M.; Kent, S. B. H. Science 1992, 256, 221–225.
(9) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776–779.
(
(
4) DeVries, A. L. Science 1971, 172, 1152–1155.
5) Prabhu, N.; Sharp, K. Chem. ReV. 2006, 106, 1616–1623.
10.1021/ja8013538 CCC: $40.75

2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 9695–9701 9 9695

A R T I C L E S
Pentelute et al.
6
Scheme 1. Predicted 81-Residue Glycine-Rich Amino Acid Sequence of sfAFP
1
0
18,19
j
molecules up to 200 or more amino acid residues in size.
in space group P1 were obtained.
These pioneering studies
Recent refinements have made total chemical synthesis an
increasingly practical way of obtaining useful amounts of protein
showed that it is possible to obtain centrosymmetric crystals
from a racemic protein mixture; however, in neither case was
crystallization of the natural L-protein limiting, so the studies
did not speak to whether or not racemic crystallization could
be used to facilitate obtaining crystals from a difficult-to-
crystallize protein target.
1
1–14
for study.
In an accompanying paper, we report the total
chemical synthesis of the mirror image forms of the snow flea
1
5
antifreeze protein: L-sfAFP and D-sfAFP; the antifreeze
activities of both of these folded, synthetic proteins were verified
15
16–22
by ice recrystallization inhibition assays. With a ready supply
of sfAFP to hand, we set out to determine the experimental
structure of sfAFP using racemic protein crystallography, a
technique that is made possible only by using chemical protein
synthesis.
In this paper we report the use of racemic crystallization
to obtain high-quality crystals of the protein sfAFP for X-ray
diffraction studies. Crystal formation occurred much more
readily from a racemic mixture of D-sfAFP and L-sfAFP than
with the L-protein 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 of diffraction data from a quasi-racemate crystal
was then used to solve the X-ray structure of the sfAFP protein
molecule. The resulting model was used to solve by molecular
replacement the structure of L-sfAFP to a resolution of 0.98 Å.
The X-ray structure of the true racemate made up of the protein
While X-ray structures have been determined for many
thousands of distinct protein molecules, it is not infrequently
observed that a particular protein may be difficult to crystallize
or may form crystals unsuitable for the collection of single-
crystal X-ray diffraction data. This has become one of the
principal obstacles to the high throughput determination of
protein X-ray structures. As will be described below, we
encountered severe difficulties in the crystallization of sfAFP,
which we were able to overcome as follows.
j
Proteins found in nature (i.e., proteins made up of L-amino
acids and glycine) are inherently chiral entities and can therefore
crystallize only in one of the 65 “biological” space groups that
lack an inversion center or mirror plane, out of the total of 230
unique space groups that describe all possible crystal sym-
enantiomers L-sfAFP and D-sfAFP in space group P1 was also
analyzed.
Results and Discussion
Crystallization of L-sfAFP. Using sfAFP prepared by total
1
6
15
metries. The frequency of protein crystallization in different
space groups is markedly nonuniform, with most protein crystals
belonging to one of just a few space groups. P212121 is by far
the most commonly observed space group for globular protein
molecules. Yeates has explained this preference for certain space
groups based on an entropic model: “the favored space groups
are simply less restrictive than others... they allow the molecules
more rigid-body degrees of freedom and can therefore be
chemical synthesis, extensive crystallization trials were carried
out over a period of six months with L-sfAFP alone, varying
temperature, protein concentration, and the concentration of
(NH4)2SO4 or other precipitants. Crystals were eventually
1
6
realized in a greater number of ways.” This same model
predicts that, for globular proteins, a racemic protein mixture
will crystallize more readily. This is so because the space group
j
most favored for globular proteins on theoretical grounds, P1,
contains inversion symmetry and is thus not accessible to natural
16,17
j
L-proteins.
However, the P1 space group would in principle
be accessible to the achiral entity made up of a D-protein and
the corresponding L-protein as a molecular pair. The more facile
crystallization of racemic protein mixtures has never been
experimentally verified. Only two proteins have been crystallized
as racemates; in both cases centrosymmetric racemic crystals
(
(
10) Dawson, P. E.; Kent, S. B. Annu. ReV. Biochem. 2000, 69, 923–960.
11) Bang, D.; Kent, S. B. Angew. Chem., Int. Ed. Engl. 2004, 43, 2534–
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).
2
538.
(
(
12) Johnson, E. C.; Kent, S. B. J. Am. Chem. Soc. 2006, 128, 6640–6646.
13) Durek, T.; Torbeev, V. Y.; Kent, S. B. Proc. Natl. Acad. Sci. U.S.A.
2
007, 104, 4846.
(
(
(
(
14) Torbeev, V. Y.; Kent, S. B. Angew. Chem., Int. Ed. 2007, 46 (10),
1
667–1670.
15) Pentelute, B. L.; Gates, Z. P.; Dashnau, J. L.; Vanderkooi, J. M.; Kent,
S. B. H. J. Am. Chem. Soc. 2008, 130, 9702–9707.
16) Wukovitz, S. W.; Yeates, T. O. Nat. Struct. Biol. 1995, 2 (12), 1062–
1
067.
17) Pellegrini, M.; Wukovitz, S. W.; Yeates, T. O. Proteins 1997, 28,
15.
5
9
696 J. AM. CHEM. SOC. 9 VOL. 130, NO. 30, 2008

X-ray Structure of Snow Flea Antifreeze Protein
A R T I C L E S
obtained, with difficulty. By screening selected sections of ∼30
crystals, grown at room temperature (∼23 °C) by vapor
diffusion, we were able to collect a synchrotron diffraction data
set to 0.98 Å resolution. These data were indexed in the P21
space group and had unit cell parameters a ) 16.7 Å, b ) 74.3
Å, c ) 17.7 Å, ꢀ ) 102.2°, and had one sfAFP molecule per
asymmetric unit. The solvent content for this crystal was
unusually low at 25%. Exhaustive attempts to crystallize heavy
atom derivatives of L-sfAFP for use in phase determination
failed, and no structures of homologous protein molecules were
available, so we were unable to solve the X-ray structure.
Racemic Crystallization. The total chemical synthesis of the
two enantiomeric molecules, D-sfAFP and L-sfAFP, was carried
1
5
out as previously described. In trials using a racemic solution
containing equal amounts of D-sfAFP and L-sfAFP we found
that crystals were obtained in ∼50% of the 194 conditions
initially explored using the commercially available Hampton
index, crystal screen and crystal screen II. This was in marked
contrast to what we had observed in attempts to crystallize
L-sfAFP. Figure 1 shows results obtained from simultaneous
crystallization experiments under representative conditions for
L-sfAFP alone, and for a racemic solution of D-sfAFP and
L-sfAFP under the same conditions.
Diffraction data from a sampling of the racemic crystals
formed in our initial screen revealed that, under all conditions
examined, the protein racemate crystallized with triclinic
symmetry and had unit cell parameters a ) 28.6 Å, b ) 32.4
Å, c ) 59.8 Å, R ) 88.7°, ꢀ ) 89.2°, γ ) 73.4° with two
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-sfAFP/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. The X-ray structure of snow flea antifreeze protein (sfAFP). Atomic resolution (1.2 Å) X-ray structure of sfAFP, obtained from quasi-racemate crystals
of D-sfAFP and L-Se-sfAFP 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.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 30, 2008 9697

A R T I C L E S
Pentelute et al.
2
sfAFP molecules in the asymmetric unit. The 〈|E - 1|〉 statistic
calculations in program XPREP indicated the centrosymmetric
j
space group P1.
Crystallization of
a
Quasi-Racemate for Phase
Determination. The next step was to solve the structure of the
sfAFP molecule. It was suggested by Berg that, because of the
quantization of phases in diffraction data obtained from a
centrosymmetric racemic protein crystal, direct methods should
2
2
be more applicable. For the sfAFP protein, our preliminary
attempts to solve the structure by direct methods failed. To speed
up the structure determination we chose to solve the structure
using experimental phases obtained using the multiple wave-
2
3
length anomalous dispersion (MAD) method. We collected
synchrotron X-ray data from a selenium-containing quasi-
racemate in which only one enantiomer contained a selenium
atom. (For centrosymmetric structures, such as a protein
racemate [pair of enantiomers], Friedel’s law holds even in the
presence of an anomalous scattering atom, and it is not possible
2
4
to measure anomalous differences. ) The quasi-racemate was
obtained by the cocrystallization of a mixture of D-sfAFP and
L-Se-sfAFP. The L-Se-sfAFP was a chemically synthesized
1
1
sfAFP analogue in which Asn was replaced by seleno-Cys
alkylated with bromoacetamide (“pseudo-Se-Gln”; see Experi-
mental Section for details). The more facile production of
crystals by racemic crystallization was critical for obtaining
crystalsswe had made extensive efforts to grow crystals of the
L-Se-sfAFP protein alone, with no success. By contrast, a quasi-
racemic solution containing equal amounts of D-sfAFP and L-Se-
sfAFP crystallized readily under a variety of conditions.
Synchrotron X-ray diffraction data to a resolution of 1.2 Å
were collected from crystals of the D-sfAFP and L-Se-sfAFP
quasi-racemate. The quasi-racemate had unit cell parameters
nearly identical to those of the true racemate. The structure was
solved in space group P1 using MAD data collected at three
wavelengths (see Experimental Section). Figure 2 shows the
arrangement of D-sfAFP and L-Se-sfAFP molecules in the quasi-
racemate.
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 1j {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.
at the surface of an ice crystal, presenting the polar (hydrophilic)
face to the surrounding solution and inhibiting further growth
25,26
of the ice crystal.
Interestingly, we observed an array of
X-ray Structure of sfAFP. The resulting high resolution X-ray
structure of sfAFP is shown in Figure 3. The protein fold
observed in the X-ray structure of sfAFP is similar to the
highly ordered water molecules on the flat, hydrophobic face
of the sfAFP protein (Figure 3). This observation is consistent
with the proposed mechanism for the binding of an sfAFP
7
7
structural model recently proposed by Davies et al. It consists
molecule to an ice crystal.
of six left-handed PP-II helices, stacked in two antiparallel
groups to form an oblong brick-shaped molecule. The turns
linking the helices are in general defined by the presence of
Pro residues, and the fold of the N-terminal half of the
polypeptide chain is stabilized by two intramolecular disulfide
bonds between residues Cys1-Cys28 and Cys13-Cys43. One
face of the molecule is hydrophobic, and the other, polar/
hydrophilic. The implications of this novel protein fold for
The molecular model from the quasi-racemate was used to
solve by molecular replacement the structure of L-sfAFP using
the original 0.98 Å diffraction data (Table 3). The L-sfAFP
molecular structure in the P21 crystal is identical to that observed
in the quasi-racemate. A portion of the 2Fo - Fc electron density
map obtained at 0.98 Å is shown in Figure 4(a).
Diffraction data were also collected from crystals of the true
racemate {D-sfAFP + L-sfAFP}. Table 4 gives the X-ray
statistics for the {D-sfAFP + L-sfAFP} data set. A portion of
the 2Fo - Fc electron density map obtained for the true racemate
7
antifreeze activity have been discussed. It has been hypoth-
esized that in antifreeze proteins a flat, hydrophobic face
interacts snugly with the highly ordered water molecules found
{
D-sfAFP + L-sfAFP} at 1.0 Å is shown in Figure 4 (b). The
j
crystals of the true racemate were refined in the P1 centrosym-
metric space group.
(
(
18) Zawadzke, L. E.; Berg, J. M. Proteins 1993, 16, 301–305.
19) Hung, L. W.; Kohmura, M.; Ariyoshi, Y.; Kim, S. H. J. Mol. Biol.
Conclusions
1
999, 285, 311–321.
(
(
20) Toniolo, C.; Peggion, C.; Crisma, M.; Formaggio, F.; Shui, X.;
Eggleston, D. S. Nat. Struct. Biol. 1994, 1, 908–914.
Crystallization of a racemic mixture of enantiomeric protein
molecules prepared by total chemical synthesis has been used to
experimentally determine the X-ray structure of the snow flea
21) Patterson, W. R.; Anderson, D. H.; DeGrado, W. F.; Cascio, D.;
Eisenberg, D. Protein Sci. 1999, 8, 1410.
(
22) Berg, J. M; Goffeney, N. W. Methods Enzymol 1997, 276, 619–627.
(23) Hendrickson, W. A.; Ogata, C. M. Macromol. Crystallogr., Pt A 1997,
2
76, 494–523.
(25) Liou, Y. C.; Tocilj, A.; Davies, P. L.; Jia, Z. Nature 2000, 406, 322–
324.
(24) Stout, G. H.; Jensen, L. H. X-Ray Structure Determination: A Practical
Guide, 2nd ed.;Wiley: New York, 1989.
(26) Leinala, E. K.; Davies, P. L.; Jia, Z. Structure 2002, 10, 619–627.
9
698 J. AM. CHEM. SOC. 9 VOL. 130, NO. 30, 2008

X-ray Structure of Snow Flea Antifreeze Protein
A R T I C L E S
greatly facilitate the X-ray structure determination of a synthetically
accessible protein, as was the case for the snow flea antifreeze
protein studied here. Moreover, we have shown that, at least for
sfAFP, the more facile crystallization also applies to quasi-racemic
mixtures in which one protein molecule contains an anomalous
scattering atom for phase determination. Including the work
reported here, only a handful of racemic protein crystallizations
have been described. Because a mixture of enantiomers can
crystallize either as a racemate or the individual enantiomers can
crystallize as a conglomerate, it is too early to say whether more
facile crystallization of racemic protein mixtures is a general
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 ×
1
50 mm column using a linear gradient of 1-61% buffer B over 15 min
16
(
buffer A ) 0.1% TFA in H2O; buffer B ) 0.08% TFA in acetonitrile).
phenomenon, as predicted by Yeates. If protein racemates
crystallize as a rule, then the chemical synthesis of mirror image
The inset is the ESMS summed over the entire LC peak. Calculated masses
were based on average isotope composition.
16,17
proteins for racemic crystallization, as suggested by Yeates,
may prove to be a powerful tool in structural biology.
antifreeze protein (sfAFP). This protein was found to have a tertiary
structure consisting only of stacked PPII helices, which represents
a unique protein fold. The experimental structure of sfAFP is similar
to a molecular model recently proposed, and has implications for
Experimental Section
Total Chemical Synthesis of sfAFP and Analogues. The 81-
residue sfAFP polypeptide chain (Scheme 1) was synthesized by
native chemical ligation of four unprotected peptides (Thz1-12-
thioester; Thz13-27-thioester; Thz28-42-thioester; Cys43-81) as
7
the molecular basis of the antifreeze activity of sfAFP.
In this paper we have experimentally demonstrated, for the first
time, that a racemic protein mixture can more readily form highly
ordered crystals suitable for X-ray diffraction studies. This can
1
5
described in an accompanying paper. L-Se-sfAFP was prepared
by incorporation of “pseudo-Se-Gln” into the Thz1-12- thioester
Table 1. X-ray Data Collection and Refinement Statistics for the Quasi-Racemate [D-sfAFP+L-Se-sfAFP]
a
b
Values for highest resolution shell are given in parentheses. Friedel mates merged.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 30, 2008 9699

A R T I C L E S
Pentelute et al.
Table 2. Backbone rmsd (Å) Deviations between Molecules in the
Quasi-Racemate: Molecules A and B are L-Se-sfAFP, and
Molecules C and D Are Inverted D-sfAFP
Table 3. X-ray Data Collection and Refinement Statistics for
L-sfAFP
A
B
C-inverted
D-inverted
A
B
0.520
0.165
0.564
0.514
0.203
0.540
C-inverted
27
peptide during stepwise solid-phase synthesis. Boc-“[4Se]-pseudo-
Gln”-OH for use in peptide synthesis was prepared by reducing
L-selenocystine with sodium borohydride, alkylating at selenium
with 2-bromoacetamide, and N-acylating with Boc O. The LC-MS
2
analysis of chemically synthesized L-Se-sfAFP synthetic protein is
shown in Figure 5.
Protein Crystallization. Lyophilized sfAFP was dissolved in
water at 10-40 mg/mL and was used directly for crystallizations.
Crystallization screening conditions were based on the commercially
available Hampton index, crystal screen and crystal screen II.
Crystals were grown by vapor diffusion in hanging drops at room
temperature (∼23 °C). The drops were generated by mixing 1 µL
of protein solution with 1 µL of reservoir solution and placed against
1
mL of reservoir solution. Racemic solutions of sfAFP were
prepared in water at 38 mg/mL (19 mg of D-sfAFP and 19 mg of
L-sfAFP) and 19 mg/mL (9.5 mg of D-sfAFP and 9.5 mg of
L-sfAFP). Simultaneous sparse matrix room temperature screens
were carried out for the racemic protein solutions at both concentra-
tions. Crystals appeared after 1 day, and after 10 days, 93 of the
1
94 sets of the conditions explored had crystals suitable for use in
diffraction experiments. No crystals were formed from the L-sfAFP
solutions using the same sets of conditions. The same approach
was used for crystallization of the quasi-racemate in which crystals
suitable for use in diffraction experiments appeared after 1 day in
a number of conditions. Diffraction data for the quasi-racemate were
collected using a crystal grown at room temperature from 0.2 M
sodium malonate, pH 7, and 20% w/v polyethylene glycol 3350.
Diffraction data for L-sfAFP were collected using a crystal grown
at room temperature from 0.1 M bis-Tris pH ) 5.5, 1.8 M
ammonium sulfate. Diffraction data for the racemate was collected
using a crystal grown at room temperature from 0.1 M bis-Tris pH
a
Values for highest resolution shell are given in parentheses.
)
6.5, 1.15 M ammonium sulfate.
Diffraction Data Collection for L-sfAFP. For low temperature
2
8
detector). Images were processed and scaled with HKL2000. Data
were collected to a resolution of 1.2 Å (Table 1).
data collection, selected crystals of L-sfAFP were transferred to
the cryoprotectant (reservoir solution plus 20% (v/v) glycerol) for
a few seconds and flash-frozen in liquid nitrogen. Synchrotron X-ray
data were collected at a temperature of 100 K at the Advanced
Photon Source, Argonne National Laboratory, beamline 19BM
equipped with a SBC2 detector. Images were processed and scaled
Determination of the sfAFP X-ray Structure from the
Quasi-Racemate. The L-Se-sfAFP/D-sfAFP quasi-racemate struc-
ture was solved by MAD phasing. The heavy atom search was
performed in SHELXD using X-ray data collected at 12.6607
keV peak energy and 3.5 Å resolution cutoff. MAD phasing and
density modification were performed with use of SOLVE and
RESOLVE, respectively. The model building performed with the
RESOLVE basic script was complete with use of ARP/WARP
program. The electron density and model examinations were done
using TURBO-FRODO. The restrained positional and anisotropic
2
3
2
9
2
8
with HKL2000. Data were collected to a resolution of 0.98 Å
30
(
Table 3).
Diffraction Data Collection for the Racemate. For low
31
temperature data collection, selected crystals were transferred to
the cryoprotectant (reservoir solution plus 20% (v/v) glycerol) for
a few seconds and flash-frozen in liquid nitrogen. X-ray data were
collected at 100 K at the Advanced Photon Source, Argonne
National Laboratory, beamline 23ID equipped with a MARCCD
32
33
B-factor refinement was done in REFMAC5.
Rfree was monitored
by setting aside 5% of the reflections as a test set. Crystal data and
X-ray data statistics for the quasi-racemate data set are listed in
Table 1. Backbone rmsd (Å) deviations between the enantiomeric
protein molecules in the quasi-racemate are presented in Table 2.
The coordinates and structure factors have been deposited in the
Protein Data Bank with entry code 3BOG.
28
3
00 detector). Images were processed and scaled with HKL2000.
Data were collected to a resolution of 1.0 Å (Table 4).
Diffraction Data Collection for the Quasi-Racemate. For low
temperature data collection, selected crystals of the L-Se-sfAFP/D-
sfAFP quasi-racemate were transferred to the cryoprotectant (reservoir
solution plus 20% (v/v) glycerol) for a few seconds and flash-frozen
in liquid nitrogen. Three-wavelengh anomalous dispersion X-ray data
were collected at 100K at the Advanced Photon Source, Argonne
National Laboratory, beamline 23ID equipped with a MARCCD 300
(
29) Schneider, T. R.; Sheldrick, G. M. Acta Crystallogr., Sect. D 2002,
58, 1772–1779.
(30) Terwilliger, T. C.; Berendzen, J. Acta Crystallogr., Sect. D 1999, 55,
8
49–861.
(
31) Perrakis, A.; Harkiolaki, M.; Wilson, K. S.; Lamzin, V. S. Acta
Crystallogr., Sec. D 2001, 57, 1445–1450.
(
(
27) Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H.
Int. J.Peptide Res. Ther. 2007, 13, 31–44.
28) Otwinowski, Z.; Minor, W. Macromol. Crystallogr., Pt A 1997, 276,
(32) Cambillau, C.; Roussel, A. Turbo Frodo, Version OpenGL.1; Uni-
versit e´ Aix-Marseille II: Marseille, France, 1997.
(33) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr.,
Sect. D 1997, 53, 240–255.
3
07–326.
9
700 J. AM. CHEM. SOC. 9 VOL. 130, NO. 30, 2008

X-ray Structure of Snow Flea Antifreeze Protein
A R T I C L E S
Table 4. X-ray Data Collection and Refinement Statistics for
Racemic sfAFP
positional and anisotropic B-factor refinement, was done in REF-
33
MAC5. The electron density and model examinations were done
3
2
using TURBO-FRODO.
Rfree was monitored by setting aside 5%
of the reflection as a test set. Crystal data and X-ray data statistics
for the L-sfAFP data set are listed in Table 3. The coordinates and
structure factors have been deposited in the Protein Data Bank with
entry code 2PNE.
Determination of the X-ray Structure of the
{L-sfAFP+D-sfAFP} True Racemate. Since the crystal packing
of the quasi-racemate is the same as the true racemate, we proceeded
directly to rigid-body refinement with two L-fAFP monomers in
the asymmetric unit (chains A and B) oriented as for the quasi-
racemate. The rigid body refinement, restrained positional and
3
3
anisotropic B-factor refinement, was done in REFMAC5. The
electron density and model examinations were done using TURBO-
32
FRODO.
Rfree was monitored by setting aside 5% of the reflection
as a test set. Crystal data and X-ray data statistics for the racemate
data set are listed in Table 4. The coordinates and structure factors
have been deposited in the Protein Data Bank with entry code 3BOI.
Acknowledgment. We thank W. DeGrado (U. Penn) for
suggesting that we work on the snow flea antifreeze protein.
We thank T. Sosnick, V. Pingali, V. Torbeev, Li-Wei Hung, C.
Weeks, S. Anderson, N. Sanishvili, M. Becker, and A. Moglich
for useful discussions and technical advice. Z.G. was supported
by the University of Chicago NIH Training Program in Physical
and Chemical Biology. This research was supported by the Office
of Science (BER), U.S. Department of Energy, Grant No. DE-
FG02-07ER64501 to S.B.H.K., and by the National Institutes
of Health, Grant No. R01 GM075993 to SBHK. The use of the
Advanced Photon Source was supported by the U.S. Department
of Energy, Basic Energy Sciences, Office of Science, under
Contract No. DE-AC02-06CH11357. Portions of this work were
performed at the SBC-CAT and GM/CA CAT located at Sectors
1
9BM and 23ID, respectively, of the Advanced Photon Source.
SBC-CAT is supported by the U.S. Department of Energy, Office
of Biological and Environmental Research, under Contract No.
W-31-109-ENG-38. Use of the GM/CA CAT Sector 23-ID was
funded with Federal funds from the National Cancer Institute
(
Y1-CO-1020) and the National Institute of General Medical
a
Values for highest resolution shell are given in parentheses.
Science (Y1-GM-1104).
Determination of the X-ray Structure of L-sfAFP. The
JA8013538
structure was determined by molecular replacement using the
34
program MOLREP with the L-Se-sfAFP molecule from the quasi-
racemate as a starting model. The rigid body refinement, restrained
(34) Vagin, A.; Teplyakov, A. J. Appl. Crystallogr. 1997, 30, 1022–1025.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 30, 2008 9701