X-ray Structure of Native Scorpion Toxin BmBKTx1 by Racemic Protein Crystallography Using Direct Methods

Abstract

Recently, we determined the structure of the protein sfAFP by means of racemic crystallization. 1 In the current work, we additionally sought to explore the use of protein racemates to enable structure solution using direct methods; there are only a handful of native L-protein structures that have been solved by direct methods and the successful application of this approach is still a real challenge in modern crystallography. 2,3 Here we report the first crystallization and the X-ray structure determination of native scorpion toxin BmBKTx1. Using racemic protein crystallization, we obtained crystals of BmBKTx1 in the tetragonal centrosymmetric spacegroup I41/a, that diffracted to atomic resolution; this enabled the structure to be determined by the use of direct methods.Difficulty in crystallization is often observed for proteins that contain numerous charged, surface-exposed amino acid residues. An example of one such protein is the lysine-rich scorpion toxin BmBKTx1, a 31 amino acid residue microprotein that is a high-conductance calcium-activated potassium channel blocker. 4-6 For BmBKTx1 it was reported that at 100 mg/mL protein concentration no crystal formation was observed over many weeks at room temperature, using extensive sparse-matrix crystallization searches. 6 In order to obtain diffraction-quality crystals, these authors found that it was necessary to use reductive dimethylation of lysine residues; this strategy resulted in an X-ray structure of methylated BmBKTx1 at 1.72 Å resolution. 6We explored the use of racemic crystallization for BmBKTx1; the protein contains six cysteine residues that form three disulfides in the folded molecule. 4 The mirror image isomer of the native protein required for racemic protein crystallization can only be prepared by chemical synthesis. We prepared the polypeptide chains of D-and L-BmBKTx1 in good yield and high purity by manual, Boc chemistry, stepwise solid-phase peptide synthesis. 7 The D-BmBKTx1 and L-BmBKTx1 polypeptides were synthesized at a scale of 0.1 and 0.2 millimole, respectively. The crude peptides were folded with concomitant disulfide formation, followed by HPLC purification; the LCMS of the folded and purified synthetic protein products are shown in Figure S1 (see Supporting Information). We obtained pure D-and L-proteins in multiple tens-of-milligram amounts.Crystallization trials of L-BmBKTx1 in our hands were not successful, as previously reported. 6 In contrast, using a racemic solution containing equal amounts of D-BmBKTx1 and LBmBKTx1 in the commercially available Hampton Research Index screen at 19 °C, crystals were obtained under multiple conditions; under some conditions, crystals appeared overnight. One set of conditions was further optimized to produce ultra-high quality crystals (Figure 1a). Diffraction data ( Figure S2) were collected at the Advanced Photon Source to a resolution of Email: skent@uchicago.edu. With atomic-resolution diffraction data in hand, we explored the use of direct methods 14, 15 to obtain phasing information for...

Determination of the X‐ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography

“…However, when a racemic protein mixture consisting of L-omwaprin þ D-omwaprin in equal amounts was used, protein crystals formed within a few days under 8 of the same 96 conditions. This is further evidence of facile crystal formation from racemic protein mixtures, 2,3 supporting an earlier prediction of such an advantage. 17 It was also predicted that for racemic protein crystals, the space group, P1, would be the one most frequently observed 17 ; crystallographic experiments by us and by others have so far confirmed this prediction.…”

“…2,4,8,[18][19][20] The present study adds another example of a racemic protein crystal that is not in P1 but in another space group, P2 1 /c. 3 The number of proteins that have been crystallized in racemic form is still small, so an accurate statistical analysis of space groups is not possible yet. Structures of additional racemic proteins should begin to reveal additional trends regarding their crystallization behavior.…”

“…We have recently shown that protein crystals formed more readily from a racemic mixture (containing equal proportion of L-and D-protein enantiomers) resulting in centrosymmetric crystals. 2,3 It has been shown that these centrosymmetric crystals facilitate ab initio protein structure solution via direct methods for a combination of reasons. The crystals tend to be well ordered and thus give high resolution diffraction data, and the phases of the X-ray reflections are all quantized (related by 0, p radians).…”

“…The crystals tend to be well ordered and thus give high resolution diffraction data, and the phases of the X-ray reflections are all quantized (related by 0, p radians). 3,4 Some of the potential advantages that have been observed for phasing and structure interpretation were anticipated by early studies on racemic macromolecules. 5,6 To obtain a racemic protein crystal, it is necessary to make the D-protein (the enantiomer of the L-protein); this can only be done by total chemical synthesis of the protein.…”

High-resolution structures of a heterochiral coiled coil

“…We wondered whether this tetrameric assembly would be maintained under the conditions of racemic crystallization, a technique first implemented by Zawadzke and Berg (29) that has become increasingly popular in recent years (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). It has been proposed that racemates are more prone to crystallization than single enantiomers because racemates can access achiral and centrosymmetric packing arrangements in the solid state (45), arrangements that are not available to a chiral entity.…”