Conserved and novel structural characteristics of enantiomorphic Leu‐enkephalin

Doi, Mitsunobu; Ishibe, Atsuo; Shinozaki, Humiyoshi; Urata, Hidehito; Inoue, Masatoshi; Ishida, Toshimasa

doi:10.1111/j.1399-3011.1994.tb00526.x

Cited by 23 publications

(10 citation statements)

References 18 publications

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“…It is noteworthy that crystals of racemic plectasin grown under completely different conditions all belonged to the P1 space group. Including the current work, to date six2, 5–9 of the seven previously reported racemic peptides or proteins have crystallized in space group P1, as predicted by Wukovitz and Yeates 24. With the high‐resolution diffraction data from crystals of racemic plectasin, we were able to use direct methods to solve the structure of the plectasin molecule to atomic resolution.…”

Section: Discussionmentioning

confidence: 80%

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Racemic crystallography of synthetic protein enantiomers used to determine the X‐ray structure of plectasin by direct methods

et al. 2009

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We describe the use of racemic crystallography to determine the X-ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers L-plectasin and D-plectasin were prepared by total chemical synthesis; interestingly, Lplectasin showed the expected antimicrobial activity, while D-plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X-ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group P1 with one L-plectasin molecule and one D-plectasin molecule forming the unit cell. Dimer-like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%-15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation.

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Section: Discussionmentioning

confidence: 80%

“…in P1, all phases are 0 or π) 4. However, of the centrosymmetric racemic protein structures reported to date,2, 3, 5, 6 only the small scorpion toxin protein BmBKTx1 has been solved by direct methods,3 although three small peptides with not more than 12 amino acid residues have also been solved as racemates by direct methods 7–9…”

Section: Introductionmentioning

confidence: 99%

Racemic crystallography of synthetic protein enantiomers used to determine the X‐ray structure of plectasin by direct methods

et al. 2009

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“…Despite suggestions that direct methods should be more feasible with racemic crystals,16, 17, 18 it is noteworthy that none of the three centrosymmetric racemic protein structures previously reported was solved using direct methods,1, 8, 12 although three small peptide racemates have been solved by direct methods with some difficulty 9–11…”

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confidence: 99%

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

et al. 2009

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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...

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“…In 1989 Alan Mackay proposed in a letter to Nature that 'co-crystallization of equal numbers of D-and L-molecules might produce crystals of a racemate, which could be centro-symmetrical with a consequent simplification in the process of solution' [70]. Subsequently, this assumption was put to test by the chemical synthesis of small D-peptides [71,72] and the 45 amino acid iron binding protein rubredoxin [73] and their L-counterpart. In a more theoretical approach on protein crystallization Wukovitz and Yeates predicted that racemic protein mixtures will be more easily to crystallize than the corresponding biological enantiomers [74].…”

Section: Native Chemical Ligation (Ncl)mentioning

confidence: 99%

Quest for the chemical synthesis of proteins

Engelhard

2016

Journal of Peptide Science

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The chemical synthesis of proteins has been the wish of chemists since the early 19th century. There were decisive methodological steps necessary to accomplish this aim. Cornerstones were the introduction of the Z-protecting group of Bergmann and Zervas, the development of Solid-phase Peptide Synthesis of Merrifield, and the establishment of Native Chemical Ligation by Kent. Chemical synthesis of proteins has now become generally applicable technique for the synthesis of proteins with tailor made properties which can be applied not only in vitro but also in vivo .Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

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Conserved and novel structural characteristics of enantiomorphic Leu‐enkephalin

Cited by 23 publications

References 18 publications

Racemic crystallography of synthetic protein enantiomers used to determine the X‐ray structure of plectasin by direct methods

Racemic crystallography of synthetic protein enantiomers used to determine the X‐ray structure of plectasin by direct methods

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

Quest for the chemical synthesis of proteins

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