Single-wavelength phasing strategy for quasi-racemic protein crystal diffraction data.

Racemic protein crystallography offers two key features: an increased probability of crystallization and the potential advantage of phasing centric diffraction data. In this study, a phasing strategy is developed for the scenario in which a crystal is grown from a mixture in which anomalous scattering atoms have been incorporated into only one enantiomeric form of the protein molecule in an otherwise racemic mixture. The structure of a protein crystallized in such a quasi-racemic form has been determined in previous work [Pentelute et al. (2008), J. Am. Chem. Soc. 130, 9695-9701] using the multiwavelength anomalous dispersion (MAD) method. Here, it is shown that although the phases from such a crystal are not strictly centric, their approximate centricity provides a powerful way to break the phase ambiguity that ordinarily arises when using the single-wavelength anomalous dispersion (SAD) method. It is shown that good phases and electron-density maps can be obtained from a quasi-racemic protein crystal based on single-wavelength data. A prerequisite problem of how to establish the origin of the anomalous scattering substructure relative to the center of pseudo-inversion is also addressed.

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers.

Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation.

Use of model peptide reactions for the characterization of kinetically controlled ligation.

Since the introduction of kinetically controlled ligation (KCL), a chemoselective reaction between a peptide-(α)thioarylester and a Cys-peptide-(α)thioalkylester, KCL has been utilized for the total chemical synthesis of large proteins (i.e., lysozyme and HIV-protease) by providing fully convergent synthetic routes. Although KCL has the potential to become an important chemistry for protein synthesis, the principle of KCL is not fully characterized. In particular, prior work on KCL has focused on the reactivity difference of the two different -(α)thioester forms-alkyl vs aryl. Another equally important feature of KCL, Xaa-Cys ligation sites, has not been investigated. The work reported here describes combinatorial KCL reactions using model peptides to dissect the interplay of the Xaa(1), Xaa(2), -(α)thioarylester, and -(α)thioalkylester. Results from these studies provide fundamental insights into the KCL reaction, and will lead to the optimal synthetic route for the routine chemical synthesis of large target protein molecules.

Total chemical synthesis and X-ray structure of kaliotoxin by racemic protein crystallography.

Here we report the total synthesis of kaliotoxin by 'one pot' native chemical ligation of three synthetic peptides. A racemic mixture of D- and L-kaliotoxin synthetic protein molecules gave crystals in the centrosymmetric space group P1 that diffracted to atomic-resolution (0.95 Å), enabling the X-ray structure of kaliotoxin to be determined by direct methods.

A semisynthesis platform for investigating structure-function relationships in the N-terminal domain of the anthrax Lethal Factor.

Many bacterial toxins act by covalently altering molecular targets within the cytosol of mammalian cells and therefore must transport their catalytic moieties across a membrane. The Protective-Antigen (PA) moiety of anthrax toxin forms multimeric pores that transport the two enzymatic moieties, the Lethal Factor (LF) and the Edema Factor, across the endosomal membrane to the cytosol. The homologous PA-binding domains of these enzymes contain N-terminal segments of highly charged amino acids that are believed to enter the pore and initiate N- to C-terminal translocation. Here we describe a semisynthesis platform that allows chemical control of this segment in LF(N), the PA-binding domain of LF. Semisynthetic LF(N) was prepared in milligram quantities by native chemical ligation of synthetic LF(N)(14-28)alphathioester with recombinant N29C-LF(N)(29-263) and compared with two variants containing alterations in residues 14-28 of the N-terminal region. The properties of the variants in blocking ion conductance through the PA pore and translocating across planar phospholipid bilayers in response to a pH gradient were consistent with current concepts of the mechanism of polypeptide translocation through the pore. The semisynthesis platform thus makes new analytical approaches available to investigate the interaction of the pore with its substrates.

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

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, L-plectasin 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.

X-ray structure of native scorpion toxin BmBKTx1 by racemic protein crystallography using direct methods.

Racemic protein crystallography, enabled by total chemical synthesis, has allowed us to determine the X-ray structure of native scorpion toxin BmBKTx1; direct methods were used for phase determination. This is the first example of a protein racemate that crystallized in space group I41/a.

Mirror image forms of snow flea antifreeze protein prepared by total chemical synthesis have identical antifreeze activities.

The recently discovered glycine-rich snow flea antifreeze protein (sfAFP) has no sequence homology with any known proteins. No experimental structure has been reported for this interesting protein molecule. Here we report the total chemical synthesis of the mirror image forms of sfAFP (i.e., L-sfAFP, the native protein, and D-sfAFP, the native protein's enantiomer). The predicted 81 amino acid residue polypeptide chain of sfAFP contains Cys residues at positions 1, 13, 28, and 43 and was prepared from four synthetic peptide segments by sequential native chemical ligation. After purification, the full-length synthetic polypeptide was folded at 4 degrees C to form the sfAFP protein containing two disulfides. Chemically synthesized sfAFP had the expected antifreeze activity in an ice recrystallization inhibition assay. Mirror image D-sfAFP protein was prepared by the same synthetic strategy, using peptide segments made from d-amino acids, and had an identical but opposite-sign CD spectrum. As expected, D-sfAFP displays the same antifreeze properties as L-sfAFP, because ice presents an achiral surface for sfAFP binding. Facile synthetic access to sfAFP will enable determination of its molecular structure and systematic elucidation of the molecular basis of the antifreeze properties of this unique protein.

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.

Selective desulfurization of cysteine in the presence of Cys(Acm) in polypeptides obtained by native chemical ligation.

Increased versatility for the synthesis of proteins and peptides by native chemical ligation requires the ability to ligate at positions other than Cys. Here, we report that Raney nickel can be used under standard conditions for the selective desulfurization of Cys in the presence of Cys(Acm). This simple and practical tactic enables the more common Xaa-Ala junctions to be used as ligation sites for the chemical synthesis of Cys-containing peptides and proteins. [reaction: see text].

Direct on-resin synthesis of peptide-alphathiophenylesters for use in native chemical ligation.

[reaction: see text] A peptide-(alpha)thiophenylester is a key reactant in native chemical ligation. Preformation of the peptide-(alpha)thiophenylester could be useful for enhancing the ligation reaction. We report the direct on-resin preparation of preformed peptide-(alpha)thiophenylesters using a simple and efficient method. The peptide-(alpha)thiophenylester reacted extremely rapidly with a Cys-peptide when compared to the peptide-(alpha)thioalkylester.