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.

Converging on the goal: New chemistry enables the key Cys‐peptide‐(αthioester) intermediate to be extended by ligation at either the N or C termini in a controlled fashion. This approach forms the basis for a set of novel tactics for the fully convergent synthesis of proteins by the chemical ligation of multiple peptide segments.

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].

Dem Ziel nahe: Mit einer neuen Strategie gelingt es, das Schlüsselintermediat Cys‐Peptid‐αThioester durch Ligation am N‐ oder am C‐Terminus kontrolliert zu verlängern. Dieser Ansatz ist die Grundlage für eine vollständig konvergente Synthese von Proteinen durch die chemische Ligation einer Vielzahl von Peptidsegmenten.

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.