Determination Of The X-ray Structure Of A Protein By Total Chemical Synthesis And Racemic Crystallography

All natural proteins are chiral.This chirality is biologically necessary and has an important impact on X-ray crystallography.It is well known that molecules can crystallize in 230 different ways,that is,they can crystallize in 230 different space groups.Notably,chiral molecules only crystallize in 65 of them,the remaining 165symmetry-centered space groups contain a symmetry center or inversion center.Thus,the molecules and their mirror enantiomers should be present in the same quantity,or the molecules themselves are centrosymmetric,whereas the latter is unlikely to exist in the natural proteins.The mixture of chiral molecules and their opposite enantiomers often tends to form racemic crystals where equimolar enantiomers are arranged orderly in the elementary cell.With the development of protein synthesis technology,racemic crystallography have gradually become a powerful and attractive alternative to the structure determination of biomacromolecules.Theory and experiment have proved that the racemic mixture crystallizes more easily than the single chiral compound.In addition,such crystals have only two possible phase angles?0 or??in the centro-symmetric space group,which has more advantages in structure determination compared to achiral space group.In order to reduce the synthetic cost or to use heavy atom derivatives,a discovery that molecules which are almost but not exactly mirror images of each other also have tendency to cocrystallize to form quasi-racemic crystals,has emerged and been widely used.For example,the quasi-racemic crystal of K27-linked di-Ubs was acquired,by mixing K27-linked diubiquitin with D-K27-linked diubiquitin whose Gly₇₆was mutated to alanine.Racemic or quasi-racemic crystallography has gradually become a useful technique for obtaining the crystal structure of biological macromolecules,however,it remains unclear how far the biomacromolecules can differ from its opposite handedness.This paper reported a new discovery that D-monoubiquitin molecules can be co-crystallized with many different L-type diubiquitin,triubiquitin and even tetraubiquitin to obtain quasi-racemic crystals.In these co-crystals,monomeric D-Ubs can form pseudomirror images within different oligomers of L-Ub self-assembly.Undoubtedly,the Monomer/Oligomer Quasi-Racemic Protein Crystallography phenomenon as mentioned above expanded the concept of racemic-crystallography.Using the Monomer/Oligomer Quasi-Racemic Protein Crystallography technology we obtained,the structures of linear tri-,tetra-Ubs as well as a K11/K63-branched tri-Ub for the first time.Furthermore,we also applied racemic crystallization strategy to obtain the crystal structure of the hard-to-crystallize peptide toxin.Polypeptide toxins have been shown to efficiently and selectively target ion channel proteins and can be used as potential drugs for the treatment of related diseases.The author synthesized peptide toxins inhibitors of L-type calcium channels in the myocardium and smooth muscle,calciseptine,FS-2,and calcicludine.The crystal structure of calciseptine,FS-2 and calcicludine was determined by racemic and quasi-racemic crystallization methods.It was also found that calciseptine and FS-2 exhibit a typical three-finger structure,and that calcicludine exhibits a typical Kunitz protease inhibitor structure.The structural information of calciseptine,FS-2,and Calciludine provides the basis for the design of future drug molecules..In summary,herein we report a new quasi-racemic crystallization phenomenon,Monomer/Oligomer Quasi-Racemic Protein Crystallography phenomenon,and applied it to obtain new ubiquitin structures.Moreover,we also obtained new toxin crystals using racemic or quasi-racemic protein crystallography,which provides a structural basis for the development of toxin drugs.