User:Abudiman99/Racemic crystallography



Racemic crystallography is a technique used in structural biology where crystals of a protein molecule are developed from the mixture of an ordinary chiral protein and its mirror image. These protein molecules consist of 'left-handed' L-amino acids that can be produced in bacteria, yeast or other cellular expression systems, whereas the mirror image of the protein molecules consist of 'right-handed' D-amino acids prepared by chemical synthesis. It has been used as a method to manufacture structures with high amounts of protein by increasing the rate of success in crystallizing proteins as well as eliminating unwanted phases in X-ray diffraction.

Manufacturing
Chemical ligation is done to prepare proteins with polypeptide chains where peptides are joined by one another to form a polypeptide chain, which is then folded to form a protein chain. The most common type of chemical ligation done towards proteins is native chemical ligation (NCL) where a C-terminal peptide thioester reacts with an N-terminal cysteinyl peptide to produce a peptide bond. Then, the purity and covalent structure of the proteins are verified by liquid chromatography–mass spectrometry (LCMS). The formation of the folded structure is verified by multidimensional NMR, whereas the structure of the synthetic protein is determined by X-ray crystallography. D-protein enantiomers can be manufactured using synthetic peptide building blocks made from protected D-amino acids and Gly once the chemical synthesis of L-protein is achieved.

Convergent synthesi s is also effective in preparing long polypeptide chains by using peptide-hydrazide, where the hydrazide is synthetically equivalent to a thioester synthon. It is stable to the conditions of NCL, and can be converted in situ to a reactive peptide-thioester for the next NCL condensation reaction.

Theory
As molecules form themselves in crystal form, they arrange themselves in space group symmetry, which describes the orientation between the molecules within the crystal. In general. proteins have a total of 230 different space groups that exist in nature. However, only 65 are chiral and can exist in nature, whereas the remaining 165 are centrosymmetric which requires the molecule and its mirror image to be the same amount inside the crystal. These proteins have preferences for certain space groups where they would have higher chances of producing more optimal chiral space groups. Wukowitz and Yeates suggested that proximity between proteins enable the molecules to have closer contact with each other in order to optimize connection within the crystal. This leaves the chiral space group to be determined by a number of degrees of freedom (D) or dimensionality to indicate the ease of how a given symmetry can be formed. Meanwhile, the number of degrees of freedom was analyzed for achiral space groups where it was found that the space group with D=8 is the most dominant space group observed, which was indicated as P1(bar). Since the achiral space group had a higher degree of freedom of compared to the chiral space groups, it was predicted that racemic proteins with P1(bar) would crystallize more easily compared to regular proteins. Space group P1(bar) is strongest, and P21/c and C2/c commonly appear more, whereas the other achiral space groups are expected to appear less. Hence, P1(bar), P21/c, and C2/c are considered as centrosymmetric space groups in racemic mixtures.

Early developments
In 1989, Alan Mackay suggested synthesizing protein enantiomers would enable the use of racemic mixtures to crystallize proteins in centrosymmetric space groups. He stated that simplifying possible phases would facilitate overcoming the phase problem in protein structure determination through X-ray crystallography. This indicated that for X-ray data obtained from a centrosymmetric crystal, the off-diagonal phases cancel and all phases differ by 180 degrees.

In 1993, Laura Zawadzke and Jeremy Berg used small (45 amino acids) protein rubredoxin and synthesized in racemic form. This was done since the structural determination would potentially be easier and more robust by using diffraction data from a centrosymmetric crystal, which requires growth from a racemic mixture. It was also pointed out that the rubredoxin crystal was achiral and had symmetrical centers, indicating that its phase in X-ray diffraction took exclusively 0 or 180 degree orientation. By having a symmetric center imposed by the racemic proteins, steps of phasing diffraction in data analysis can be further simplified. In 1995, Stephanie Wukovitz and Todd Yeates tested to see why protein molecules tend to crystallize more optimally in certain symmetries, where they predict that protein can crystallize more easily in racemic form where favored racemic crystal symmetries can only be obtained when using a racemic protein mixture. Hence, they inferred that racemic proteins are much easier to crystallize than natural proteins due to the theoretical increase in the number of feasible protein crystalline forms.

Recent applications
Racemic crystallography has been studied further to determine the structures of different proteins.

Rv1738, a protein of Mycobacterium tuberculosis that enables the bacteria to enter dormancy, typically resists extensive attempts to crystallize in order to expand this protein. However, crystals were able to be obtained from a racemic mixture of D and L-proteins of Rv1738 where the crystal forms under the centrosymmetric space group C2/c using racemic crystallization. The structure, containing L- and D-dimers in a centrosymmetric space group, revealed bacterial hibernation-promoting characteristics that can bind to ribosomes and suppress translation.

Crystallization of ubiquitin protein was successfully done using racemic crystallography. Crystallization of either D or L-ubiquitin had failed, whereas a racemic mixture of D-ubiquitin and L-ubiquitin was crystallized where its diffraction quality crystals were obtained overnight in almost half the conditions tested in a standard commercial crystallization screen.

Crystallization of disulfide microprotein molecules was done to determine the structure of trypsin inhibitor SFTI-1 (14 amino acids,1 disulfide), conotoxin cVc1.1 (22 amino acids, 2 disul-fides) and cyclotide kB1 (29 amino acids, 3 disulfides). Using X-ray diffraction to characterize, it was found that the racemates crystallized in the centrosymmetric spacegroups P3(bar), Pbca and P1 (bar).

A high-resolution crystal structure of the racemate of a heterochiral D-protein complexes used in vascular endothelial growth factor A (VEGF-A). D-protein form of VEGF-A was used to identify a 56 residue L-protein binder with nanomolar affinity. A mixture of chemically synthesized proteins consisting of D-VEGF-A, L-VEGF-A, and two equivalents each of the D-protein binder and L-protein binder, gave racemic crystals in the centrosymmetric space group P21/n.