Chemical Synthesis of Polyubiquitin Chains for Structure and Crystallization Studies

Author:Zheng Yong

Supervisor:tian chang zuo


Degree Year:2019





Protein chemical synthesis is an important pathway to obtain homogeneous proteins.Although traditional biological expression strategy can be applied to obtain varieties of target proteins,some characteristic proteins,including toxin peptides or poly-disulfide proteins,post-translational modification proteins,membrane proteins,and natural activity-based protein probes are still rigorously limited,which highlight the necessity for the development of protein chemical synthesis.Chemical synthesis has three major advantages over biological expression:a)High degree of environment compatibility.b)Precise control at the atomic level.c)Providing protein functional extension.Ubiquitin acts as a novel post-translational modification protein that is involved in all aspects of the cell’s life cycle.The process of ubiquitination is mainly carried out by the cooperation of E1 activating enzyme,E2 binding enzyme and E3 ligase.Since ubiquitin has multiple self-ubiquitinated sites(M1,K6,K11,K27,K29,K33,K48,K63),it can form polyubiquitin chains with different linkage types and chain lengths.The difference in chain length and chain type results in a difference in the structural diversity,which can be recognized by different receptor proteins to execute divergent functions.To elucidate the molecular mechanism of polyubiquitin chain regulation,a key requirement is to obtain different types of homogeneous polyubiquitin chain.In virtue of chemical synthesis,we obtained different linkage types of polyubiquitin chains,and carried out two aspects of work.In the first part of the work,we obtained the K27 linkage type of diubiquitin and triubiquitin by total chemical synthesis.Using quasi-racemic crystallization,we successfully obtained and resolved the crystal structure of K27-linked di-and tri-ubiquitin for the first time.We found that the isopeptide bond of the K27 ubiquitin chain is embedded inside ubiquitin,which is different from the other seven types of ubiquitin chains.In addition,we used OTUD2 Deubiquitinase to test the hydrolysis of these two ubiquitin chains,and found that the hydrolysis of triubiquitin is significantly faster than diubiquitin,indicating the existence of different binding patterns of receptor protein towards different chain length.This finding is important for us to study the functional properties of the K27 ubiquitin chain.In the second part of our work,we reported a crystallization method named monomer/oligomer quasi-racemic proteins for ubiquitin chain crystallization.By using chemically synthesized monomeric D-type ubiquitin,we co-crystallize it with different types of L-type diubiquitin,triubiquitin or even tetraubiquitin.We were surprised to find that this newly racemic crystallization can assist to obtain crystal faster and easier.Using this novel crystallization-assisted strategy,we obtained the crystal structure of a linear M1-linked tri-and tetra-ubiquitin chain and a K11/K63 branched triubiquitin for the first time.This strategy extends the definition of quasi-racemic crystalgraphy and is expected to be further applied to the crystallization of multi-domain proteins.In the third part of the work we shifted our focus to the synthesis of post-translationally modified histones.By inserting the serine-serine pseudo dipeptide into the fragment synthesis of histone H3,we successfully improved the efficiency of H3 with total chemical synthesis on tens of milligram scale.By analyzing the secondary structure of the bivalently modified H3 with circular dichroism spectroscopy,we found that the trimethylation and acetylation changed the histone H3 a-helix structure compared to the recombinant histone H3,which was demonstrated by short-wavelenth shift.Finally,we successfully assembled this bivalently modified histone H3 into nucleosomes.Here we developed an improved three-segment ligation strategy for the efficient synthesis of bivalently modified histone H3,and this strategy can provide a valuable tool for the synthesis of other histone H3 with multiple post-translational modifications.