Solution Structure Of Saccharomyces Cerevisae Urm1 And Its Evolutionary And Functional Significance

Our work focuses on the cloning, expression, purification and solving the solution structure of S. cerevisiae Urm1 protein by NMR method. Structure of Urm1 is based to infer the evolutionary relationships between the protein modifiers and sulfur carrier proteins and explore the function and interaction pattern of the Urm1 conjugation system. We use both structural comparison and phylogenetic analysis of the ubiquitin superfamily to test the hypothesis that Urm1 is a "molecular fossil" in the superfamily and has the most conserved structural features of the family's common ancestor. We presumed a possible partner-binding interface of Urm1 to Uba4. Besides, we are studying the targets of Urm1 conjugation system using proteomic strategy and methods.First chapter is the brief review of protein modifiers. Classification of protein modifiers and their biological functions were introduced. Till now, 11 protein conjugation systems have been identified, include: Ubiquitin, NEDD8/Rubl, SUMO, ISG15/UCRP, FAT10, FUB1, UBL5/Hub1, Atg8, Atg12, Ufm1 and Urm1 conjugation systems. We focused on introducing the investigation background of Urm1 conjugation system. The gene for all urmylation pathway proteins, Urm1 and Uba4, is essential for S. cerevisiae viability during budding in vegetative growth and is shown to play a role in invasive growth into agar in the haploid state and in pseudohyphal growth and cell elongation under starvation conditions in the diploid state. A functional cross-link between the TOR (target of rapamycin) signaling pathway and the urmylation pathway was also detected, in which Urm1 was shown to be involved in nutrient sensing. It was found that Urm1 covalently attaches to antioxidant protein Ahp1 to modulate its activity in oxidant-stress response. However, much still remains to be learned about the biological function and functional mechanism of the Urm1 conjugation system. We also introduced the evolutionary study on protein modifiers in this chapter.The second chapter introduced the primary theory and study method on molecular evolution. 'Molecular clock' and 'Neutral theory' based the investigation on molecular evolution. Construction of evolutionary and phylogenetic tree is usually used to study molecular evolution. The steps of constructing of evolutionary and phylogenetic tree include: 1. sequence alignment orientated data model. 2. Determination of substitute model. 3. Selective constructing. 4. Query of phylogenetic tree. 5. Root determination. 6. Estimation of phylogenetic tree. We also introduced the characters of protein evolution and identification of protein evolutionary relationships.In the third chapter, we solved the solution structure of S. cerevisae Urm1 and analyzed its evolutionary implications. The gene coding full length Urm1 protein was obtained by PCR from Saccharomyces cerevisae(S288C) genome. Recombinant Urm1 was cloned and expressed in E.coli and was purified using Ni-chelating column. The solution structure of S. cerevisae Urm1 was obtained by heteronuclear three-dimensional spectroscopy and calculated with the supplement of computer softwares. The structure of Urm1 contains one five-strandβ-sheet and fourα-helices. Theβ-sheet is arranged in the order 21534, as in ubiquitin; four helices together back the curvedβ-sheet on the concave side; Interaction between the inner face of theα2-helix and the concave face of theβ-sheet form a hydrophobic core, which is essential to maintain the compact fold. The short one-turnα4-helix follows theβ4-strand tightly. Long-range NOEs were observed from the N terminus (residues 33-35) of a2-helix to the residues (80-83) after the C terminus ofα4-helix. The C terminus (residues 95-99) is flexible and protrudes from the globular fold as a tail. Structural comparison indicates that Urm1 shares higher structural similarity with ThiS and MoaD than with other protein modifiers. The similarities of 3D structure and hydrophobic and electrostatic surface features between Urm1 and MoaD suggest that they may interact with partners in a similar manner. Both structural comparison and phylogenetic analysis of the ubiquitin superfamily, with emphasis on the Urm1 family, indicate that Urm1 is the unique 'molecular fossil' that has the most conserved structural and sequence features of the common ancestor of the entire superfamily. On the other hand, it is proposed that, analogy to MoaD, the partner-binding interface of Urm1 may consist of a hydrophobic region exposed to solvent and some charged residues. We believe that the solved Urm1 protein structure, which is considered to be a critical piece of evidence for inferring the evolutionary origin of the ubiquitin superfamily, would also be extremely informative for further investigation of the complex function and mechanism of the modification pathway during the evolution of protein modifiers.The fourth chapter introduced the work processed on identifying the modification targets of Urm1 conjugation system. The concepts of proteomics and the study strategy and methods were introduced firstly. Then the strategy for proteomic studies of protein modifiers was discussed. To identify potential targets of Urm1 conjugation system is significant for uncovering the biological process the system involved and its functional mechanism. Till now, we have successfully constructed the yeast strain which can be induced to express His-tagged Urm1 protein. We also have set up a primary strategy for purifying and enriching Urm1 and Urm1 conjugated proteins. However, we can not detect the Urm1 protein expressed by wild-type strains and the Urm1 conjugated proteins seem to be missed during purification according to Western blot results. In the future work, purification strategy and detection method should be optimized and we will try to analysis the purified product by 2DLC-MS/MS method.