Structural Studies Of Proteins By Single Particle EM

EM single particle technique has been developed vastly to become a mainstream structural biology method in recent years.In this dissertation two proteins were investigated via EM single particle analysis.In part?,the structure of Urea Amidolyase holoenzyme was solved.Urea is a metabolite of many nitrogen-containing biological molecules（such as arginine,purine,or ureide,etc.）,which may be converted to inorganic ammonia by Urea Amidolyase（UA）to reenter the anabolic pathway.By this way nitrogen is recycled in the bio-sphere.UA is composed of urea carboxylase（UC）and allophanate hydrolase（AH）,breaking down urea in two steps.UC carboxy-lates urea to generate allophanate which is further hydrolysed by AH into NH₃and CO₂.UC con-sists of biotin carboxylase（BC）domain,carboxyl transferase（CT）domain and biotin-carboxylcarrier protein（BCCP）domain,belonging to biotin-dependent carboxylase family.It catalyzes a two-step reaction of urea carboxylation.Firstly,biotin linked to BCCP is carboxylated in BC.Then carboxylated BCCP-biotin is transferred to CT where it transfers its carboxyl to ureaproducing allophanate.AH hydrolyses allophanate via two steps as well in its N-domain and C-domain respectively.So,as a multi-domain enzyme with multi-activity,there must be close cross-talks in between components of UA for effective transfer of intermediates.Particularly,unlikeother family members being active as oligomers,UC is active as monomer.Crystal structures of UC and AH have been resolved now,with the former in a conformation of BCCP-biotin binding to the CT active site.However,the architecture of UA holoenzyme is still unknown and the confor-mational changes in the process of activity unravelled.In this study,we determine the architecture and oligomeric mechanism of UA holoenzyme by the EM single particle technique for furtherinvestigation the conformational changes of UA in the process activation.In part II,the architectural plasticity of AMPK protein revealed by single particle EM and X-ray crystallography was analyzed.Mammalian AMP-activated protein kinase（AMPK）acts as an important sensor of cellular energy homeostasis related with AMP/ADP to ATP ratio.Theoverall architecture of AMPK has been determined in either homotrimer or monomer form byelectron microscopy（EM）and X-ray crystallography successively.Accordingly proposed models have consistently revealed a key role of theαsubunit linker in sensing adenosine nucleosidebinding on theγsubunit and mediating allosteric regulation of kinase domain（KD）activity,whereas there are vital differences in orienting N-terminus ofαsubunit and locating carbohydrate-binding module（CBM）ofβsubunit.Given that Mg²⁺,an indispensable cofactor of AMPK waspresent in the EM sample preparation buffer however absent when forming crystals,here wecarried out further reconstructions without Mg²⁺to expectably inspect if this ion may contribute to this difference.However,no essential alteration has been found in this study compared to ourearly work.Further analyses indicate that the intra-molecular movement of the KD and CBM are most likely due to the flexible linkage of the disordered linkers with the rest portion as well as a contribution from the plasticity in the inter-molecular assembly mode,which might ulteriorlyreveal an architectural complication of AMPK.