Structural And Functional Study Of Biomacromolecules By Synchrotron Radiation Circular Dichroism

This thesis presents the investigation on the structure and conformational transition of biomacromolecules in solution by synchrotron radiation circular dichroism (SRCD) spectroscopy, combined with X-ray crystallography, dynamic light scattering (DLS), atomic force microscopy (AFM), etc. It involves three different aspects:(1) Identifying point-mutations of human phosphoribosyl pyrophosphate synthetase 1 (PRS1) in cells of patients with primary gout; (2) Observing effects of ATP on the conformation and thermal unfolding of the hen egg white lysozyme (HEWL); and (3) Investigating the conformational transitions of beta sheet proteins: N domain of the silk fibroin heavy chain (Fib-H N domain). The secondary structure analysis for biomacromolecules presented in this thesis is helpful for understanding the structure-biofunction relationship, and additionally demonstrates the valuable applications of SRCD spectroscopy in the biological science.1. Mechanism of PRS1 superactivity caused by the point-mutationsSome cases of point mutation of PRS1 have long been thought to cause PRS1 superactivity, which is one reason of primary gout. In this work, the differences in secondary structure and aggregation state between recombinant wild-type PRS1 and six point-mutations are studied by spectroscopy methods combining with the crystal structure analysis of wild-type PRS1. It is found that the activity of PRS1 does not depend on the subunit-aggregation that has been thought to be necessary for activated state; however, the substrate ATP can induce monomeric wild-type PRS1 to form hexamer which is the structural basis for the ADP binding. Under physiological conditions, the production of ADP downstream is a negative feedback inhibitor that controls the catalytic activity of PRS1 at the normal level. When point mutation occurs, the enzymatic activity dramatically increases, and the aggregation ability of PRS1 is greatly weakened or even lost. Furthermore, SRCD reveals that the point-mutations result in the alteration of ATP-binding conformation. Examination of the crystal structure of wild-type PRS1 indicates that the alteration of ATP-binding conformation can lead to the breakage of the PO43- binding site. Therefore, in the cases of point-mutants, these factors result in an increased concentration of the product PRPP in the patient, and may finally cause gout.2. ATP-induced instability, noncooperative melting and aggregation of HEWL To gain insight into the fibril formation mechanism of proteins, the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light scattering, and DSC, is used to investigate the conformational changes and thermal unfolding of the hen egg white lysozyme (HEWL) in the presence of ATP, ADP, AMP, and Mg2+-ATP complex. The results indicate that the ATP can bind to HEWL, and the strong electrostatic effect of ATP phosphate groups changes tryptophan residues into more hydrophobic environments, and alters the secondary structures of HEWL. This effect decreases the thermal stability of HEWL, induces a noncooperative melting of secondary structures in the unfolding process of HEWL, and sequentially produces a partially unfolded intermediate which contains relatively rich helical structures and lessβ-sheet structures. This study suggests that the extent of denaturation of the amyloidogenic fragments, rather than monomericβ-sheet enriched intermediate, is critical for the fibril formation of proteins.3. Conformational transitions of Fib-H N domain (FN)Four FN segments with different length are prepared for exploring the function of FN in silk spinning process. The SRCD spectroscopy and AFM are used to investigate the conformational and morphological changes of FN in different conditions. This study reveals that FN has the similar structure transition phenomena as regenerated silk functions. All four FN can form the nanoglobular structure with an average diameter dimension ranging from 50 to 100 nm providing that the second structure of the proteins is changed toβ-sheet. These nanoglobular can fuse into each other to form an irregular reticulation. Moreover, the longer FN segment is much easier to have conformational transition and to form the nanoglobular structure. All these results indicate that FN can involve in the silk spinning as a folding promoter instructing the fibroin structure transition.