Protein Conformational Transition And The Accumulation Of Molecular Mechanism

Correctly folded protein is the basis of normal biological function. Some mis folding diseases, especially neurodegene rative diseases are thought to be closely associated with conform a tional transformation and abnorm alaggregation of proteins. IgGbinding domain B1 of protein G(PGBD), which has compact folded structure and strong stability , is an ideal model for sequence insertion and displa cem ent. W e have designed two PG BD chim eras, in which th e prim itive α helix segm ent of PGBD was replaced by the α helix sequence or β 2sheet sequence of IgGbinding dom a in B1 of pr otein L (PLBD). The resultant proteins were nam e d PGBDL α a nd PGBDL β resp ective l y . Rela tive to wild type PGBD, the α helix con t ent of PGBDL α decreases and the overall stru cture becom e s unstable, while PGBDL β is unstructured. Accordingly , it is demonstrative that dif f erent secondary structural elem ent m a y a dopt di f f erent secondary structural confor m a tion despite bein g accomm odated in th e sam e st ru ctural env i ronm ent. The stru ctu r al propensity of a segm ent as well as the inte r actio ns between side chain s within the protein ensem b le can significantly influence its secondary structural form ation. By sequence alignm ent of various a ggregationprone proteins, such as β a m y loid peptide, prion protein and α sy nuclein ( α Syn), a hydrophobic hom o logous sequence VGGA V V A GV , na m e ly GA V motif, has been identified. W e constructed two variants that accom m odate the GA V seque nce in the cen t ra l loop reg i on (nam ed GA V INS) or the Cterm inus (na m ed GA V CT) of PGBD. Biophysical analysis, particularly atom ic force m i croscopic im aging show that GA V INS but not GA V CT , for m s abundant am orphous aggregates. The results indicate that aggregation f o rm ation of the f u sion proteins is rela ted to n o t only the self assem b ly ability of GA V segm e n t but also the structural environm ent nearby and the position of GA V sequence in PGBD. By sitedirected mutagenesis combinated with thioflavin T fluorescence detection, atomic force microscop icimaging as well as cytotoxic ity assays, we report at the first time that anineresidue segment VGGAVVT GV,is essential for f i brilliza tion and cytoto xicity of α Syn. Deletion of the c o re sequence im pedes f i brillar f o rm ation and inhibits toxicity to PC12 cells. Me anwhile, su bstitu tion of Gly68 with Ala or Cterm inal truncation of α Syn accelerates the fibrillizatio n processes. Circular d i chroism stu d ies su ggest that β sheet form ation is ofte n concom itant with the filam e nt for m ation. More details about α Syn aggregation were also inves t igated by seeded aggreg ation experim e nt, incorporation of fluorescence probes, TFEinduced structural transfor m a tion and solidstate CD technique. Taken together, this finding may pr ovide further insight into the understanding of the molecular m echanism underlying the fibrillog e nes i s im plicated in neurodegeneration as well as developm ent of transgenic m odels and aid drug design. The GAV p e ptide (VGGAVVAGV) was also chem ically synthesized and its property of aggregation was studied in vitro . Under a certain condition, GAV peptide not only selfassem b lies into filam e nts but also facilitates the aggregation of α Syn , which dem onstrates that the GAV sequence plays a vital role in am yloidogenesis.