Investigation of the Unfolding and Folding Pathway(s) of Snake Toxins From the Taiwan Cobra (Naja naja atra)

• Main Authors: Sivaraman-T, 席爾曼
• Other Authors: Chin Yu
• Format: Others
• Language: en_US
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/26842147854297627136

博士 === 國立清華大學 === 化學系 === 87 === Cardiotoxin III (CTX III) is a small, an all b-sheet protein, purified from Taiwan Cobra (Naja naja atra). In the present study, the structure, stability and folding pathway(s) of this protein has been systematically probed. The first chapter gives a brief overview on many concepts that exist in the problem of protein folding. In addition, this chapter traces the developments that have occurred in the pursuit of understanding the rules of the protein folding game. In chapter 2 of the thesis, a description of the purification protocol is described in detail. This chapter also brings to fore the raging controversy that persists in the nomenclature of cardiotoxins derived from snake venom sources. An attempt has been made in this chapter to offer our stand on this controversial issue of nomenclature of cardiotoxins. Chapter 3 of this dissertation provides a vivid description of the solution structure of cardiotoxin III obtained by refinement of the co-ordinates estimated previously. Chapter 4 of this thesis a repository of data obtained on the equilibrium unfolding of CTX III under a variety of denaturing conditions. At the out set this chapter describes the effect of various aliphatic alcohols on the structure of CTX III. A thorough analysis of the modes of action of various alcohols interms of induction of helical conformation (in proteins) is also provided in this chapter. This chapter also describes the structural features of partially structured intermediate states (akin to molten globule states) along the acid and alcohol-induced unfolding pathway of CTX III. A section of this chapter has been devoted to describe the interlink between acid (2,2,2-trichloroacetic acid) induced protein precipitation and induction of ''molten globule''''-like states in acid mediated unfolding of proteins. The hallmark of this chapter is the description of the structural stability and dynamics of the protein (CTX III) as probed by native state amide exchange kinetics. The concluding portion of this chapter evaluates the relationship between the structure and the temperature co-efficient of individual residues in CTX III. This chapter provides a detailed account of the methodology and the logistics involved in the use of quenched-flow hydrogen-deuterium techniques to monitor folding events of proteins at the atomic level. Using a variety of biophysical techniques in conjunction with quenched-flow hydrogen-deuterium exchange, frame the events in the refolding pathway have been traced This chapter also provides a comparative the folding kinetics of other all b-sheet proteins with that of CTX III. An analysis of the relationship between hydrogen exchange and the chronology of events in the folding pathway(s) is also given in this chapter. The concluding chapter (chapter 6) provides on interesting comparison of the folding kinetics of two homologous proteins, cobrotoxin (CBTX) and CTX III, isolated from the Taiwan cobra. An exhaustive analysis of the structural stability of CBTX and CTX III can also discerned in this chapter. The chapter concludes with the evaluation of the applicability of the ''first-in - last-out'' hypothesis to describe the folding and stability of CBTX. Finally, results described in this thesis, I believe is just a trigger for the sequence of thought-provoking studies on the all b-sheet snake toxins in the near future.