Study Of The Folding Behavior Of Insulin Family Members

This thesis consists of two parts. The first part is the identification of the sequence basis of different folding behavior of insulin and IGF-1. The second part is the investigation of the oxidative folding and reductive unfolding pathway of amphioxus insulin like peptide (AILP).Part one: Insulin folds into one thermodynamically stable structure, while IGF-1-folds into two thermodynamically stable structures. By exchanging the N-terminal sequences of mini-IGF-1 and recombinant porcine insulin precursor (PIP), we prepared four peptide models: [1-9]PIP, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1 by means of protein engineering. Only [1-9]-PIP folds into two thermodynamically stable structures, while the other peptide models, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1, fold into one stable structure as PIP does. The results demonstrated that sequences of B-chain/domain 1-10/1-9 of insulin and IGF-1 determine their different folding behavior, possible mechanism was discussed.Part two: During oxidative refolding process of AILP, besides swap AILP, four major intermediates, P1, P2, P3, and P4, were captured, which were almost identical to those intermediates (U1, U2, U3, and U4) captured during AILP reductive unfolding process. P4(U4) with the native disulfide A20-B19; P1(U1), P2(U2) and P3(U3) with two disulfide bonds which include A20-B19. A schematic flow chart of major oxidative folding and reductive unfolding pathway of AILP was proposed. Three features of the folding behavior of insulin family proteins were proposed as: (1) disulfide A20-B19 is the most important one and formed during the initial stage; (2) formation of the second disulfide is likely random; (3) the in vitro folding/unfolding pathway may be similar and share some common intermediates but reverse. Furthermore, swap AILP was revealed as both thermodynamically final product and productive folding intermediate in AILP folding process, which behavior was between swap insulin and swap IGF-1.