Dissecting The Role Of Disulfide Bonds On The Amyloid Formation Of Insulin

Disulfide bonds play critical role in the stability and folding of proteins. Here, weused insulin as a model system, to investigate the role of its individual disulfide bondduring the amyloid formation of insulin. Tris(2-carboxyethyl)phosphine (TCEP) wasapplied to reduce two of the three disulfide bonds in porcine insulin and the reduceddisulfide bonds were then alkylated by iodoacetamide. Three disulfide bond-modifiedinsulin analogues, INS-2(lack of A6-A11), INS-3(lack of A7-B7) and INS-6(lack ofboth A6-A11and A7-B7), were obtained. Far-UV circular dichroism (CD)spectroscopy results suggested the secondary structure of INS-2was the closest toinsulin under neutral condition, followed by INS-3and INS-6, whereas in acidsolution most analogues were essentially unfolded. To test how these modificationsaffect the amyloidogenecity of insulin, thioflavin-T (ThT) fluorescence, transmissionelectronic microscopy (TEM) were performed. Our results suggested that allanalogues were more prone to aggregate than insulin, the aggregation rates fall in theorder of INS-6> INS-3> INS-2. Cross-Linking of unmodified proteins (PICUP)assay showed that analogues without A6-A11(INS-2and INS-6) have higherpotential for oligomerization than insulin and INS-3, which is accompanied withhigher cytotoxicity as the hemolytic assays of human erythrocytes suggested. Theresults indicated that breakage of A7-B7induced more insulin structure unfolded andhigher amyloidogenicity than breakage of A6-A11, but breakage of A6-A11causedsignificant cytotoxicity increase and a higher potency to form high order toxicoligomers.