Human insulin (HI) is a small globular protein which regulates the blood sugar level. The formation of insulin fibrils is an intensively studied problem, since it has a dual implication in science: on one side, HI is used as a model peptide to understand the mechanism of fibril formation; on the other, HI based drugs are used to treat diabetic people. In the latter case, fibril formation should be avoided since it reduces the effectiveness of the medication, and it might have negative counter effects. The picture of insulin fibril formation is further complicated by its oligomeric forms such as monomers, dimers and hexamers found in nature. The relative concentration of each species can be controlled by the solution concentration and the addition of metal ions. We here focus on the role of monomers, since they are the only species that can undergo denaturation [1]. During the production and storage of insulin based drugs the air/water interface is omnipresent under conditions of liquid flow and/or agitation. Previous studies showed that insulin solutions become more stable upon agitation, if dimers are present [2] (e.g. high concentration). So far, the role of the air/water interface has not been clarified yet. While insulin has been studied intensively in bulk solution, using e.g. infrared spectroscopy and circular dichroism, the role of surface interactions for the structure of protein and/or protein aggregation remains a challenge to overcome. Here we propose to use sum frequency generation (SFG) spectroscopy to study the oligomeric composition of HI at the air/water interface. SFG is a second order nonlinear optical technique which provides surface specific vibrational spectra. We combined SFG experiments, with calculations of the second order nonlinear susceptibility of HI dimers and monomers and we found that the experimental results can only be explained by the exclusive presence of monomers at the interface. We show that independent of the oligomeric composition of the bulk HI solutions, only monomers segregate at the air/water interface. The hydrophobic nature of the air/water interface favors monomers - which are prone to aggregation and fibrillation - to the water surface. This finding is an important step towards solving the long lasting puzzle of insulin fibrils formation. [1] Brange J. et al. Journal of Pharmaceutical Sciences, 86(5), 1997 [2] Sluzky, V. et al. PNAS, 88(21), 1991

Structure of insulin at the air/water interface: monomers or dimers?

249th National Meeting (2015)

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