In situ Measurement of Human islet Amyloid Polypeptide Misfolding at Lipid/Water Interfaces Probed by sum Frequency Generation Spectroscopy

Abstract

Kinetic analysis of conformational changes of proteins at interfaces is crucial for understanding many biological processes at membrane surfaces. We applied surface-selective sum frequency generation (SFG) spectroscopy to investigate the kinetics of conformational changes of an intrinsically disordered protein, human islet amyloid polypeptide (hIAPP), which is known to misfold into β-sheet upon interaction with the lipid/water interface. We observed changes in the amide I spectra of hIAPP in the presence of dipalmitoylphosphoglycerol (DPPG) that correspond to lipid-induced changes in protein secondary structures. We obtained both the achiral and chiral SFG spectra. Using the achiral-sensitive ssp (s-polarized SFG, s-polarized 800 nm, p-polarized infrared) polarization, we observed a gradual shift of the amide I spectrum of hIAPP. Using the chiral-sensitive psp polarization, we observed a gradual buildup of the chiral structures that display characteristics of parallel β-sheets, including a dominant peak at ∼1620 cm−1 and a shoulder at 1660 cm−1, which is in good agreement with the theoretical prediction based on the hIAPP parallel β-sheet structure. Moreover, we used ab inito calculation to obtain the hyperpolarizibility of the hIAPP β-sheet aggregates, from which we determined the orientational angle of the β-sheet strand to be ∼45° at interfaces. We discuss the implication of such insertion of the β-sheet aggregates into the membrane in relation to the hIAPP induced permeability in cell membrane. Also, we propose that the second-order chiral-optical response from the hIAPP β-sheet aggregates could be a highly characteristic optical property for other amyloid, and thus potentially useful for in situ amyloid detection. Our study demonstrates that SFG is an in situ and label-free technique, which can provide both kinetic and structural information to probe protein conformational changes at interfaces.