A Conventional and 2DCOS Infrared Study of Amyloid Fibril Formation

Abstract

Amyloidosis is characterized by the abnormal self-assembly and deposition of proteinaceous material into insoluble ordered aggregates. There are several pathologies associated with this aggregates, known as “protein misfolding” disorders. In many proteins, when heated at high temperature and low pH, a series of structural changes resulting in the formation of fibrillar structures are produced. Insulin is a model of fibril formation that has produced a wealth of biochemical and structural data. The time-course of fibril formation can be followed by infrared spectroscopy looking at the appearance of a characteristic band in the amide I region. The kinetics is triggered by temperature at 70 °C and pH at 2.3. The infrared spectrum shows, that after a lag time (concentration-dependent), α-helix and β-turns bands decrease first whereas the random coil component increases. Later, a band at 1626 cm-1, associated with extended chains, replaces the random coil component. Infrared 2D-COS has been applied to different stages of the process. Maps have been formed at different incubation times: before random coil formation and at different stages in the random coil-fibril exchange. The synchronous maps do not give in principle much information because they are dominated by the appereance of the band due to the fibril. In order to extract such information we have developed an approach in which we take a window and move it along the perturbation (2D-moving lapse). This, together with the asynchronous maps indicate a two-step process with a first stage associated with an opening of the protein driven by destabilization of β-turns, located in the outer part of the protein, and a second part where the fibril is formed.This work was supported in part by the Spanish Ministerio de Educacion y Ciencia (grant No. BFU 2010-22103).