Intermediates caught in the act: tracing insulin amyloid fibril formation in time by combined optical spectroscopy, light scattering, mass spectrometry and microscopy.

Interest in the topic of amyloid formation by peptides and proteins has increased dramatically in recent years, transforming it from a puzzling phenomenon associated with a small number of diseases into a major subject of study in disciplines ranging from material science to biology and medicine. The tendency of numerous (also non-pathogenic) proteins such as insulin to self-assemble into amyloid-like fibrils is well known. While fibrils are usually easily detected, the observation of transient intermediates is a big challenge in general. They are the key and the 'holy grail' for a molecular understanding of mechanisms in this context. Here we show that intermediates, i.e. oligomers, can be detected and their hydrodynamic radius RH as well as their overall conformation and structure can be monitored and the aggregation dynamics as well as structure formation can be detected in time with a suitable combination of experimental techniques. We have observed transient intermediates that resemble large oligomers held together in solution by non-covalent forces. The oligomers appear to convert into building blocks for mature fibrils with largely beta-sheet conformation resembling key players in a mechanism, which is termed 'nucleated conformation conversion' in the literature. Structural transformations of oligomers in time towards dominant beta-sheet conformations have been observed for the first time. The structures can even be observed in liquid phase AFM experiments. With this approach we have successfully shed new light into the aggregation and fibrilization process of insulin being possibly a model system for other amyloid systems.

Hydrogen bond dynamics of superheated water and methanol by ultrafast IR-pump and EUV-photoelectron probe spectroscopy.

Supercritical water and methanol have recently drawn much attention in the field of green chemistry. It is crucial to an understanding of supercritical solvents to know their dynamics and to what extent hydrogen (H) bonds persist in these fluids. Here, we show that with femtosecond infrared (IR) laser pulses water and methanol can be heated to temperatures near and above their critical temperature Tc and their molecular dynamics can be studied via ultrafast photoelectron spectroscopy at liquid jet interfaces with high harmonics radiation. As opposed to previous studies, the main focus here is the comparison between the hydrogen bonded systems of methanol and water and their interpretation by theory. Superheated water initially forms a dense hot phase with spectral features resembling those of monomers in gas phase water. On longer timescales, this phase was found to build hot aggregates, whose size increases as a function of time. In contrast, methanol heated to temperatures near Tc initially forms a broad distribution of aggregate sizes and some gas. These experimental features are also found and analyzed in extended molecular dynamics simulations. Additionally, the simulations enabled us to relate the origin of the different behavior of these two hydrogen-bonded liquids to the nature of the intermolecular potentials. The combined experimental and theoretical approach delivers new insights into both superheated phases and may contribute to understand their different chemical reactivities.