Analysis of turbidity progress curves from protein aggregation reactions

Abstract

To investigate the individual and combined effects of protein molecular chaperone abilities on the aggregation of proteins a vital first step is the development of a robust and simple assay for examining the aggregation of proteins. By far the most common in vitro method to monitor protein aggregation is the turbidity assay. All protein aggregates scatter light in the visible wavelength region since their size ranges from nanometer to micrometers. This characteristic combined with a lack of absorption in the visible wavelength region makes the low-cost turbidity assay a particularly attractive method for monitoring protein aggregation. Colloidal solution turbidity is generally considered to exhibit a linear relationship with the aggregation reaction. However, this assumption is usually not based on convincing supporting experiments or theory. The turbidity of a colloidal solution is not only determined by the size, but also the shape of the particles. As a result, analyzing the relationship between solution turbidity and protein aggregation can be quite challenging. In my postgraduate research I examined and developed improved methods for simulating and analyzing turbidity profiles of mixed protein aggregation reactions, which will greatly facilitate the understanding of protein aggregation and the effect of molecular chaperone reactions. In my first paper I contributed to developing a hybrid method for simulating turbidity of protein aggregates of different sizes in the low concentration limit. This simulation utilises a combination of the Rayleigh, the Rayleigh-Gans-Debye (RGD) and approximate forms of the Mie scattering equations. This hybrid approach was used to generate empirical interpolating functions, which may be used for both simulation and analysis of turbidity profiles. In my second paper, I helped to develop a method for quantifying the variability in the amyloid aggregation assay. We investigated the variability in the amyloid aggregation kinetics, and developed methods for its simulation, identification and analysis. Rather unexpectedly, such an analysis had not been previously developed despite it being the fundamental cornerstone of all differential analyses of drug and condition effects upon the protein aggregation reaction. In my third paper, I reviewed the physical chemistry of the turbidimetric assay methodology, investigating the reviewed information with a series of pedagogical kinetic simulations. We particularly focused upon recent literature relating to ultra-microscope image analysis light scattering and turbidity development by protein aggregates and computer simulation of the kinetics of amyloid and other aggregate types.