Molecular mechanisms of protein aggregation from global fitting of kinetic models

Developers

Georg Meisl, Julius B. Kirkegaard, Sara Linse, Tuomas P. J. Knowles, etc.

Description of the technology

The elucidation of the molecular mechanisms by which soluble proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer’s and Parkinson’s diseases. However, because of the complexity associated with aggregation reaction networks, the analysis of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task.

Here we describe a framework, using quantitative kinetic assays and global fitting, to determine and to verify a molecular mechanism for aggregation reactions that is compatible with experimental kinetic data. The approach is implemented in a web-based software, AmyloFit. The procedure starts from the results of kinetic experiments that measure the concentration of aggregate mass as a function of time. The approach is illustrated with results from the aggregation of the β-amyloid peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic experiment measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-separated text file. Also the general experimental strategies is outlined, as well as practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions.

For the core part of the analysis, the authors have developed an online platform (see Links) that enables robust global analysis of kinetic data without the need for extensive programming or detailed mathematical knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic analysis: determination of constraints to be placed on the aggregation mechanism based on the concentration dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and fitting the chosen models using an advanced minimization algorithm to yield the reaction orders and rate constants. Protocol of the technology also describes how to use this approach to investigate targets for potential inhibitors of amyloid formation and their participation in reaction mechanism. The protocol, from processing data to determining mechanisms, can be completed in <1 d.

Practical application

The technology provides a framework and the software for determination of the functional roles of linear protein filaments and molecular mechanisms of protein aggregation into linear assemblies, which underlies pathogenesis of numerous diseases, associated with amyloid formation, such as Alzheimer’s and Parkinson’s diseases. These framework, software and kinetic models of aggregation processes can also be used to study how various compounds (drugs for treatment of degenerative disease, inhibitors of aggregation, etc) affect the aggregation processes and, therefore, pathogenesis of the mentioned diseases.

Laboratories

• Department of Chemistry, University of Cambridge, Cambridge (UK)
• Department of Biochemistry and Structural Biology, Lund University, Lund (Sweden)

Links

Publications

• Meisl, G. et al. «Molecular mechanisms of protein aggregation from global fitting of kinetic models." 11 Nature Protocols (2016): 252–272.
• Knowles, T.P.J., Vendruscolo, M. & Dobson, C.M. «The physical basis of protein misfolding disorders." 68 Phys. Today (2015): 36.
• Meisl, G. et al. «Differences in nucleation behavior underlie the contrasting aggregation kinetics of the aβ40 and aβ42 peptides." 111 Proc. Natl. Acad. Sci. USA (2014): 9384–9389.