Modulation of electrostatic interactions to reveal a reaction network unifying the aggregation behaviour of the Ab42 peptide and its variants† Georg Meisl, a Xiaoting Yang, b Christopher M. Dobson, * a Sara Linse * b and Tuomas P. J. Knowles * a The aggregation of the amyloid b peptide (Ab42), which is linked to Alzheimer's disease, can be altered significantly by modulations of the peptide's intermolecular electrostatic interactions. Variations in sequence and solution conditions have been found to lead to highly variable aggregation behaviour. Here we modulate systematically the electrostatic interactions governing the aggregation kinetics by varying the ionic strength of the solution. We find that changes in the solution ionic strength induce a switch in the reaction pathway, altering the dominant mechanisms of aggregate multiplication. This strategy thereby allows us to continuously sample a large space of different reaction mechanisms and develop a minimal reaction network that unifies the experimental kinetics under a wide range of different conditions. More generally, this universal reaction network connects previously separate systems, such as charge mutants of the Ab42 peptide, on a continuous mechanistic landscape, providing a unified picture of the aggregation mechanism of Ab42. Introduction Most functional proteins have a net charge under normal physi- ological conditions, which helps confer solubility, 1–3 and is gov- erned by the protein sequence and structure, as well as the solution conditions such as pH, salt concentration and the concentration of other charged species. 4–7 The interactions involving charged and polar groups modulate properties such as solubility, stability and reaction rates. 2,8–12 In addition to their importance in the functional interactions of proteins, electrostatic interactions play a key role in the formation of aberrant protein aggregates. 13–16 In particular, charged proteins with embedded hydrophobic segments can be highly aggregation-prone and their assembly into amyloid brils is associated with Alzheimer's disease (the Ab peptide), Parkinson's disease (the protein a-syn- uclein) and a range of other debilitating human diseases. The aggregation kinetics of these proteins are strongly inuenced by electrostatic interactions and therefore depend on solution conditions and the presence of species able to shield charges. 17,18 Recent years have seen a signicant advance in the mecha- nistic understanding of the aggregation of disease-associated proteins under controlled conditions in vitro. 19–21 The mechanistic effects of variations in solution conditions, however, have oen not been characterised in detail and therefore only the part of the overall reaction network relevant under a given set of conditions has been investigated. The individual systems under different conditions are not linked together into a continuous mechanistic picture. A more complete reaction network will be particularly important in vivo where aggregation-prone proteins are found in the presence of a large number of other molecules, which modulate their interactions. Here, we present a method of sampling a large region of the reaction network of an aggregating system by modulating electrostatic interactions. This approach provides a means of altering the relative importance of different processes contrib- uting to the overall reaction network and thereby allows the sampling of a broad range of macroscopic behaviour that can be explained by a single reaction network. In the present work we investigate the aggregation kinetics of the 42-residue amyloid b peptide, Ab42, at different peptide and salt concentrations under quiescent conditions. We develop a model that quanti- tatively accounts for the observed lag times, kinetic proles and peptide concentration dependences over the range of ionic strengths studied and rationalizes the interplay of the indi- vidual microscopic rates and their dependence on the magni- tude of the electrostatic screening. Results and discussion Monomeric Ab42 has a net charge of between 3 and 4 at pH 8.0 where the C-terminus and Asp, Glu, Lys and Arg side chains a Department of Chemistry, University of Cambridge, Lenseld Road, Cambridge CB2 1EW, UK. E-mail: tpjk2@cam.ac.uk; cmd44@cam.ac.uk b Chemistry Department and Molecular Protein Science, Lund University, P. O. Box 124, SE221 00 Lund, Sweden. E-mail: sara.linse@biochemistry.lu.se † Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc00215g Cite this: Chem. Sci. , 2017, 8, 4352 Received 15th January 2017 Accepted 3rd April 2017 DOI: 10.1039/c7sc00215g rsc.li/chemical-science 4352 | Chem. Sci. , 2017, 8, 4352–4362 This journal is © The Royal Society of Chemistry 2017 Chemical Science EDGE ARTICLE Open Access Article. Published on 26 April 2017. Downloaded on 23/09/2017 07:10:10. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue