Effect of agitation on the peptide fibrillization: Alzheimer’s amyloid-b peptide 1-42 but not amylin and insulin fibrils can grow under quiescent conditions Ann Tiiman, a * Andra Noormägi, a,b Merlin Friedemann, a Jekaterina Krishtal, a Peep Palumaa a,b and Vello Tõugu a Many peptides and proteins can form fibrillar aggregates in vitro, but only a limited number of them are forming pathological amyloid structures in vivo. We studied the fibrillization of four peptides – Alzheimer’s amyloid-b (Ab) 1-40 and 1-42, amylin and insulin. In all cases, intensive mechanical agitation of the solution initiated fast fibrillization. However, when the mixing was stopped during the fibril growth phase, the fibrillization of amylin and insulin was practically stopped, and the rate for Ab 40 substantially decreased, whereas the fibrillization of Ab 42 peptide continued to proceed with almost the same rate as in the agitated conditions. The reason for the different sensitivity of the in vitro fibrillization of these peptides towards agitation in the fibril growth phase remains elusive. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Keywords: Alzheimer’s amyloid-b; insulin; amylin; fibrillization; agitation Introduction The formation of amyloid aggregates by peptides and proteins has attracted a great deal of attention because the presence of the resulting proteinaceous deposits with a fibrillar structure is characteristic to over 25 human diseases. Some of these amyloidogenic diseases, such as AD, Parkinson’s disease and type II diabetes exert remarkable clinical relevance because of their dramatic prevalence in the elderly population [1]. The number of proteins that can misfold and aggregate into amyloid assemblies in vitro is considerably larger than the number of proteins involved in amyloid diseases. Moreover, it has been shown that most proteins can form fibrils under specific experimental conditions and fibrillization is suggested to be a generic property of polypeptide chains [2]. One of the most im- portant questions in the studies of amyloid peptides/proteins is to find molecular and physicochemical characteristics that distinguish the peptides and proteins that are causing severe amyloid pathology from those that can fibrillize only in the in vitro conditions. As a rule, protein fibrillization in vitro is characterized by a sigmoidal growth curve typical to autocatalytic processes [3,4]. The autocatalysis means that the addition of monomers to the ends of an existing fibril is faster than the formation of new fibrils from the monomers and, accordingly, the rate of the process depends on the number of fibril ends. Analysis of the kinetics of the self assembly of filamentous structures demonstrates that amyloid growth can often be dominated by secondary rather than primary nucleation events, for instance, fragmentation of early fibrils [5]. One of the important environmental factors that has a significant impact on the fibrillization kinetics and can also enhance fibril fragmentation is the agitation of the reaction mixture [6–8]. In this paper, we determined the effect of agitation on the different stages of fibrillization of four amyloidogenic peptides in vitro. The experimental conditions were similar for all four peptides, but optimized individually for each peptide, to obtain adequate fibrillization in the same time scale. For all the peptides studied, intensive agitation was required in the initial exponential phase of the reaction where the fibrillar ‘seeds’ and primary fibrils are formed that grow during the next elongation phase. Ab 42 was the only peptide that did not require agitation during this elongation phase as stopping of the agitation did not affect fibril elongation rate. Fibrillization of insulin and amylin stopped when agitation was stopped in this phase and fibrillization of Ab 40 proceeded with a significantly lower rate. We assume that the profound ability of Ab 42 fibrils to grow under quiescent * Correspondence to: Ann Tiiman, Department of Gene Technology, Tallinn University of Technology, Akadeemia 15, Tallinn 12618, Estonia. E-mail: ann.tiiman@ttu.ee a Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia b Competence Center on Reproductive Medicine and Biology, Tiigi 61 b, Tartu 50410, Estonia Abbreviations: Ab, amyloid-b peptide; AD, Alzheimer’s disease; ThT, Thioflavin T; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol J. Pept. Sci. 2013; 19: 386–391 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Research Article Received: 1 March 2013 Accepted: 11 March 2013 Published online in Wiley Online Library: 23 April 2013 (wileyonlinelibrary.com) DOI 10.1002/psc.2513 386