Nnoiophysicl explortion of trnsthyretin myloid firils Finl report Summry In this work we used AFM to follow the myloidogenetic pthwy of trnsthyretin (TTR) y imging the events leding to the formtion of myloid protofilments. Singlemolecule force spectroscopy (SMFS) of protofilments ws compred to nïve TTR in order to proe dynmic nd structurl differences. We oserved tht the pthwy proceeds through the formtion of trnsient morphous ggregtes, followed y the occurrence of nnulr oligomers (rings or doughnuts). In other types of myloidoses similr ring structures hve een implicted in cytoxicity, ut their properties nd involvement in the myloid pthwy re poorly understood. We show tht the rings hve tendency to stck, forming tuulr protofilments. These tuulr protofilments precede the ppernce of myloid protofilments. Their height nd pitch resemle those of previous structurl models for the TTR myloid protofilment. Upon solvent exchnge we lso oserved myloid protofilment dissocition. The dissocition ppers to proceed through n unzipping mechnism, reveling structures reminiscent of the TTR nnulr oligomers. SMFS of protofilments reveled time-dependent increse in the length of the mnipulted structure, suggesting tht ssocitions etween monomers stilize with time. Force spectr of ntive TTR nd protofilments contined trnsitions spced 4 nm prt, indicting tht the component β- strnds unfold sequentilly. Bsed on our results model of TTR protofilment ssemly is proposed. Results nd Discussion The firillogenesis of trnsthyretin myloid firils ws explored in this proposl s prt of collortive effort etween the reserch groups of Professor An M. Dms of Porto (Portugl) nd of the University of Pécs, Hungry. Atomic force microscopic methods were utilized to gin insight into the mechnisms of TTR firil structure nd ssemly. Although the effect of toxic muttions ws not resolved in lieu of time, we succeeded in otining novel oservtions out the structurl chnges ssocited with the formtion of TTR firils nd protofilments. The structurl chnges during the formtion of firils ws followed y exmining the topogrphicl structure s function of incution time. In ddition, y mechniclly mnipulting the firils, the rrngement of TTR monomers within the firil were investigted. To our knowledge, our experiments re pioneering in such nnomnipultion of TTR firils. In the following, the time-dependent structurl chnges nd the results of the nnomechnicl experiments re presented. Dy 1: Ntive TTR Smples of ntive TTR were investigted following incution on mic surfce. The results re shown in Figure 1. Gloulr structures with n verge height of 4 nm were oserved. The height vlues correspond reltively well with the dimensions of the tetrmeric ntive TTR units. Firil formtion ws induced y lowering the ph (ph 3) nd incuting the smples t 37 C. At different time points liquots from the smple were removed nd nlyzed y AFM. 1
c Figure 1. Imging of ntive TTR smple with AFM.. AFM imge.. Height profile long the line drwn in (). c. Distriution of topogrphicl height of ntive TTR. 3 to 5-dy-old TTR In smples 3 to 5 dys old, curviliner, firil-like ggregtes were oserved (Figure 2). The firils were 50-100 nm long nd 3 nm in height. Quite interestingly, the firillr structures ppered to e constructed of nnulr oligomers. Such nnulr intermedites hve een shown to pper in the cse of other types of myloid firils. Most likely, protofirillr ggregtes result from oligomer lterl ssocition. c Figure 2. Imges of nnulr oligomeric structures.. AFM imge of firillr structures.. Enlrged view of two doughnut-shped oligomers. (c) Height profile long the line drwn in (). Figure 3. Annulr TTR oligomer structure nd its schemtic interprettion.. High-mgnifiction AFM imge.. Structurl model of ntive TTR. Inset, schemtics of nnulus formtion. 2
OTKA IN 70320 Averge dimensions of the nnulr oligomeric structures were (Figure 3): dimeter: 15.8 ± 2.3 nm, height: 0.97± 0.04 nm. Ech unit of the TTR nnulr oligomers could then e composed of monomer or n orgnized cluster of monomer molecules. 5 to 7-dy-old-smples Annulr structures were lso oserved in older smples s well. Apprently, the rings cn ind to ech other so s to form stcks (Figure 4). The stcking of TTR nnulr structures my e n importnt step in the process of TTR firil formtion. Figure 4. AFM imges of 5-7 dy old TTR smples. The firils prently grow y the stcking of nnuilr structures (), which merge to form continully growing firils (). Nnomnipultion of TTR firils In order to gin further insight into the structurl rrngement within the TTR firils, we crried out nnomechnicl mnipultions. The AFM cntilever ws lowered onto specific points long firillr structures; susequently, the cntilever ws pulled wy t predjusted, constnt velocity. The forces during the procedures were mesured y using cntilevers clirted for stiffness. Exmples of force curves re shown in Figure 5. In the force curves, trnsitions re oserved in teh form of swtooth-like peks. Such force trnsitions correspond to structurl trnsitions within the firil evoked y the imposed mechnicl forces. Force Figure 5. Collection of force versus extension curves otined during nnomnipultion experiments of TTR firils. Extension 3
From the force curves nlyses were mde ccording to the mximum stretch (totl extension) nd the seprtion etween the consecutive force peks (Figure 6). Totl extension (nm) Force pek seprtion (nm) Figure 6. Histogrms of totl extension nd force pek seprtion otined from force versus extension curves of TTR firils. Ntive TTR nd erly ggregtes (4 dys) were stretched up the the unfolded contour length of the TTR monomer. In ggregtes of 14 dys, 19% of records show pullings ove the TTR monomer unfolded contour length. Possily, monomer-monomer interfce stiliztion within the firils resulted in the longer stretches. In the force curves 4 nm pek seprtion dominted. The 4 nm corresponds well to the length of single ß-strnd. Thus, the nnomechnicl perturtions likely cused sequentil unfolding of the ß-strnds tht uild up the TTR structure. The presence of force ptterns with n underlying 4 nm repet oserved in more mture firillr ggregtes indicte firly regulr structure of these TTR ggregtes. Model of TTR myloid ssemly Bsed on our findins model of TTR ssemly my e proposed (Figure 7). In this model ntive TTR ssemles, vi prtilly unfolded intermedites, into morphous ggregtes, followed y the formtion of solule oligomeric species. Solule oligomers then ssemle into nnulr structures which form the sis of susequent ssemly steps. The nnulr structures colesce y stcking into protofirillr ssemlies. Conceivly, structurl rerrngements t this stge permit the formtion of helicl protofilments. Understnding the topologicl nd energetic constrints of such structurl rerrngement wit further experimenttion. 4
Figure 7. Schemtics of the structurl model of trnsthyretin myloid firil ssemly. Pulictions 1. Pires RH, Krsi A, Sriv MJ, Kellermyer MSZ, Dms (2008) Understnding the Trnsthyretin Amyloidogenesis Pthwy. Revist Portugues de Frmáci LII(3): 36. 2. Pires RH, Krsi A, Sriv MJ, Kellermyer, MSZ nd Dms AM. "Understnding the Trnsthyretin Amyloidogenesis Pthwy. I Ntionl Meeting on Medicinl Chemistry, 13-15th Nov. 2008, Porto - PORTUGAL 3. Pires RH, Krsi A, Sriv MJ, Dms AM nd Kellermyer, MSZ. Structure Of Trnsthyretin Amyloid Firils Explored With AFM Imging nd Nnomnipultion. XVI Ntionl Congress of Biochemistry, 22-26th Octoer 2008, Azores - PORTUGAL 4. Pires RH, Krsi A, Sriv MJ, Kellermyer, MSZ nd Dms AM. Trnsthyretin Amyloidogenesis Pthwy: insights from AFM nd force spectroscopy. EURAMY Meeting, 17-18th Oct. 2008. Póvo do Vrzim - PORTUGAL 5. Pires RH, Krsi A, Sriv MJ, Dms AM nd Kellermyer, MSZ. "Assemly nd Structure of Trnsthyretin Amyloid Protofilments reveled y AFM Imging nd Single Molecule Force Spectroscopy". EBSA Congress 6-9 July, 2009, Genov, Itly. 5