Structural studies of amyloidogenic peptides and proteins Annette - PowerPoint PPT Presentation

Structural studies of amyloidogenic peptides and proteins Annette Eva Langkilde Dept. Of Drug Design and Pharmacology / Dept. Of Chemistry University of Copenhagen, Denmark

Amyloid fibrils • Disease Related • Unbranched • Extracellular • In vivo • Green birefringance upon Congo Red binding • Cross- β fiber diffraction pattern •… and lots of amyloid -like fibrils E. coli Biofilm AJC1/Flickr www.alzheimersinfo.info Am. Soc.Hematology pdb-code 1d0r, GLP1

� � Fibril Structure Jimenez et al, 2002, PNAS Human Insulin GNNQQNY fibrils � Nelson et al, 2005, Nature Fitzpatrick et al, 2013, PNAS aSN Peptide/yeast prion protein TTR 105-115 A. van Maarschalkerweerd

The fibrillation process Stationary phase (maturation) Jimenez et al, 2002, PNAS Growth phase Human Insulin (elongation) Grønning, Vestergaard Human Insulin Lag-phase (nucleation) Lashuel et al. JMB Hua & Weiss (2004) a-synuclein Insulin, pH~1 Nelson et al, 2005, Nature Losic et al. 2006, J. Langkilde, Vestergaard Peptide/yeast prion protein Struct. Biol A β (1-40) Peptide/yeast prion protein

Method? SAXS! Amorphous aggregate ? Native folded Native oligomer monomer Partially Fibrillation (un)folded related off- (membrane pathway vicinity) oligomer Native interactions Fibrillation prone Nucleus? conformation fibril On-pathway oligomer Native unfolded monomer Amorphous FIBRIL strain 1 aggregate Alternative fibrillation pathways FIBRIL strain 2

Time resolved SAXS during fibrillation ( α -synuclein) 6000 y = (m1)+((m3)/(1+exp(-(m0-m... Value Error m1 -119.03 207.13 5000 m3 4315.1 262.24 m5 8.1073 0.34187 m6 1.5765 0.30351 Chisq 9.4279e+6 NA ThT-emission [rfu] 4000 R 0.96673 NA 3000 2000 1000 0 0 5 10 15 20 Time [h] Giehm L, Svergun DI, Otzen DE, Vestergaard B (2011) PNAS 108, 3246-3251 Langkilde AE, Vestergaard B. (2012) Meth.Mol. Biol. 849, 137-55

n k : volume fraction I k (q) : scattering intensity from SAXS data – additive the k-th type of particle K : number of components *

How many species? Normalized eigenvectors Singular Value Decomposition (SVDplot) Log(eigenvalue) Momentum transfer 3 components: I tot =xI a +yI b +zI c Component

6000 y = (m1)+((m3)/(1+exp(-(m0-m... Value Error m1 -119.03 207.13 5000 m3 4315.1 262.24 m5 8.1073 0.34187 m6 1.5765 0.30351 Chisq 9.4279e+6 NA ThT-emission [rfu] 4000 R 0.96673 NA Isolating the scattering curves - OLIGOMER 3000 2000 1000 3 components: 3 components: 0 0 5 10 15 20 Time [h] I tot =xI a +yI b +zI c I tot =xI native +yI ??? +zI fibril I tot =xI native +zI fibril Get residuals, as first estimate I tot =xI native +yI unknown +zI fibril Get x, y and z estimates Recalculate and refine using residuals

Decomposition of species ( α -synuclein) Fibril Monomer/dimer Oligomer ThT Giehm L, Svergun DI, Otzen DE, Vestergaard B (2011) PNAS 108, 3246-3251

Compare typical distances Mature fibril cross-section Oligomer Mature fibril Giehm L, Svergun DI, Otzen DE, Vestergaard B (2011) PNAS 108, 3246-3251

Ab initio models of α Synuclein fibrillar species Corresponding sizes… Do the oligomers build the fibrils? 180Å Giehm L, Svergun DI, Otzen DE, Vestergaard B (2011) PNAS 108, 3246-3251

When it doesn’t work….

α SN E46K I tot =xI native +zI fibril Get residuals, as first estimate I tot =xI native +yI unknown +zI fibril Get x, y and z estimates A. Van Maarschalkerweert, PhD thesis, 2014 Recalculate and refine using residuals

α SN E46K A. Van Maarschalkerweert, PhD thesis, 2014

Development of an OBJECTIVE ROUTINE for the decomposition Chemometric method: Multivariate Curve Resolution using Alternating Least Squares (MCR-ALS) Fátima Herranz-Trillo & Prof. Pau Bernado, U. Montpellier; Prof. Roma Tauler, U. Barcelona Bente Vestergaard, U. Copenhagen Reduce Ambiguities: Holtzer Absolute Use of multiple data plot scale matrices (I Vs s) (I·s Vs s) χ 2 〉 = χ 2 = 1.16 3.98 χ 2 〉 = Kratky Porod 1.04 χ 2 = plot plot (I·s 2 Vs (I·s 4 Vs 2.08 s) s) χ 2 〉 = 1.03

Insulin fibrils ~ 5-6 monomers 1240 1 monomer monomers Vestergaard B, Groenning M, Roessle M, Kastrup JS, van de Weert M, Flink JM, Frokjaer S, Gajhede M, Svergun DI (2007) PLoS Biol, 5, e134

Insulin & E46 revisited Coming soon to a journal near you… Structural Analysis of Multicomponent Amyloid Systems by Chemometric SAXS Data Decomposition Herranz-Trillo F, Groenning M, van Maarschalkerweerd A, Tauler R, Vestergaard B and Bernadó P Structure (2016) Accepted

Full-length proteins vs peptide fragments Amorphous aggregate ? Native folded GNNQQNY Native monomer oligomer Partially Fibrillation (un)folded related off- (membrane pathway vicinity) oligomer Native Fibrillation interactions Nucleus? prone fibril On-pathway conformation oligomer Native unfolded Amorphous FIBRIL strain 1 monomer aggregate Alternative fibrillation pathways FIBRIL strain 2 Nelson et al (2005) Nature, 435, 774-449

GNNQQNY Fibril cross-section P(r) • No lag-phase • No oligomer build-up • Monomeric starting state • Ribbon like fibrils • Bragg peak (late fibril state) Langkilde, Morris, Serpell, Svergun, & Vestergaard, Acta Cryst. (2015). D71, 882 – 895

Fibre diffraction Method and sample prep: Morris, K. L. & Serpell, L. C (2012) Meth.Mol. Biol. 849, 121-35.

SAXS vs FD - complementarity SAXS data recorded at ID14-EH3, ESRF Detector distance: 2.34 m Wavelength: 0.931 Å Resolution range :~ 1000 - 10 Å FD recorded at 911:2, MAXlab Detector distance: 0.22 m Wavelength: 1.04 Å Resolution range :~ 50 - 3 Å A. E. Langkilde

Small-angle X-ray scattering (in solution) Log I SAXS data recorded at ID14-EH3, ESRF Detector distance: 2.34 m Wavelength: 0.931 Å Resolution range :~ 1000 - 10 Å q (s) nm -1 A. E. Langkilde

Combining SAXS, FD, and TEM 48 Å a=31 Å c=48 Å b=4.7 Å Langkilde, Morris, Serpell. Svergun, & Vestergaard, Acta Cryst. (2015). D71, 882 – 895

Packing model Langkilde, Morris, Serpell. Svergun, & Vestergaard, Acta Cryst. (2015). D71, 882 – 895

Fiber diffraction simulation - CLEARER Experimental diffraction image Simulated diffraction image c a Langkilde, Morris, Serpell. Svergun, & Vestergaard, Acta Cryst. (2015). D71, 882 – 895

Transthyretin (TTR) • Native state: 55 kDa Homotetramer • TTR misfolding and aggregation  amyloid diseases • wildtype ATTR amyloidosis (senile systemic amyloidosis) • familial amyloid polyneuropathy • familial amyloid cardiomyopathy Protofibrils/ Fibrils Native Native Misfolded tetramer monomer monomer

SAXS on TTR fibrillation • Pre-ini MW ≈ 3.3 monomers • Pre-ini ≠ xtal • Pre-ini ≠ xtal+dimer/monomer • Pre-ini = 85% xtal + 15% unfolded TTR dH 2 O ___ TTR 3.5 min ….. TTR 10 min TTR unfolded (11% ac.ac.) ~2900 kDa ~215 protomers Groenning et al. & Vestergaard (2015) Scientific Reports

TTR toxicity

The unfolded state *

Some practical aspects • Test your system, find optimal conditions • Know your system • complementary methods, e.g. TEM and FD (”SAXS and …” talks tomorrow) • Consider beamline stability, time frames, additional equipment etc • Check 2D images • Check buffers, basic parameters • ...and double check! • Test different inputs and parameters (consistent solutions) Langkilde AE, Vestergaard B (2012) Methods Mol Bio, 849, 137-155

Acknowledgements University of Copenhagen • Bente Vestergaard • Minna Grønning • Andreas van Maarschalkerweerd EMBL, Hamburg DE CBS, Montpellier, FR Beamlines and local contacts Funding • Dmitri Svergun, • Fatima Herranz-Trillo • Manfred Rössle • Pau Bernado The Lundbeck Foundation DESY/PETRAIII, Hamburg, DE • BioSAXS group X33, P12 Carlsberg Foundation IDAEA-CSIC, Barcelona, ES ESRF, Grenoble, FR • Roma Tauler Aarhus University, DK ID14-3, BM29 The Danish Council for • Lise Giehm Independent Research | MAX-lab, Lund, SE Medical Sciences • Danial Otzen Linköping University, SE 911:2, 911:3 • Per Hammarström CoNEXT – University of Copenhagen Univesity of Sussex, UK • Raul I Campos • Louise Serpell • Daniel Hirschberg Danscatt • Kyle Morris BiostructX