Structural investigation of protein fibrillation · SCC ~ 8.5-11mg/mL 0 20 40 60 80 100 1,6 2 22 32...

Structural Investigation of Protein Fibrillation without

disturbing the inherent equilibriums

-Towards structural and Biophysical Analysis of the Autocatalytic

Effect of Fibrillogenesis

Bente Vestergaard

University of Copenhagen

Yeast prion protein derived peptide fibril, Annette Eva Langkilde, KU-FARMA

Bente Vestergaard - BioSAXS

BioSAXS group @ University of Copenhagen

Current members:Annette E. LangkildeKatrine N. ToftMinna Grønning Malene H. JensenCharlotte R. PetersenMagda MøllerPaola SciortinoBjörg EythorsdottirHelle MulvadLotte Solvang ChristensenThea WindSine NørgaardMartin Nors

Funding:Danish Strategic Research CouncilDanish Medical Research CouncilLundbeck FoundationCarlsberg FoundationNovo Nordisk A/SDrug Research Academy

Former members:Mads G. JeppesenVito FoderaMagnus AnderssonDavid NolanJesper Neergaard


• Amyloidosis

• Systemic - Build-up of amyloid deposits

• Organ-specific amyloidosis

• E.g. Alzheimers disease, Parkinsons disease...

• Protein re/mis-folding and aggregation

• General feature of all proteins

(’the other side of folding’)?

• From native/soluble to

non-native/cytotoxic, massive insoluble

• Functional Fibrils

• Antimicrobial

• Anti-cancer (suicide)

• Biofilm

• Spider silk

• Biodrug instability

• Insulin, glucagon...

Protein Fibrillation

pdb-code 1d0r, GLP1

Novopen®4

E. coli Biofilm AJC1/Flickr

Am. Soc. Hematology

www.alzheimersinfo.info


Mechanism includes multiple states in equilibrium

Structural elucidation of intermediates paramount! - An inherent analytical challengeBente Vestergaard - BioSAXS

www.alzheimersinfo.info

Which species is cytotoxic?

Bente Vestergaard - BioSAXS Nelson et al, 2005, NaturePeptide/yeast prion protein

Lashuel et al. J.Mol.Biol.

a-synucleinHua & Weiss (2004)Insulin, pH~1

Langkilde, VestergaardPeptide/yeast prion protein

Losic et al. 2006,

J. Struct. Biol Aβ(1-40)

Jimenez et al, 2002, PNASHuman Insulin

Grønning, VestergaardHuman Insulin

Bits of information – Challenging the equilibrium


SAXS – Monitoring the equilibrium

0

1000

2000

3000

4000

5000

6000

0 5 10 15 20

Th

T-e

mis

sio

n [rf

u]

Time [h]

y = (m1)+((m3)/(1+exp(-(m0-m...

ErrorValue

207.13-119.03m1

262.244315.1m3

0.341878.1073m5

0.303511.5765m6

NA9.4279e+6Chisq

NA0.96673R

SAXS data – addi(c)tive Itot=xIa+yIb


Powers & Powers, Biophys. Rev. 2006xINuc=ITotal-(yIMon+zIFib)

Volume fractions: ProcessSignal from nucleus: StructureData from mature Solution structure offibrils: fibril (no surface

effects but average)


The thermodynamic/structural nucleus and the supercritical concentration

10

100

1 10

t 50

% (

hrs

)

[aSN] [mg/mL]

A

aSN wt and mutant – experimental design

Plate reader installed on the beamline, individual samples (no stirring, no touching, complete emptying of the sample chamber each timeDirect determination of ThT-signal before measurement (+ standard curve)Samples were measured each 30 min, and a buffer scan was done before and after measuring the sample.

0

1000

2000

3000

4000

5000

6000

0 5 10 15 20T

hT

-em

issio

n [rf

u]

Time [h]

y = (m1)+((m3)/(1+exp(-(m0-m...

ErrorValue

207.13-119.03m1

262.244315.1m3

0.341878.1073m5

0.303511.5765m6

NA9.4279e+6Chisq

NA0.96673R

Experimental: 12 mg/ml protein, 20mM phosphate buffer, 150mM NaCl, 40µM ThT, pH7.4Sample volume 150µL, three replicates, glass beads, 30min/h orbital shaking, 37°C. SCC ~ 8.5-11mg/mL

0

20

40

60

80

100

1,6 2

22

32

56

271

300

982

Mass D

istr

ibu

tio

n [

%]

Hydrodynamic Radius Rh [nm]

0 hours

5 hours

10 hours

24 hours

BIC 35° BIC 90° UV CH 1 280

Time [min]

605040302010

Dete

cto

r S

ignals

[V

]

0.2

0.18

0.16

0.14

0.12

0.1

0.08

0.06

0.04

0.02

Monomer ~66% Dimer

~5%17-mer ~29%


L. Giehm, D.E. Otzen; Inano, Aarhus University, Denmark; D.I. Svergun, EMBL-Hamburg


0

1000

2000

3000

4000

5000

6000

0 5 10 15 20T

hT

-em

issio

n [rf

u]

Time [h]

y = (m1)+((m3)/(1+exp(-(m0-m...

ErrorValue

207.13-119.03m1

262.244315.1m3

0.341878.1073m5

0.303511.5765m6

NA9.4279e+6Chisq

NA0.96673R

aSN wt

Structural change before ThT signal

Structural change after ThT stabilizes (repacking?)

1st and last curves cannot be linearly combined to fit data at

intermediate time points

Decompose the data

0

1000

2000

3000

4000

5000

6000

0 5 10 15 20

Th

T-e

mis

sio

n [r

fu]

Time [h]

y = (m1)+((m3)/(1+exp(-(m0-m...

ErrorValue

207.13-119.03m1

262.244315.1m3

0.341878.1073m5

0.303511.5765m6

NA9.4279e+6Chisq

NA0.96673R

How many species?

Singular value decomposition(here: SVD-plot) of the entiredataset

Vary: First/last curves

(Low-)/high-s datapoints

Then decompose

– which program?

OLIGOMER

Method 1:

3 species with ’initial model’

a) Exp. curve from monomer

b) Theoretical curves from multiple ’random’ models of different size and shape

c) Exp. curve from fibril

Then: Use the calculated volume fractions for monomers and fibrils and RECALCULATE the scattering from the third species

Method 2:

2 species + ’the rest’

a) Exp. curve from monomer

b) Exp. curve from fibril

c) Isolate the residual from this (expected!) poor fit in OLIGOMER

Then: Use the average of the residuals as a third species and recalculate the volume fractions and scattering from the third species

Decomposition result (volume fractions) andThT

FibrilMonomer/dimerOligomerThT


Next:Compare the isolated scattering curves atall time points from the ’unknown’ species

What do we expect?

Decomposition result (volume fractions) andThT


Next:Compare the isolated scattering curves atall time points from the ’unknown’ species

What do we expect?

Average stable solutions

Refine(input 3 components-add residuals-recheck )

-Then Guinier, GNOM,DAMMIF etc

Corresponding sizes…Do the oligomers build the fibrils?

Ab initio models of αSynuclein fibrillar species

180Å


Compare typical distances

Mature fibril

Mature fibrilcross-section

Oligomer


0

20

40

60

80

100

0 5 10 15 20 25 30

Calcein release Oligomer [%]Calcein release Monomer [%]

Calc

ein

re

lea

se [%

]

Time [min]

Vesicle permeabilisation

First structural elucidationof a toxic species?


~3Å

7-10Å

25-35Å

Theory of ’beads on a string’ and the early/late fibrils

0

1000

2000

3000

4000

5000

6000

0 5 10 15 20

ThT

-em

issio

n [rf

u]

Time [h]

y = (m1)+((m3)/(1+exp(-(m0-m...

ErrorValue

207.13-119.03m1

262.244315.1m3

0.341878.1073m5

0.303511.5765m6

NA9.4279e+6Chisq

NA0.96673R


Facts versus open questions

Solution structure of an oligomeric species accumulating during fibrillation

Exists in equilibrium (disturbed when attempting insolation)

Coincides with strong vesicle permeabilizing

Solution structure (no surface) of mature fibrils

Coinciding volume fractions of fibrils and ThT

On- or off-pathway?

I.e. corresponding to building block?

Corresponding to nucleus?

The toxic species? The hole in the ring? Vesicles versus cells?

Structure of individual protomers in oligomer and in fibril?


1240 monomers

1 monomer

Repeating unit of mature insulin fibrils


The Structural Nucleus elongates fibrils

Time (hours)

0 2 4 6 8 10

Fra

ctio

ns

0.0

0.2

0.4

0.6

0.8

1.0



MW~5.6

monomers

Fibrillar Precursor -> Protofilament

B. Vestergaard M. Grønning et al. (2007) PLoS Biology


α


Nelson et al. (2005) Nature

Annette Eva Langkilde

N-terminal Yeast Prion Protein

GNNQQNY

Seems to fit with monomer + endin linear combinations

…but…Bragg spacing?

Crystallized peptides

Nelson et al. (2005) Nature

Annette Eva Langkilde

N-terminal Yeast Prion Protein

GNNQQNY

Crystallized peptides

~34 nm

.....


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Structural investigation of protein fibrillation · SCC ~ 8.5-11mg/mL 0 20 40 60 80 100 1,6 2 22 32...

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Transcript of Structural investigation of protein fibrillation · SCC ~ 8.5-11mg/mL 0 20 40 60 80 100 1,6 2 22 32...