Perturbation of the Stability of Amyloid Fibrils through Alteration of

Electrostatic Interactions

Shammas SL, Knowles TPJ, Baldwin AJ, MacPhee CE, Welland ME, Dobson CM, and Devlin GL

Biophysical Journal 100, 2783-2791 (2011)

Na Young KimCHEM 645

Amyloid Fibrils

2

Info: Shammas SL et al. Biophys. J. 100, 2783-2791 (2011)Nelson R et al., Nature 435, 773-778 (2005)Picture: Ross CA, Poirier MA. Nature Medicine 10, S10-S17 (2004)

Structure of Amyloid Fibrils

3

Nelson R et al., Nature 435, 773-778 (2005)

Structure of Amyloid Fibrils

4

Nelson R et al., Nature 435, 773-778 (2005)

Insulin• Readily forms amyloid fibrils at acidic pH• These dissociate under alkaline conditions, but by what

mechanism?– Dissociation due to charge change at higher pH– Effects of partial distruption of beta-sheet on dissociation via

denaturant addition– Kinetics of dissociation

Bovine Insulin (Hexameric form) PDB: 2ZP6

Dissociation of Amyloid Fibrils at High pH

• Amyloid fibrils formed at pH 2.0 by seeding• Incubated amyloid fibrils at room temperature and various pH for

48h

Driving Force Behind Dissociation?

• These simple electrostatic considerations accurately described the dissociation of these charged fibrils

• •

€

Uch =Q2

4πε0εr0e−r0 rD

ΔGtot = ΔG0 +Uch Q( )

€

ΔG0 = −39.4 ±1.3kJ /mol

€

r0 =1.48nm

Is Nothing Happening Below pH 8?

Structural changes apparent above pH 4, but no dissociation of insoluble fibrils until above pH 8 (from TEM)

Structural Changes Below pH 8

Fourier transform infrared spectroscopy (FTIR) amide I’ region (1600-1700 cm-1) used to determine secondary structure content (pH* 1.6-3.6-5.6-7.6)

Beta-sheet Content Correlates Well to Fibril Stability

pH deltaG (kJ/mol)

2 -56.3 +/- 1.2

4 -55.4 +/- 1.6

6 -43.2 +/- 0.9

8 -33.3 +/- 0.6

Kinetics of Dissociation

€

%T =I

I0⋅100

OD = −log%T

100

⎛

⎝ ⎜

⎞

⎠ ⎟= −log

I

I0

⎛

⎝ ⎜

⎞

⎠ ⎟

Picture: http://en.wikipedia.org/wiki/AbsorbanceEquations: http://www.chroma.com/knowledge/introduction-fluorescence/percent-transmission-and-optical-density

Kinetics of Dissociation Continued

12

Kinetics of Dissociation Continued

13

Conclusions

Significance

• Significance of beta-sheet content on thermodynamic stability of amyloid fibrils

• “Fibrillar state can be thermodynamically more stable than the native state”

15

Questions?

16

17

Outline

• “Fibril stability is susceptible to electrostatic repulsion between constituent polypeptide chains” (pH 2 to 12)

• “Thermodynamic stability of fibrils is reduced by partial disruption of beta-sheet structure” (pH 4 to 8)

• “The kinetics of insulin fibril dissociation is dependent on the ionization state of a single side chain” (pH>10)

Explanation of Eq 2

Assumptions:• “Each monomer in the fibril experiences a repulsive elctrostatic

interaction with its nearest neighbors”• Dynamic equilibrium exists between the fibrillar and soluble states

of insulin– This can be described by classical linear polymerization theory

19

FTIR of Proteins

20

Gallagher, W. “FTIR Analysis of Protein Structure” www.chem.uwec.edu/Chem455_S05/Pages/.../FTIR_of_proteins.pdf