The cumulative effect of macromolecular crowding and surface adsorption on protein fibrillation



NIH, NIDDK, LBG
Travis Hoppe, Allen Minton, Di Wu
https://github.com/thoppe/Presentation_NIST_crowding



Hoppe and Minton, Biophysical Journal (2015) Vol. 108, Issue 4

Biochemistry in the lab setting


Folding
Binding, dimerization
Aggregation, Fibril formation

Biochemistry in a
crowded environment



Background image from Harvard University, XVIVO Scientific Animation

Biochemistry in a
crowded environment

(as an analytical model)


Background interactions: Weak, nonspecific interactions between macromolecular species and local constituents. Does not depends on detailed structure of interaction but only on gross physical properties like size, shape, hydrophobicity, net charge, dipole moment, ...

  • What are the dominant interactions?
  • What length scales are important?
  • Dynamic or equilibrium?
  • Experimental scope?

What is crowding?

(usually) repulsive nonspecific interactions with other soluble macromolecules

Relative free volume decreases when inserting a larger macromolcule into the same solution of hard-spheres.


Annual Review of Biophysics (Vol. 37: 375-397) 2008, Zhou, Rivas & Minton

Experimental Evidence

Direct observation of the enhancement of non-cooperative protein
self-assembly by macromolecular crowding


Hemoglobin (Hb) enhances fiber formation of FtsZ


PNAS Vol. 98(6), Rivas, Fernandez, Minton

Experimental Evidence

Macromolecular Crowding Accelerates Amyloid Formation
of apoC-II as a function of Dextran T10 concentration


Turbidity
Non-aggregated protein
Thioflavin-T binding


JBC 277(7824-7830), Hatters, Minton, Howlett

Experimental Evidence

Accelerated α-synuclein fibrillation from crowding


α-synuclein fibrillation (alone, Dextran, Ficoll-70, Ficoll-400)
Electron micrographs of synuclein aggregates


FEBS Lett. Vol. 515(1-3), Uversky, et al.

Model outline

restrict the domain



  • Fiber formation is isodesmic & linear
  • Dilute macromolecular concentration
  • Steric crowding interactions
  • Spherical and sphereocylinder geometries
  • Linear, isotropic adsorption
  • Thermodynamic equilibrium

Model schematic

Adsorption only happens over a narrow volume near the surface.

Partitioning between

bulk (b) and surface (s) compartments


Three terms: change of chemical potential & enthalpy/entropy of absorption



concentration, thermodynamic activity coefficient, equilibrium constant,
change in enthalpy during adsorption per monomer, and change in entropy upon adsorption.

Isodesmic fiber formation

cost of fibrillation independent of length




thermodynamic activity, thermodynamic activity coefficient, equilibrium constant,
change in enthalpy during adsorption per monomer, and change in entropy upon adsorption.

Scaled particle theory


Thermodynamic cost of cavity formation

equivalently the cost of inserting a solute molecule of radius a into the solvent of radius b.


Hard-sphere case: Integrand can be evaluated exactly for close distances,
and approximated with the Percus-Yevick closure
of the Orstein-Zernike equation.



is the average density of solvent molecules in contact with the solvent,
Reiss, Frisch, and Lebowitz, J. Chem. Phys. 31, 369 (1959)

Scaled particle theory

Can be extended to non-spherical geometries:


Sphereocylinder into hard-spheres



is the packing fraction of the crowders, is the length of the macromolecule.

Adsorption

Estimate the entropic & enthalpic cost of adsorption

Loss of entropy upon adsorption


Free rotation in bulk


Free rotation near the surface


rotational states available to bulk/surface solute of length n.
is the width of the bounding surface.

Activity coefficient

two-body approximation


Activity coefficients depend only on excluded volume


Sear, Phys. Rev. E. (1998)

Activity coefficient

two-body approximation for all when .

Activity coefficient

multi-body approximation

is not constant near the surface (boundary effects)
dependent on oligomer too (solute effects)
Effects can be real and measurable!

Interaction of charged colloidal spheres near a charged wall
Radial distribution function and PMF

Behrens, Grier, Phys Rev E. 2001, 64(5)

Activity coefficient

multi-body approximation


Use Monte-Carlo to estimate , i.e. the
dependence of crowding concentration.


Sample conformational states of hard-spheres near a boundary.


Two approaches to improve sampling, DMD (Discrete molecular dynamics) and particle displacement.

Widom sampling


Split into , sample over "test" particle:

is the chemical potential, is the free-energy, and is the partition function with solute molecules.

Widom sampling (hard-spheres)

For hard-potentials and .

computationally sampling the ratio of insert probabilities
(monomers, dimers, trimers, and tetramers from black to red)

Linear dependence of on

Results

Critical crowding concentration leads to unbounded oligomer
growth on the surface ... even in the absence of surface attraction!


Bulk/Surface (Solid/Dashed) ratio of species growth.
Red, ; Black .
Average size of absorbed oligomers.
Left to right .

Qualitative predictions

Model predicts a critical "falling-out" of fiber growth in the solution at moderate, neutral and even repulsive surfaces.


Location of critical phenomena and extent of cooperatively is
sensitive to details of the model (adsorption/association).


Biological relevance?

The presence of large fibers in solution is unfavorable thermodynamically.


If fibrillation and/or adsorption is linked to biological function,
crowding can act as a sensitive form of biochemical regulation.

Thanks, you.



Laboratory of Biochemistry and Genetics
Physical Biochemistry Section

Allen Minton
Di Wu
Travis Hoppe


Support provided by the Intramural Research Division of the NIDDK, NIH.