Tag Archives: Birefringence

Amyloid Structure at Last ! – 2 Birefringence

This was the state of the art 19 years ago in a PNAS paper (vol. 99 pp. 16742 – 16747 ’02).  “Amyloid fibrils are filamentous structures with typical diameters of 10 nanoMeters and lengths up to several microns.  No high resolution molecular structure of an amyloid fibril has yet been determined experimentally because amyloid fibrils are noncrystalline solid materials and are therefore incompatible with Xray crystallography and liquid state NMR.”

Well solid state NMR and cryo electron microscopy have changed all that and we now have structures for many amyloids at near atomic resolution.  It’s probably behind a pay wall but look at Cell vol. 184 pp. 4857 – 4873 ’21 if you have a chance.  I’ve spent the last week or so with it, and a series of posts on various aspects of the paper will be forthcoming.  The paper contains far too much to cram into a single post.

So lacking an Xray machine to do diffraction, what did we have 57 years ago when I started getting seriously interested in neurology?  To find amyloid we threw a dye called Congo Red on a slide, found that it bound amyloid and became birefringent when it did so.

Although the Cell paper doesn’t even mention Congo Red, the structure of amyloid they give explains why this worked.

What is birefringence anyway?  It means that light moving through a material travels at different speeds in different directions.  The refractive index of a material is the relative speed of light through that material versus the speed of light in a vacuum.   Stand in a shallow pool.  Your legs look funny because light travels slower in water than in air (which is nearly a vacuum).

Look at the structure of Congo Red — https://en.wikipedia.org/wiki/Congo_red.  It’s a long thin planar molecule, containing 6 aromatic rings, kept planar with each other by pi electron delocalization.

The previous post contained a more detailed description of amyloid — but suffice it to say that instead of wandering around in 3 dimensional space, the protein backbone in amyloid is confined to a single plane 4.8 Angstroms thick — here’s a link — https://luysii.wordpress.com/2021/10/11/amyloid-structure-at-last/

Plane after plane stacks on top of each other in amyloid.  So a micron (which is 10,000 Angstroms) can contain over 5,000 such planes, and an amyloid fibril can be several microns long.

It isn’t hard to imagine the Congo Red molecule slipping between the sheets, making it’s orientation fixed.  Sounds almost pornographic doesn’t it? This orients the molecule and clearly light moving perpendicular to the long axis of Congo Red will move at a different speed than light going parallel to the long axis of Congo Red, hence its birefringence when the dye binds amyloid.

Well B-DNA (the form we all know and love as the double helix) has its aromatic bases stacked on top of each other every 3.4 Angstroms.  So why isn’t it birefringent with Congo Red?  It has a persistence length of 150 basePairs or about .05 microns, which means that the average orientation is averaged out, unlike the amyloid in a senile plaque

There is tons more to come.  The Cell paper is full of fascinating stuff.

Amyloid

Amyloid goes way back, and scientific writing about has had various zigs and zags starting with Virchow (1821 – 1902) who named it because he thought it was made out of sugar.  For a long time it was defined by the way it looks under the microscope being birefringent when stained with Congo red (which came out 100 years ago,  long before we knew much about protein structure (Pauling didn’t propose the alpha helix until 1951).

Birefringence itself is interesting.  Light moves at different speeds as it moves through materials — which is why your legs look funny when you stand in shallow water.  This is called the refractive index.   Birefringent materials have two different refractive indexes depending on the orientation (polarization) of the light looking at it.  So when amyloid present in fixed tissue on a slide, you see beautiful colors — for pictures and much more please see — https://onlinelibrary.wiley.com/doi/full/10.1111/iep.12330

So there has been a lot of confusion about what amyloid is and isn’t and even the exemplary Derek Lowe got it wrong in a recent post of his

“It needs to be noted that tau is not amyloid, and the TauRx’s drug has failed in the clinic in an Alzheimer’s trial.”

But Tau fibrils are amyloid, and prions are amyloid and the Lewy body is made of amyloid too, if you subscribe to the current definition of amyloid as something that shows a cross-beta pattern on Xray diffraction — https://www.researchgate.net/figure/Schematic-representation-of-the-cross-b-X-ray-diffraction-pattern-typically-produced-by_fig3_293484229.

Take about 500 dishes and stack them on top of each other and that’s the rough dimension of an amyloid fibril.  Each dish is made of a beta sheet.  Xray diffraction was used to characterize amyloid because no one could dissolve it, and study it by Xray crystallography.

Now that we have cryoEM, we’re learning much more.  I have , gone on and on about how miraculous it is that proteins have one or a few shapes — https://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/

So prion strains and the fact that alpha-synuclein amyloid aggregates produce different clinical disease despite having the same amino acid sequence was no surprise to me.

But it gets better.  The prion strains etc. etc may not be due to different structure but different decorations of the same structure by protein modifications.

The same is true for the different diseases that tau amyloid fibrils produce — never mind that they’ve been called neurofibrillary tangles and not amyloid, they have the same cross-beta structure.

A great paper [ Cell vol. 180 pp. 633 – 644 ’20 ] shows how different the tau protofilament from one disease (corticobasal degeneration) is from another (Alzheimer’s disease).  Figure three shows the side chain as it meanders around forming one ‘dish’ in the model above.  The meander is quite different in corticobasal degeneration (CBD) and Alzheimers.

It’s all the stuff tacked on. Tau is modified on its lysines (some 15% of all amino acids in the beta sheet forming part) by ubiquitination, acetylation and trimethylation, and by phosphorylation on serine.

Figure 3 is worth more of a look because it shows how different the post-translational modifications are of the same amino acid stretch of the tau protein in the Alzheimer’s and CBD.  Why has this not been seen before — because the amyloid was treated with pronase and other enzymes to get better pictures on cryoEM.  Isn’t that amazing.  Someone is probably looking to see if this explains prion strains.

The question arises — is the chain structure in space different because of the modifications, or are the modifications there because the chain structure in space is different.  This could go either way we have 500+ enzymes (protein kinases) putting phosphate on serine and/or threonine, each looking at a particular protein conformation around the two so they don’t phosphorylate everything — ditto for the enzymes that put ubiquitin on proteins.

Fascinating times.  Imagine something as simple as pronase hiding all this beautiful structure.