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.

Baudelaire comes to Chemistry

Could an evil molecule be beautiful? In Les Fleurs du Mal, a collection of poems, Baudelaire argued that there was a certain beauty in evil. Well, if there ever was an evil molecule, it’s the Abeta42 peptide, the main component of the senile plaque of Alzheimer’s disease, a molecule whose effects I spent my entire professional career as a neurologist ineffectually fighting. And yet, in a recent paper on the way it forms the fibrils constituting the plaque I found the structure compellingly beautiful.

The papers are Proc. Natl. Acad. Sci. vol. 113 pp. 9398 – 9400, E4976 – E4984 ’16. People have been working on the structure of the amyloid fibril of Alzheimer’s for decades, consistently stymied by its insolubility. The authors solved it not by Xray crystallography, not by cryoEM, but by solid state NMR. They basically looked at the distance constraints between pairs of isotopically labeled atoms, and built their model that way. Actually they built a bouquet of models using computer aided energy minimization of the peptide backbone. Another independent study produced nearly the same set.

The root mean square deviation of backbone atoms of the 10 lowest energy models of the bouquets in the two studies was small (.89 and .71 Angstroms). Even better the model bouquets of the two papers resemble each other.

There are two chains of Abeta42, EACH shaped like a double horseshoe (similar to the letter S). The two S’s meet around a twofold axis. The interface between the two S’s is form by two noncontiguous areas on each monomer (#15 – #17) and (#34 – #37).

The hydrophilic amino terminal residues (#1 – #14) are poorly ordered, but amino acids #15 – #42 are arranged into 4 short beta strands (I only see 3 obvious ones) that stack up and down the fibril into parallel in register beta-sheets. Each stack of double horseshoes forms a thread and the two threads twist around each other to form a two stranded protofilament.

Glycines allow sharp turns at the corners of the horseshoes. Hydrogen bonds between amides link the two layers of the fibrils. Asparagine side chains form ladders of hydrogen bonds up and down the fibrils. Water isn’t present between the layers because the beta sheets are so close together (counterintuitively this decreases the entropy, because water molecules don’t have to align themselves just so to solvate the side chains).

Each of the horseshoes is stabilized by hydrophobic interactions among the hydrophobic side chains buried in the core. Charged residues are solvent exposed. The interface between the two horsehoes is a hydrophobic interface.

Many of the famlial mutations are on the outer edges of double S structure — they are K16N, A21G, D23N, E22A, E22K, E22G, E22Q.

The surface hydrophobic patch formed by V40 and A42 may explain the greater rate of secondary nucleation by Abeta42 vs. Abeta40.

The cryoEM structures we have of Abeta42 are different showing the phenomenon of amyloid polymorphism.

The PNAS paper used reombinant Abeta and prepared homogenous fibrils by repeated seeding of dissolved Abeta42 with preformed fibrils. The other study used chemically synthesized Abeta and got fibrils without seeding. Details of pH, peptide concentration, salt concentration differed, and yet the results are the same, making both structures more secure.

The new structure doesn’t immediately suggest the toxic mechanism of Abeta.

To indulge in a bit of teleology — the structure is so beautiful and so intricately designed, that the aBeta42 peptide has probably been evolutionarily optimized to perform an (as yet unknown) function in our bodies. Animals lacking Abeta42’s parent (the amyloid precursor protein) don’t form neuromuscular synapses correctly, but they are viable.