Tag Archives: alpha helix

4 diseases explained at one blow said the protein chemist — part 1

A brilliant paper [ Science vol. 377 eabn5582 pp. 1 –> 20 ’22 ] explains how changing a single amino acid (proline) to another  can cause 4 different diseases, depending on the particular protein it is found in (and which proline of many is changed).

There is so much in this paper that it will take several posts to go over it all.  The chemistry in the paper is particularly fine.  So it’s back to Biochemistry 101 and the alpha helix and the beta sheet.

Have a look at this


If you can tell me how to get a picture like this into a WordPress post please make a comment.

The important point is that hydrogen bonds between the amide hydrogen of one amino acid and the carbonyl group of another hold the alpha helix and the beta pleated sheet together.

Enter proline : p//en.wikipedia.org/wiki/Proline.  Proline when not embedded in a protein has a hydrogen on the nitrogen atom in the ring.  When proline is joined to another amino acid by a peptide bond in a protein, the hydrogen on the nitrogen is no longer present.  So the hydrogen bond helping to hold the two structures (alpha helix and beta sheet) is no longer present at proline, and alpha helices and beta sheets containing proline are not has stable.  Prolines after the fourth amino acid of the alpha helix (e. g. after the first turn of the helix) produce a kink.  The proline can’t adopt the alpha helical configuration of the backbone and it can’t hydrogen bond.

But it’s even worse than that (and this observation may even be original).  Instead of a hydrogen bonding to the free electrons of the oxygen in the carbonyl group you have the two electrons on the nitrogen jammed up against them.  This costs energy and further destabilizes both structures.

Being a 5 membered ring which contains the alpha carbon of the amino acid, proline in proteins isn’t as flexible as other amino acids.

This is why proline is considered to be a helix breaker, and is used all the time in alpha helices spanning cellular membranes to cause kinks, giving them more flexibility.

There is much more to come — liquid liquid phase separation, prion like domains, low complexity sequences, frontotemporal dementia with ALS, TDP43, amyloid, Charcot Marie Tooth disease and Alzheimer’s disease.

So, for the present stare at the link to the diagram above.

Apologies for another posting delay

Hopefully the post on the paper I’m so impressed with will be out in the next few days.  I’ve been clearing away the underbrush in Needham’s Visual Differential Geometry and Forms before the final push on the Einstein field equation and Riemannian geometry.

Apologies for the delay

Here’s a clue for you all to think about — what effects does proline have on (1) the alpha helix (2) the beta pleated sheet?

Amyloid structure at last !

As a neurologist, I’ve been extremely interested in amyloid  since I started in the late 60s.  The senile plaque of Alzheimers disease is made of amyloid.  The stuff was insoluble gunk. All we had back in the day was Xray diffraction patterns showing two prominent reflections at 4 and 9 Angstroms, so we knew there was some sort of repetitive structure.

My notes on papers on the subject over the past 20 years contain  about 100,000 characters (but relatively little enlightenment until recently).

A while ago I posted some more homework problems — https://luysii.wordpress.com/2021/09/30/another-homework-assignment/

Homework assignment #1:  design a sequence of 10 amino acids which binds to the same sequence in the reverse order forming a plane 4.8 Angstroms thick.

Homework assignment #2 design a sequence of 60 amino acids which forms a similar plane 4.8 Angstroms thick, such that two 60 amino acid monomers bind to each other.

Feel free to use any computational or theoretical devices currently at our disposal, density functional theory, force fields, rosetta etc. etc.

Answers to follow shortly

Hint:  hundreds to thousands of planes can stack on top of each other.


If you have a subscription to Cell take a look at a marvelous review full of great pictures and diagrams [ Cell vol. 184 pp. 4857 – 4873 ’21 ].


Despite all that reading I never heard anyone predict that a significantly long polypeptide chain could flatten out into a 4.8 Angstrom thick sheet, essentially living in 2 dimensions.  All the structures we had  (alpha helix, beta pleated sheet < they were curved >, beta barrel, solenoid, Greek key) live in 3 dimensions.



So amyloid is not a particular protein, but a type of conformation a protein can assume (like the structures mentioned above).



So start with NH – CO – CHR.  NH  CO and C in the structure all lie in the same plane (the H and the side chain of the amino acid < R >  project out of the plane).


Here’s a bit of elaboration for those of you whose organic chemistry is a distant memory.  The carbon in the carbonyl bond (CO) has 3 bonding orbitals in one plane 120 degrees apart, with the 4th orbital perpendicular to the plane — this is called sp2 hybridization.  The nitrogen can also be hybridized to sp2.  This lets the pair of electrons above the plane roam around moving toward the carbon.  Why is this good?  Because any time you let electrons roam around you increase their entropy (S) and anything increasing entropy lowers their free energy (F)which is given by the formula F = H – TS where H is enthalpy (a measure of bond strength, and T is the absolute temperature in Kelvin.



So N and CO are in one plane, and so are the bonds from  N and C to the adacent atoms (C in both cases).


You can fit the plane atoms into a  rectangle 4.8 Angstroms high.  Well that’s one 2 dimensional rectangle, but the peptide bond between NH and CO in adjacent rectangles allows you to tack NH – CO – C s together while keeping them in a 3 dimensional parallelopiped 4.8 Angstroms high.



Notice that in the rectangle the NH and CO bonds are projecting toward the top and bottom of the rectangle, which means that in each plane  NH – CO – CHR s, the NH and CO are pointing out of the 2 dimensional plane (and in opposite directions to boot). This is unlike protein structure in which the backbone NHs and COs hydrogen bond to each other.  There is nothing in this structure for them to bond to.



What they do is hydrogen bond to another 3 dimensional parallelopiped (call it a sheet, but keep in mind that this is NOT the beta sheet you know about from the 3 dimensional structures of proteins we’ve had for years).



So thousands of sheets stacked together form the amyloid fibril.


Where does the 9 Angstrom reflection of cross beta come from?  Consider the  [ NH – CHR – CO ]  backbone as it lies in the 4.8  thick plane (I never thought such a thing would be even possible ! ).  It curves around like a snake lying flat.  Where are the side chains?  They are in the 4.8 thick plane, separating parts of the meandering backbone from each other — by an average of 9 Angstroms


Here is an excellent picture of the Alzheimer culprit — the aBeta42 peptide as it forms the amyloid of the senile plaque



You can see the meandering backbone and the side chains keeping the backbone apart.



That’s just the beginning of the paper, and I’ll have lots more to say about amyloid as I read further.   Once again, biology instructs chemistry and biochemistry giving it more “things in heaven and earth, Horatio, than are dreamt of in your philosophy.”

Maybe the backbone is more important than the side chains

I’m really embarrassed that I was unaware of the following work on protein design from Japan. Apparently, they were able to design proteins stable at 100 Centigrade using a methodology of which I was completely in the dark (N. Koga et al., Principles for designing ideal protein structures. Nature 491, 222–227 (2012), Y. R. Lin et al., Control over overall shape and size in de novo designed proteins. Proc. Natl. Acad. Sci. U.S.A. 112, E5478–E5485 (2015)). I read those journals but must have skipped the articles — I’ll have to go back and have a look.

A recent article (PNAS 117 31149 – 31156 ’20) brought it to my attention. Here’s what they say they’ve done.

“We proposed principles for designing ideal protein structures stabilized by completely consistent local and nonlocal interactions , based on a set of rules relating local backbone structures to preferred tertiary motifs (7, 10 — given above). These design rules describe the relation of the lengths or torsion patterns of two secondary structure elements and the connecting loop to favorable packing geometries . The design principles enable to encode strongly funneled energy landscapes into amino acid sequences, by the stabilization of folded structures (positive design) and by the destabilization of nonnative conformations (negative design) due to the restriction of folding conformational space by the rules”

Hard to believe but it works apparently. The paper also stands an idea about protein structure and stability on its head — the hydrophobic core of a compact protein, in this case a designed protein with a Rossmann fold (two pairs of alpha helices sandwiching a beta sheet is absolutely crucial to the ultimate 3 dimensional conformation of the protein backbone.

The protein is quite stable, not denaturing at 100 C. So then they mutated 10 of the large hydrophobic amino acids (leucine, isoleucine) to a small one (valine) so that 30 of the 34 amino acids in the core were valine and watched what happened.

What’s your guess? Mine would have been that the core was in a molten globule state and that backbone structure was lost.

That’s not what happened at all. The resulting protein was still stable over 100 C (although not quite as much by 5 KCal/mole)

To quote the authors again — “This result indicates that hydrophobic tight core packing may not be quite important for designed proteins: The folding ability and extremely high stability may arise from the restriction of conformational space to be searched during folding by the local backbone structures. This can lead to an exceptionally stable protein even in the absence of precise core packing.”

Astounding. However, this may not be true for proteins ‘designed’ by natural selection.
It’s time to try the same trick on some of them.

The world’s longest allosteric effect

I think there is some very interesting protein physical chemistry to be discovered/worked out based on a recent report [ Nature vol. 537 pp. 107 – 111 ’16 ]. It involves a long (2,200 Angstrom) coiled coil protein called EEA1 (Early Endosome Antigen 1). It contains 1,400 amino acids 1,275 of which form a coiled coil.

If you are conversant with the alpha helix and how two of them form a coiled coil, jump to ****. Otherwise here is some background and links to pictures which should help.

The alpha helix is a type of protein secondary structure in which the protein backbone assumes the shape of a coiled spring. There are 3.64 amino acids per turn. A single turn is 5.4 Angstroms high and 11 Angstroms wide. The alpha helix is right handed. That is to say, that if you orient the chain so that your thumb points from the N terminal to C terminal amino acid, the chain will twist in the direction of the fingers of the right hand as it rises. For some reason I can’t provide a link to a very large number of images for you hit. However, when I go to Google and type images of alpha helices you see them immediately — you’ll have to do the same to get there.

Coiled coils have two alpha helices winding around each other. This means that for secure interactions, the same types of amino acids must repeat again and again. A 7 residue periodicity (abcdefg)n in the distribution of nonpolar and charged amino acid residues is a feature characteristic of proteins which form alpha helices coiled about each other (coiled coil molecules). The 7 amino acids are lettered a – g from amino to carboxy. Positions a and d are usually hydrophobic amino acids (Leu, Ile, Val, Ala), positions e and g are usually polar or charged. The nonpolar a and d side chains associate by means of complementary knobs into holes packing. Each individual alpha helix is right handed, but the two helices wind around each other with a left handed turn. There are 3.64 amino acids per turn of an alpha helix, so for a regular repeating structure an amino acid should appear at the same position in space on the alpha helix (which forms a rigid rod). To see all the pictures you want — go to Google and type “Images of the Alpha Helix”.

To get the number of amino acids down so there are 3.5 per/turn (so the structure can repeat exactly every 7 amino acids –e.g. after 2 alpha helical turns) left handed supercoiling of each helix occurs (it’s a chicken and the egg situation). The helices are at an angle of 18 degrees to each other, and every 3.5 amino acids still form a 5.4 Angstrom (when one helix is viewed in isolation), but due to the tilt, they take up 5.1 Angstroms. This means that the same type of amino acid is found at positions 1, 8, 15, 22 etc. All intermediate filament proteins (keratin, neurofilaments, vimentin, etc.) contain a coiled coil structure. So to see all the pictures you could want — go to Google and type “Images of coiled coil proteins”

So the 1,275 amino acids of EEA1 divided by 3.5 and multiplied by 5.1 give you a coiled coil of fairly enormous length for a protein (1,858 Angstroms) — average protein diameter (if there is such a thing) is under 50 Angstroms

Functionally, EEA1 seems to be used as a tether with one end free and the other end hooked to a target membrane which wants to ‘catch’ the early endosome. The target membrane isn’t specified in the paper. Apparently EEA1 when not binding the endosome, is in a fully extended state, at around 2,000 Angstroms.

A protein called Rab5 is found on the early endosome membrane, and when EEA1 contacts it, the long coiled coil helix collapses, dragging the endosome toward the target membrane.  This is entropy in action, there being far more configurations of a collapsed protein than a rigidly extended one. To feel entropy for yourself, just pull on a rubber band, entropic effects just like this one are what you feel pulling back.

The collapse of EEA1  is an allosteric effect and a very long one, although the authors note long range allosteric effects are “not uncommon among coiled coil proteins”.

EEA1 is more complicated than initialy described. It contains amino acids which disrupt the 7 amino acid periodicity of the coiled coil (making it a jointed structure). The authors then made an EEA1 protein without the joints (so it was a perfect very long coiled coil). Binding of this protein to Rab5 on an endosome doesn’t result in collapse. So clearly normal EEA1 collapses at the ‘joints’.

The authors talk about some hypotheses as to how this happens in the Supplementary material (but I was unable to find).

So here’s a good research proejct for an enterprising grad student: either find out why and how a protein with multiple joints should exist in a fully extended configuration, or figure out how binding of Rab5 at one end of EEA1 produces such profound allosteric changes through this long linear protein. Happy hunting and thinking.

I must say it’s a pleasure to get back to chemistry after writing about the neurologic and medical issues of the presidential candidates.

Addendum 29 September — I wrote one the following to one of the authors (Dr. Grill) sending him the post above

Dr. Grill

Greatly enjoyed the paper.  I could never find the discussion of possible mechanism in the supplementary material.  You might enjoy the following post written about the paper

He replied as follows:

“Dear Luysii thank you very much for the kind words, and I really like your title!

With the supplementary discussion, besides the method part there is an additional supplement file on the Nature website that is easy to miss…I attach it here for you. We discuss this a bit more, but I must admit that this is not very satisfactory at the moment. We just don’t know how this works, and much of our efforts at the moment are dedicated to understand”
So for other readers of the original paper who also can’t find the supplement with the authors’ speculations as to what is going on– here  is what he sent.

” A key question is how Rab5 can induce such a long-range global molecular transition in flexibility of EEA1. Indeed, long-range allosteric effects have been observed for other coiled-coil proteins. In the case of myosin, the presence of discontinuities in the coiled-coil heptads drive structural changes to flexibility. Other tethering factors may bend through large breaks in coiled-coil structure acting as joints, although it remains to be shown whether and how conformational changes are triggered by Rab binding, as shown for EEA1.

Furthermore, a dynamically flexible coiled-coil is mostly extended, provided its ends are free60. However, when the ends of this coiled coil are tethered, bent, or when torsion is locally applied, compensatory structural changes are propagated and even amplified through the length of the structure. Our results suggest that a change in intrinsic static curvature may contribute but is not the major cause for the reduction in end-to-end distance. However, a more rigorous assessment would require visualizing the thermal fluctuations of the bound and unbound EEA1 very rapidly and in three dimensions.

Force generation due to entropic effects plays a key role in many processes in biology ranging from DNA cytoskeletal filaments to motor proteins. Switching a molecule from stiff to flexible could be an effective and general mechanism of many coiled-coil proteins for generating an attractive force, thereby pulling two objects together or allowing reactions otherwise hindered by polymer rigidity. Future experiments will test to what extent the entropic collapse is a general mechanism used not only by membrane tethers but also in other biological processes.”