Category Archives: Chemistry (relatively pure)

The silence is deafening

3 weeks ago I published a post about a paper that I thought would be a real bombshell, in effect contradicting a paper in a prestigious journal, and strongly arguing from real data that the pandemic virus could have been made in a lab, quite possibly Wuhan.  .

Absolutely nothing has happened. No letters to PNAS (the source of the article) to Cell (the source of the criticized study).  With a question of this magnitude and importance  you’d think Nature or Science would weigh in about it.  The origin of the pandemic virus is certainly they’ve covered extensively.

So I’m going to send this to all concerned and see if I get any feedback.

Here is the original post.

Evidence that the pandemic virus was made in a lab

 

Everyone knows that the Chinese have been less than forthcoming about the origin of the pandemic virus (SARS-CoV-2).  An article in the current Proceedings of the National Academy of Sciences — https://doi.org/10.1073/pnas.2202769119 arguesthat US data, which hasn’t been released, and some 290 pages of which has been redacted could shed a good deal of light on the subject (without any help from China).  One of the authors is an economist, but the other has serious biochemical chops — https://www.pharmacology.cuimc.columbia.edu/profile/neil-l-harrison-phd.

Basically a variety of US institutions (see the paper — it’s freely available) have been working with the lab at Wuhan for years modifying the virus, long before the pandemic.  The paper names the names etc. etc. and is quite detailed, but I want to explain the evidence that the virus could have been produced (by human modification) at the Wuhan lab.  It has to do with a site in a viral protein which says ‘cut here’.

Here is more background than many readers will need, but the virus has affected us all and I want to make it accessible to as many as possible.

Proteins are linear strings of amino acids, just as this post is a linear sequence of letters, spaces and punctuation.

We have fewer amino acids (20 to be exact) than letters  and to save space each one has a one letter abbreviation (A for alanine V for valine, etc. etc.).  The spike protein (the SARS-CoV-2 protein binding to the receptor  for it on our cells) is quite long (1,273 amino acids all in a row).

Our genome codes for 588  proteins (called proteases) whose job it is to cut up other proteins. Obviously, it would be a disaster if they worked indiscriminately.  So each cuts at a particular sequence of amino acids. Think of the protease as a key and the sequence as a lock.  One protease called furin cuts in the middle of an 8 amino acid sequence RRAR’SVAS (R stands for aRginine and S for Serine).  This is called the furin cleavage site (FCS)

A paper (The origins of SARS-CoV-2: A critical review. Cell 184, 4848–4856 (2021) argued that the amino acid sequence of the FCS in SARS-CoV-2 is an unusual, nonstandard sequence for an FCS and that nobody in a laboratory would design such a novel FCS.  So, like many, I skimmed the paper and accepted its conclusions, as Cell is one of the premier molecular biology journals.

One final quote “The NIH has resisted the release of important evidence, such as the grant proposals and project reports of EHA, and has continued to redact materials released under FOIA, including a remarkable 290-page redaction in a recent FOIA release.”

Sounds like Watergate doesn’t it?

 

Watch this space

Brilliant structural work on the Arp2/3 complex with actin filaments and why it makes me depressed

The Arp2/3 complex of 5 proteins forms side branches on existing actin filaments.  The following paper shows its beautiful structure along with movies.  Have a look — it’s open access. https://www.pnas.org/doi/10.1073/pnas.2202723119.

Why should it make me depressed? Because I could spend the next week studying all the ins and outs of the structure and how it works without looking at anything else.  Similar cryoEM studies of other multiprotein machines are coming out which will take similar amounts of time.  Understanding how single enzymes work is much simpler, although similarly elegant — see Cozzarelli’s early work on topoisomerase.

So I’m depressed because I’ll never understand them to the depth I understand enzymes, DNA, RNA etc. etc.

Also the complexity and elegance of these machines brings back my old worries about how they could possibly have arisen simply by chance with selection acting on them.  So I plan to republish a series of old posts about the improbability of our existence, and the possibility of a creator, which was enough to me get thrown off Nature Chemistry as a blogger.

Enough whining.

Here is why the Arp2/3 complex is interesting.  Actin filaments are long (1,000 – 20,000 Angstroms and thin (70 Angstroms).  It you want to move a cell forward by having them grow toward its leading edge, growing actin filaments would puncture the membrane like a bunch of needles, hence the need for side branches, making actin filaments a brush-like mesh which could push the membrane forward as it grows.

The Arp2/3 complex has a molecular mass of 225 kiloDaltons, or probably 2,250 amino acids or 16 thousand atoms.

Arp2 stands for actin related protein 2, something quite similar to the normal actin monomer so it can sneak into the filament. So can Arp3.  The other 5 proteins grab actin monomers and start them polymerizing as a branch.

But even this isn’t enough, as Arp2/3 is intrinsically inactive and multiple classes of nucleation promoting factors (NPFs) are needed to stimulate it.  One such NPF family is the WASP proteins (for Wiskott Aldrich Syndrome Protein) mutations of which cause the syndrome characterized by hereditary thrombocytopenia, eczema and frequent infections.

The paper’s pictures do not include WASP, just the 7 proteins of the complex snuggling up to an actin filament.

In the complex the Arps are in a twisted conformation, in which they resemble actin monomers rather than filamentous actin subunits which have a flattened conformation.  After activation arp2 and arp3 mimic the arrangement of two consecutive subunits along the short pitch helical axis of an actin filament and each arp transitions from a twisted (monomerLike) to a flattened (filamentLike) conformation.

So look at the pictures and the movies and enjoy the elegance of the work of the Blind Watchmaker (if such a thing exists).

If the right hand don’t get you, the left hand will

Do you know the source of the title?  I found it surprising.  Answer at the end.

Some cancer cells have elevated levels of an enzyme called PHosphoGlyceride DeHydrogenase (PHGDH, others have decreased levels.  Many cancers contain both types of cells.  Neither is good news.

Those cancers  with low levels of PHGDH  have slower growth.  That’s good news isn’t it?  No.  Such cells are more likely to metastasize.

Those with high levels of PHGDH are less likely to metastasize.  That’s good news isn’t it?  No. such cells grow faster.

So cancers with both types of cells are more aggressive.

Here’s how it works [ Nature vol. 605 pp. 617 – 617, 747 – 753 ’22 ].

PHGDH is on the pathway for synthesis of serine, an amino acid required for protein synthesis (like all of them).  So low levels of the enzyme result in less protein synthesis and less tumor growth.

So how is this bad?  PHGDH binds to another enzyme PFK (PhosphoFructoKinase) stabilizing it.  When PHGDH is low PFK enzyme levels are low, so the subsrate of PFK (fructose 6 phosphate) is diverted to making sialic acid, which modifies cell surface proteins making them more likely to migrate.

So blocking sialic acid synthesis reverses the effects of low PHGDH on cancer migration and metastasis — but it does potentiate cell proliferation.

You just can’t win

Things like this may explain other paradoxic and unexpected effects of enzyme blockade.

16 Tons by Tennessee Ernie Ford

Evidence that the pandemic virus was made in a lab

 

Everyone knows that the Chinese have been less than forthcoming about the origin of the pandemic virus (SARS-CoV-2).  An article in the current Proceedings of the National Academy of Sciences — https://doi.org/10.1073/pnas.2202769119 argues that US data, which hasn’t been released, and some 290 pages of which has been redacted could shed a good deal of light on the subject (without any help from China).  One of the authors is an economist, but the other has serious biochemical chops — https://www.pharmacology.cuimc.columbia.edu/profile/neil-l-harrison-phd.

Basically a variety of US institutions (see the paper — it’s freely available) have been working with the lab at Wuhan for years modifying the virus, long before the pandemic.  The paper names the names etc. etc. and is quite detailed, but I want to explain the evidence that the virus could have been produced (by human modification) at the Wuhan lab.  It has to do with a site in a viral protein which says ‘cut here’.

Here is more background than many readers will need, but the virus has affected us all and I want to make it accessible to as many as possible.

Proteins are linear strings of amino acids, just as this post is a linear sequence of letters, spaces and punctuation.

We have fewer amino acids (20 to be exact) than letters  and to save space each one has a one letter abbreviation (A for alanine V for valine, etc. etc.).  The spike protein (the SARS-CoV-2 protein binding to the receptor  for it on our cells) is quite long (1,273 amino acids all in a row).

Our genome codes for 588  proteins (called proteases) whose job it is to cut up other proteins. Obviously, it would be a disaster if they worked indiscriminately.  So each cuts at a particular sequence of amino acids. Think of the protease as a key and the sequence as a lock.  One protease called furin cuts in the middle of an 8 amino acid sequence RRAR’SVAS (R stands for aRginine and S for Serine).  This is called the furin cleavage site (FCS)

A paper (The origins of SARS-CoV-2: A critical review. Cell 184, 4848–4856 (2021) argued that the amino acid sequence of the FCS in SARS-CoV-2 is an unusual, nonstandard sequence for an FCS and that nobody in a laboratory would design such a novel FCS.  So, like many, I skimmed the paper and accepted its conclusions, as Cell is one of the premier molecular biology journals.

One final quote “The NIH has resisted the release of important evidence, such as the grant proposals and project reports of EHA, and has continued to redact materials released under FOIA, including a remarkable 290-page redaction in a recent FOIA release.”

Sounds like Watergate doesn’t it?

 

Watch this space

 

Another neuropharmacologic surprise.

Our genome contains 826 different genes for G Protein Coupled Receptors (GPCRs) which are targeted by at least 475 FDA approved drugs (Nature vol. 587 p. 553 ’20 ). Yet part of the fascination of reading the current literature is the surprises it brings.

Our basic understanding was that the GPCRs sit on the surface of the cell waiting for ligands outside the cell to bind to it, which produces a conformational change on the cytoplasmic side of the cell membrane, changing the way the GPCR binds to the G protein, triggering all sorts of effects inside the cell.

As far as I recall, we never thought that different GPCRs would bind to each other in the cell membrane, even though a single cell can express ‘up to’ 100 different GPCRs [ Mol. Pharm. vol. 88 pp. 181 – 187 ’15 ].  Neurons express GPCRs and some are thought to be involved in neuropathic pain

But that’s exactly what Proc. Natl. Acad. Sci. vol. 119 e2123511119  ’22  is saying.

First a few definitions, if you’re as rusty about them as I was

A cytokine is an extracellular protein or peptide  helping cells to communicate with each other.  A chemokine is an extracellular protein which attracts cells.

Our genome has over 50 chemokines.  Most are  proteins with about 70 amino acids. The are classified by where the cysteines lie in them.  We have 23 receptors for chemokines, 18 of which are GPCRs.   Binding is promiscuous — a given chemokine binds to multiple receptors, and a given receptor binds to multiple chemokines.

Clearly the chemokines and their receptors are intimately involved in inflammation which always involves cell migration.  Neurons express chemokine receptors GPCRs and some are thought to be involved in neuropathic pain.

We also know that the nervous system is involved in immune function, particularly inflammation.  One prominent neurotransmitter is norepinephrine, and a variety of receptors bind to it.  There are 3 alpha1 norepinephrine receptors (a, b and d), all of which are GPCRs.

What is so shocking is that alpha1 GPCRs bind to chemokine receptors (forming heteromers), and that this binding is required for chemokines to have any effect on cell migration.  Even more interesting is that binding of norepinephrine to the alpha1 component of the heteromer INHIBITs cell migration.

So how many of our 826 GPCRs bind to each other, and what effects do they have?

Reading the literature is like opening presents, you find new fascinating toys to play with, some of which may actually benefit humanity

 

A new way to look at ALS (thank God)

It’s always good when a new way to look at a basically untreatable disease comes along.  We’ll know soon if looking at filamin A will be useful for Alzheimer’s disease.  Here’s another:  something we’ve known about for years (polyphosphate) may be important in Amyotrophic Lateral Sclerosis (ALS).   I used riluzole for ALS, but never saw any benefit.  It may have slowed the decline, but riluzole never stopped disease progression.

It is stated that 10% of ALS is familial, but I think this is an overstatement.  Even so mutations in a variety of proteins(superoxide dismutase 1 (SOD1) TDP43, C9orf72) do cause ALS, and studying them has taught us a lot about ALS.  There is plenty of work to do.  In 2016 a mere 160 mutations in the 153 amino acids of SOD1 had been found, but we still don’t know how they cause ALS despite hundreds of papers on the subject.  The proteins have allowed us to make mouse models of ALS, by putting in one or the other of mutated SOD1, TDP43, C9orf72 in motor neurons (or in whole animals)

Some real gumshoe work led to polyphosphate [ Neuron vol. 110 pp. 1603 – 1605 ’22 ].  Obviously in ALS, the motor neurons die, but recent work has shown that motor neurons are killed by neighboring astrocytes (containing any of the 3 the mutant proteins), when they are cultured together.   Normal astrocytes don’t do this.

So a lot of hard work found that it was polyphosphate in the supernatant fluid that was the killer.

So what is polyphosphate?  It’s been known for years, and is found in ALL cells — bacterial, plant, animal.  It also produced abiotically in volcanic exudates and deep sea steam vents.  No one knows what it does, so it has been called a molecular fossil.  Again teleology should inform biologic research (but it doesn’t).  Polyphosphate must be doing something useful or it wouldn’t be present in all living cells.

Chemically, polyphosphate is a chain of HUNDREDS to THOUSANDS of phosphate residues linked by high energy phosphoanhydride bonds.

Like this —

HO – PO2 – OH  + HO -PO2 -OH –>  HO – PO2 – 0 – PO2 – OH + H20

— the – O – in the middle is the phosphoanhydride bond

The authors treated motor neurons in culture with polyphosphate and found that it killed 40% of them.  So what?  Schmidt’s law of pharmacology, says that enough of anything will do anything,  So they looked at the spinal cords of patients dying of ALS and found that polyphosphate levels were higher than in neurologically normal controls.

So it’s open season on polyphosphate. Finding out what it does in normal cells, finding out how it kills motor neurons, finding out if decreasing its levels will help ALS (it does in cultures of motor neurons but that’s a long way from a living patient).  It’s an entirely new angle on an awful disease, with no useful treatment.  There is simply an enormous amount of work to be done.

Watch this space.

 

 

The cell is not a bag of water

We have over 800 G protein coupled receptors (GPCRs).  We have not found 800 distinct intracellular messengers (such cyclic adenosine monophosphate — aka CAMP).  A single cell can express up to 100 GPCRS — Mol. Pharm. vol. 88 pp. 181 – 187 ’15.  Some of them raise CAMP levels, others decrease it.  CAMP is supposed to diffuse freely within the cell.  If so, different GPCRs which change cellular CAMP levels to the same extent they should produce identical effects. But they don’t.

One example — Isuprel stimulation of beta adrenergic GPCRs increases cardiac contractile force and activates glycogen metabolism.  Prostaglandin E1 (PGE1) GPCR causes the same CAMP increase without this effect.

A recent fascinating paper may explain why [ Cell vol. 185 pp. 1130 – 1142 ’22 ]  The authors had previously done work showing that under basal conditions CAMP is mostly bound to a protein (regulatory protein kinase A subunit — aka PKA RIalpha ) leading to very low concentrations of free CAMP.

So free diffusion occurs only if CAMP levels are elevated well above the number of binding sites for it.

As usual, to get new interesting results, new technology had to be used.  A biosensor for CAMP based on Forster Resonance Energy Transfer aka FRET —  https://en.wikipedia.org/wiki/Förster_resonance_energy_transfer, was added to two different GPCRs — one for the Glucagon Like Peptide 1 (GLP1) and the other for the beta2 adrenergic receptor.

Even better, they fused the biosensor to the GPCRs using rulerlike  spacers each 300 Angstroms (30 nanoMeters) long.  So they could measure CAMP levels at 30 and 60 nanoMeters from the GPCR.  Levels were highest close to the receptor, but even at 30 and 60 nanoMeters away they were higher than the levels in the cytoplasm away from the cell membrane.  So this is pretty good evidence for what the authors call RAINs (Receptor Associated Independent camp Domains — God they love acronyms don’t they?).

Similar localized responses were seen with the beta2 adrenergic receptors, suggesting that RAINs might be a general phenomenon of GPCRs — but a lot more work is needed.

Even more interesting was the fact that there was no crosstalk between the RAINS of GLP1R and the beta2 adrenergic receptor.  Stimulation of one GPCR changed only the RAIN associated with it and didn’t travel to other RAINs

So the cell with its GPCRs resembles a neuron with its synapses on dendritic spines, where processing at each synapse remains fairly local before the neuron cell body integrates all of them.  It’s like Las Vegas — what happens at GPCR1 (synapse1) stays in GPCR1 (synapse1).  Well not quite, but you get the idea.

Why there’s more to chemistry than quantum mechanics

As juniors entering the Princeton Chemistry Department as majors in 1958 we were told to read “The Logic Of Modern Physics” by P. W. Bridgeman — https://en.wikipedia.org/wiki/The_Logic_of_Modern_Physics.   I don’t remember whether we ever got together to discuss the book with faculty, but I do remember that I found the book intensely irritating.  It was written in 1927, in early hay day of quantum mechanics.  It  said that all you could know was measurements (numbers on a dial if you wish) without any understanding of what went on in between them.

I thought chemists knew a lot more than that.  Here’s Henry Eyring — https://en.wikipedia.org/wiki/Henry_Eyring_(chemist)https://en.wikipedi developing transition state theory a few years later in 1935 in the department.  It was pure ideation based on thermodynamics, which was developed long before quantum mechanics and is still pretty much a quantum mechanics free zone of physics (although people are busy at work on the interface).

Henry would have loved a recent paper [ Proc. Natl. Acad. Sci. vol. 118 e2102006118 ’21 ] where the passage of a molecule back and forth across the free energy maximum was measured again and again.

A polyNucleotide hairpin of DNA  was connected to double stranded DNA handles in optical traps where it could fluctuate between folded (hairpin) and unfolded (no hairpin) states.  They could measure just how far apart the handles were and in the hairpin state the length appears to be 100 Angstroms (10 nanoMeters) shorter than the unfolded state.

So they could follow the length vs. time and measure the 50 microSeconds or so it took to make the journey across the free energy maximum (e.g. the transition state). A mere 323,495 different transition paths were studied.  You can find much more about the work here — https://luysii.wordpress.com/2022/02/15/transition-state-theory/

Does Bridgeman have the last laugh — remember all that is being measured are numbers (lengths) on a dial.

Here’s another recent paper Eyring would have loved — [ Proc. Natl. Acad. Sci. vol. 119 e2112372118 ’22  — ] https://www.pnas.org/doi/epdf/10.1073/pnas.2112382119  ]

The paper studied Barnase, a 110 amino acid protein which degrades RNA (so much like the original protein Anfinsen studied years ago).  Barnase is highly soluble and very stable making it one of the E. Coli’s of protein folding studies.

The new wrinkle of the paper is that they were able to study the folding and unfolding and the transition state of single molecules of Barnase at different temperatures (an experiment which would have been unlikely for Eyring to even think about doing in 1935 when he developed transition state theory, and yet this is exactly the sort of thing what he was thinking about but not about proteins whose structure was unknown back then).

This allowed them to determine not just the change in free energy (deltaG)  between the unfolded (U) and the transition state (TS) and the native state (N) of Barnase, but also the changes in enthalpy (delta H) and entropy (delta S) between U and TS and between N and TS.

Remember delta G = Delta H – T delta S.  A process will occur if deltaG is negative, which is why an increase in entropy is favorable, and why the decrease in entropy between U and TS is unfavorable.   You can find out more about this work here — https://luysii.wordpress.com/2022/03/25/new-light-on-protein-folding/

So the purely mental ideas of Eyring are being confirmed once again (but by numbers on a dial).  I doubt that Eyring would have thought such an experiment possible back in 1935.

Chemists know so much more than quantum mechanics says we can know.  But much of what we do know would be impossible without quantum mechanics.

However, Eyring certainly wasn’t averse to quantum mechanics, having written a text book Quantum Chemistry with Walter and Kimball on the very subject in 1944.

A new metabolite modifying proteins

I stopped counting when I got up to 28 mutations capable of causing clinical Parkinson’s disease (each in a different protein).  Many of them point to the mitochondrion with production of reactive oxygen species, but figuring out how they produce disease has kept armies of researchers busy.

Particularly fascinating is DJ-1, (aka PARK7) discovered in 2003 in two Dutch families when mutations  produced early onset Parkinsonism.  In December 2019 I noted that I’d taken 15 K of notes about PARK7, but that we still didn’t know how mutations cause Parkinsonism.  The mutation deletes some 14,082 nucleotides and the first 5 exons of the gene essentially destroying it.

Well, we still don’t but a new paper [ Proc. Natl. Acad. Sci. vol. 119 e2111338119 ’22 ] showed that it destroys a metabolite that no one had ever heard of.

The metabolite arises from a glycolytic intermediate (1, 3 diphospho glyceric acid).   The oxygen of the 3 phosphate attacks the carbonyl group of carbon 1 displacing the other phosphate forming a 6 membered ring containing all 3 carbons and PO2.

Here’s a link — https://www.pnas.org/doi/10.1073/pnas.2111338119

The structure is given in figure 7 D.

But there’s more, much more.  The intermediate reacts with the amino groups in proteins forming a glyceric acid modification.  PARK7 destroys the intermediate.  The protein modification was found on 80 different proteins in the brains of mice deficient in PARK7.  So now we have 80 new leads to follow, and we’ve found a completely new protein modification.

As far as we know, PARK7 is the only protein destroying the intermediate.  This explains why PARK7 is so abundant, and why its concentration is kept the same across different cell types and organisms.

Who knows how many more metabolic intermediates are out there waiting to be discovered.  Molecular biology (and drug development)  is hard because we don’t know all the players.  Here is yet another.

You can’t go wrong quoting Shakespeare

There are more things in heaven and earth, Horatio, Than are dreamt of in your philosophy.” – Hamlet (1.5.167-8),

Protein breathing — take II

As soon as the first 3 dimensional structure of a protein (sperm whale myoglobin) was determined, it became obvious that the structure couldn’t be the only one.  The whole point of the protein is to carry oxygen when the whale dives (whales aren’t fish, they are mammals).  Oxygen isn’t that big — an oxygen atom has a diameter of 1.2 Angstroms, so 02 still has a smallest diameter of 1.2 and a larger diameter of 2.9 Angstroms.  Even so, the side chains of the protein were so tightly packed that there was no way for O2 to worm its way through the protein structure they found to get to the embedded porphyrin ring which binds it.  So it became obvious that the protein had to breathe to let the oxygen in.

Well the benzene ring is quite a bit bigger.  All angles in the hexagon are 120 degrees, and the carbon carbon bond length is 1.39 Angstroms — so across the ring from carbon 1 to carbon 4 it is

1.39 * sine 30 + 1.39 + 139 * sine 30

139 * .89 + 1.39 + 1.39 * 89 = 3.8 Angstroms

Now throw in 1 Angstrom for the hydrogens on carbons 1 and 4 and you get a  diameter of about 6 Angstroms for benzene.

Now the aromatic amino acid side chains  (phenylalanine, tyrosine, tryptophan, histamine) have only one oxygen atom between them, so they don’t fit into water very well, and zillions of proteins structures have shown that they are usually found buried in the core of the protein, far away from water.  Given the tight packing of side chains in sperm whale myoglobin and most proteins, you wouldn’t expect them to move much, but move they do and 180 degree ring flips have been known to occur for 50 years.  Not only that but aromatic ring structure are quite rigid, as moving atoms out of the plane breaks up the resonance stabilization of the molecule taking quite a bit of energy.

A truly beautiful paper [ Nature vol. 602 pp. 695 – 700 ’22 ] Showed how one aromatic side chain (tyrosine) in one protein (JIP1) undergoes the 180 degree flip.  Very fancy nuclear magnetic resonance (NMR) was used (15-N  relaxation dispersion) at multiple temperatures, aromatic 1H–13C heteronuclear single quantum coherence (HSQC) spectra etc. etc.

Some 5 amino acids had to move their side chains, but the overall protein fold was maintained.

The pictures are fascinating, and there is a lot more work in the paper, using mutations, looking at other proteins in this category (SH3 proteins).

Clearly you could spend your life studying proteins, and many have done just that to our benefit.

It is therapeutic to read this elegant work and even more elegant protein dynamics and think about these matters, taking me far away from the horror going on in the Ukraine.  Everyone should have something like this.