Category Archives: Chemistry (relatively pure)

The chemical ingenuity of the AIDs virus

Pop quiz:  You are a virus with under 10,000 nucleotides in your genome.  To make the capsid enclosing your genome, you need to make 250 hexamers of a particular protein.  How do you do it?

 

Give up?

 

You grab a cellular metabolite with a mass under 1,000 Daltons to bind the 6 monomers together.  The metabolite occurs at fairly substantial concentrations (for a metabolite) of 10 – 40 microMolar.

What is the metabolite?

Give up?

 

It has nearly perfect 6 fold symmetry.

 

Still give up?

[ Nature vol. 560 pp. 509 – 512 ’18 ]  https://www.nature.com/articles/s41586-018-0396-4 says that it’s inositol hexakisphosphate (IP6)  — nomenclature explained at the end. http://www.refinebiochem.com/pages/InositolHexaphosphate.html

Although IP6 looks like a sugar (with 6 CHOH groups forming a 6 membered ring), it is not a typical one because it is not an acetal (no oxygen in the ring).  All 6 hydroxyls of IP6 are phosphorylated.  They bind to two lysines on a short (21 amino acids) alpha helix found in the protein (Gag which has 500 amino acids).  That’s how IP6 binds the 6 Gag proteins together. The paper has great pictures.

It is likely that IP6 is use by other cellular proteins to form hexamers (but the paper doesn’t discuss this).

IP6 is quite symmetric, and 5 of the 6 phosphorylated hydroxyls can be equatorial, so this is likely the energetically favored conformation, given the bulk (and mass) of the phosphate group.

I think that the AIDS virus definitely has more chemical smarts than we do.  Humility is definitely in order.

Nomenclature note:  We’re all used to ATP (Adenosine TriPhosphate) and ADP (Adenosine DiPhosphate) — here all 3 or 2 phosphates form a chain.  Each of the 6 hydroxyls of inositol can be singly phosphorylated, leading to inositol bis, tris, tetrakis, pentakis, hexakis phosphates.  Phosphate chains can form on them as well, so IP7 and IP8 are known (heptakis?, Octakis??)

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When the dissociation constant doesn’t tell you what you want to know

Drug chemists spend a lot of time getting their drugs to bind tightly to their chosen target.  Kd’s (dissociation constants) are measured with care –https://en.wikipedia.org/wiki/Dissociation_constant.  But Kd’s are only  a marker for the biologic effects that are the real reason for the drug.  That’s why it was shocking to find that Kd’s don’t seem to matter in a very important and very well studied system.

It’s not the small molecule ligand protein receptor most drug chemists deal with, it’s the goings on at the immunologic synapse between antigen presenting cell and T lymphocyte (a much larger ligand target interface — 1,000 – 2,000 Angstroms^2 — than the usual site of drug/protein binding).   A peptide fragment lies down in a groove on the Major Histocompatibility Complex (pMHC) where it is presented to the T lymphoCyte Receptor (TCR) — another protein complex.  The hope is that an immune response to the parent protein of the peptide fragment will occur.

 

However, the Kd’s (affinities)of strong (e.g. producing an immune response) peptide agonist ligands and those producing not much (e.g. weak) are similar and at times overlapping.  High affinity yet nonStimulatory interactions occur with high frequency in the human T cell repertoire [ Cell vol. 174 pp. 672 – 687 ’18 ].  The authors  determined the structure of both weak and strong ligands bound to the TCR.  One particular TCR had virtually the same structure when bound to strong and weak agonist ligands. When studied in two dimensional membranes, the dwell time of ligand with receptor didn’t distinguish strong from weak antigens (surprising).

In general the Kds  pMHC/TCR  are quite low — not in the nanoMolar range beloved by drug chemists (and found in antigen/antibody binding), but 1000 times weaker in the micromolar range.  So [ Proc. Natl. Acad. Sci. vol. 115 pp. E7369 – E7378 ’18 ] cleverly added an extra few amino acids which they call molecular velcro, to boost the affinity x 10 (actually this decreases Kd tenfold).

One rationale for the weak binding is that it facilitates scanning by the TCR of  the pMHC  repertoire allowing the TCR to choose the best.  So they added the velcro, expecting the repertoire to be less diverse (since the binding was tighter).  It was just the same. Again the Kd didn’t seem to matter.

https://en.wikipedia.org/wiki/Catch_bond

Even more interesting, the first paper noted that productive TCR/pMHC bonds had catch bonds — e.g. bonds which get stronger the more you pull on them. The authors were actually able to measure the phenomenon. Catch bonds been shown to exist in a variety of systems (white cells sticking to blood vessel lining, bacterial adhesion), but their actual mechanism is still under debate.  The great thing about this paper (p. 682) is molecular dynamics simulation showed the conformational changes which occurred during catch bond formation in one case..   They even have videos.  Impressive.

This sort of thing is totally foreign to all solution chemistry, as there is no way to pull on a bond in solution.  Optical tweezers allow you to pull and stretch molecules (if you can attach them to large styrofoam balls).

A creation myth

Sigmund Freud may have been wrong about penis envy, but most lower forms of scientific life (chemists, biologists) do have physics envy — myself included.  Most graduate chemists have taken a quantum mechanics course, if only to see where atomic and molecular orbitals come from.  Anyone doing physical chemistry has likely studied statistical mechanics. I was fortunate enough to audit one such course given by E. Bright Wilson (of Pauling and Wilson).

Although we no longer study physics per se, most of us read books about physics.  Two excellent such books have come out in the past year.  One is “What is Real?” — https://www.basicbooks.com/titles/adam-becker/what-is-real/9780465096053/, the other is “Lost in Math” by Sabine Hossenfelder whose blog on physics is always worth reading, both for herself and the heavies who comment on what she writes — http://backreaction.blogspot.com

Both books deserve a long discursive review here. But that’s for another time.  Briefly, Hossenfelder thinks that physics for the past 30 years has become so fascinated with elegant mathematical descriptions of nature, that theories are judged by their mathematical elegance and beauty, rather than agreement with experiment.  She acknowledges that the experiments are both difficult and expensive, and notes that it took a century for one such prediction (gravitational waves) to be confirmed.

The mathematics of physics can certainly be seductive, and even a lowly chemist such as myself has been bowled over by it.  Here is how it hit me

Budding chemists start out by learning that electrons like to be in filled shells. The first shell has 2 elements, the next 2 + 6 elements etc. etc. It allows the neophyte to make some sense of the periodic table (as long as they deal with low atomic numbers — why the 4s electrons are of lower energy than the 3d electons still seems quite ad hoc to me). Later on we were told that this is because of quantum numbers n, l, m and s. Then we learn that atomic orbitals have shapes, in some wierd way determined by the quantum numbers, etc. etc.

Recursion relations are no stranger to the differential equations course, where you learn to (tediously) find them for a polynomial series solution for the differential equation at hand. I never really understood them, but I could use them (like far too much math that I took back in college).

So it wasn’t a shock when the QM instructor back in 1961 got to them in the course of solving the Schrodinger equation for the hydrogen atom (with it’s radially symmetric potential). First the equation had to be expressed in spherical coordinates (r, theta and phi) which made the Laplacian look rather fierce. Then the equation was split into 3 variables, each involving one of r, theta or phi. The easiest to solve was the one involving phi which involved only a complex exponential. But periodic nature of the solution made the magnetic quantum number fall out. Pretty good, but nothing earthshaking.

Recursion relations made their appearance with the solution of the radial and the theta equations. So it was plug and chug time with series solutions and recursion relations so things wouldn’t blow up (or as Dr. Gouterman, the instructor, put it: the electron has to be somewhere, so the wavefunction must be zero at infinity). MEGO (My Eyes Glazed Over) until all of a sudden there were the main quantum number (n) and the azimuthal quantum number (l) coming directly out of the recursion relations.

When I first realized what was going on, it really hit me. I can still see the room and the people in it (just as people can remember exactly where they were and what they were doing when they heard about 9/11 or (for the oldsters among you) when Kennedy was shot — I was cutting a physiology class in med school). The realization that what I had considered mathematical diddle, in some way was giving us the quantum numbers and the periodic table, and the shape of orbitals, was a glimpse of incredible and unseen power. For me it was like seeing the face of God.

But what interested me the most about “Lost in Math” was Hossenfelder’s discussion of the different physical laws appearing at different physical scales (e.g. effective laws), emergent properties and reductionism (pp. 44 –> ).  Although things at larger scales (atoms) can be understood in terms of the physics of smaller scales (protons, neutrons, electrons), the details of elementary particle interactions (quarks, gluons, leptons etc.) don’t matter much to the chemist.  The orbits of planets don’t depend on planetary structure, etc. etc.  She notes that reduction of events at one scale to those at a smaller one is not an optional philosophical position to hold, it’s just the way nature is as revealed by experiment.  She notes that you could ‘in principle, derive the theory for large scales from the theory for small scales’ (although I’ve never seen it done) and then she moves on

But the different structures and different laws at different scales is what has always fascinated me about the world in which we exist.  Do we have a model for a world structured this way?

Of course we do.  It’s the computer.

 

Neurologists have always been interested in computers, and computer people have always been interested in the brain — von Neumann wrote “The Computer and the Brain” shortly before his death in 1958.

Back in med school in the 60s people were just figuring out how neurons talked to each other where they met at the synapse.  It was with a certain degree of excitement that we found that information appeared to flow just one way across the synapse (from the PREsynaptic neuron to the POST synaptic neuron).  E.g. just like the vacuum tubes of the earliest computers.  Current (and information) could flow just one way.

The microprocessors based on transistors that a normal person could play with came out in the 70s.  I was naturally interested, as having taken QM I thought I could understand how transistors work.  I knew about energy gaps in atomic spectra, but how in the world a crystal with zillions of atoms and electrons floating around could produce one seemed like a mystery to me, and still does.  It’s an example of ’emergence’ about which more later.

But forgetting all that, it’s fairly easy to see how electrons could flow from a semiconductor with an abundance of them (due to doping) to a semiconductor with a deficit — and have a hard time flowing back.  Again a one way valve, just like our concept of the synapses.

Now of course, we know information can flow the other way in the synapse from POST synaptic to PREsynaptic neuron, some of the main carriers of which are the endogenous marihuana-like substances in your brain — anandamide etc. etc.  — the endocannabinoids.

In 1968 my wife learned how to do assembly language coding with punch cards ones and zeros, the whole bit.  Why?  Because I was scheduled for two years of active duty as an Army doc, a time in which we had half a million men in Vietnam.  She was preparing to be a widow with 2 infants, as the Army sent me a form asking for my preferences in assignment, a form so out of date, that it offered the option of taking my family with me to Vietnam if I’d extend my tour over there to 4 years.  So I sat around drinking Scotch and reading Faulkner waiting to go in.

So when computers became something the general populace could have, I tried to build a mental one using and or and not logical gates and 1s and 0s for high and low voltages. Since I could see how to build the three using transistors (reductionism), I just went one plane higher.  Note, although the gates can be easily reduced to transistors, and transistors to p and n type semiconductors, there is nothing in the laws of semiconductor physics that implies putting them together to form logic gates.  So the higher plane of logic gates is essentially an act of creation.  They do not necessarily arise from transistors.

What I was really interested in was hooking the gates together to form an ALU (arithmetic and logic unit).  I eventually did it, but doing so showed me the necessity of other components of the chip (the clock and in particular the microcode which lies below assembly language instructions).

The next level up, is what my wife was doing — sending assembly language instructions of 1’s and 0’s to the computer, and watching how gates were opened and shut, registers filled and emptied, transforming the 1’s and 0’s in the process.  Again note that there is nothing necessary in the way the gates are hooked together to make them do anything.  The program is at yet another higher level.

Above that are the higher level programs, Basic, C and on up.  Above that hooking computers together to form networks and then the internet with TCP/IP  etc.

While they all can be reduced, there is nothing inherent in the things that they are reduced to which implies their existence.  Their existence was essentially created by humanity’s collective mind.

Could something be going on in the levels of the world seen in physics.  Here’s what Nobel laureate Robert Laughlin (he of the fractional quantum Hall effect) has to say about it — http://www.pnas.org/content/97/1/28.  Note that this was written before people began taking quantum computers seriously.

“However, it is obvious glancing through this list that the Theory of Everything is not even remotely a theory of every thing (2). We know this equation is correct because it has been solved accurately for small numbers of particles (isolated atoms and small molecules) and found to agree in minute detail with experiment (35). However, it cannot be solved accurately when the number of particles exceeds about 10. No computer existing, or that will ever exist, can break this barrier because it is a catastrophe of dimension. If the amount of computer memory required to represent the quantum wavefunction of one particle is Nthen the amount required to represent the wavefunction of k particles is Nk. It is possible to perform approximate calculations for larger systems, and it is through such calculations that we have learned why atoms have the size they do, why chemical bonds have the length and strength they do, why solid matter has the elastic properties it does, why some things are transparent while others reflect or absorb light (6). With a little more experimental input for guidance it is even possible to predict atomic conformations of small molecules, simple chemical reaction rates, structural phase transitions, ferromagnetism, and sometimes even superconducting transition temperatures (7). But the schemes for approximating are not first-principles deductions but are rather art keyed to experiment, and thus tend to be the least reliable precisely when reliability is most needed, i.e., when experimental information is scarce, the physical behavior has no precedent, and the key questions have not yet been identified. There are many notorious failures of alleged ab initio computation methods, including the phase diagram of liquid 3He and the entire phenomenonology of high-temperature superconductors (810). Predicting protein functionality or the behavior of the human brain from these equations is patently absurd. So the triumph of the reductionism of the Greeks is a pyrrhic victory: We have succeeded in reducing all of ordinary physical behavior to a simple, correct Theory of Everything only to discover that it has revealed exactly nothing about many things of great importance.”

So reductionism doesn’t explain the laws we have at various levels.  They are regularities to be sure, and they describe what is happening, but a description is NOT an explanation, in the same way that Newton’s gravitational law predicts zillions of observations about the real world.     But even  Newton famously said Hypotheses non fingo (Latin for “I feign no hypotheses”) when discussing the action at a distance which his theory of gravity entailed. Actually he thought the idea was crazy. “That Gravity should be innate, inherent and essential to Matter, so that one body may act upon another at a distance thro’ a Vacuum, without the Mediation of any thing else, by and through which their Action and Force may be conveyed from one to another, is to me so great an Absurdity that I believe no Man who has in philosophical Matters a competent Faculty of thinking can ever fall into it”

So are the various physical laws things that are imposed from without, by God only knows what?  The computer with its various levels of phenomena certainly was consciously constructed.

Is what I’ve just written a creation myth or is there something to it?

Will acyclovir be a treatment for Alzheimer’s ?

When I was a first year medical student my aunt died of probable acute herpes simplex encephalitis at Columbia University Hospital in New York City.  That was 55 years ago and her daughters (teenagers at the time) still bear the scars.  Later, as a neurologist I treated it, and after 1977, when acyclovir, which effectively treats herpes encephalitis came out, I would always wonder if acyclovir would have saved her.

The drug is simplicity itself.  It’s just guanosine (https://en.wikipedia.org/wiki/Guanosine) with two of the carbons of the ribose missing.  Herpesviruses have an enzyme which forms the triphosphate incorporating it into its DNA killing the virus.  Well, actually we have the same enzyme, but the virus’s enzyme is 3,000,000 times more efficient than ours, so acyclovir is relatively nontoxic to us.  People with compromised renal function shouldn’t take it.

What does this have to do with Alzheimer’s disease?  The senile plaque of Alzheimers is mostly the aBeta peptide (39 – 43 amino acids) from the amyloid precursor protein (APP).  This has been known for years, and my notes on various papers about over the years contain 150,000 characters or so.

Even so, there’s a lot we don’t understand about APP and the abeta peptide — e.g. what are they doing for us?  You can knockout the APP gene in mice and they appear normal and fertile.  The paper cited below notes that APP has been present in various species for the past 400,000,000 years of evolutionary time remaining pretty much unchanged throughout, so it is probably doing something useful

A recent paper in Neuron (vol. 99 pp. 56 – 63 ’18) noted that aBeta is actually an antimicrobial peptide.  When exposed to herpes simplex it binds to glycoproteins on its surface and then  oligomerizes forming amyloid (just like in the senile plaque) trapping the virus.  Abeta will protect mice against herpes simplex 1 (HSV1) encephalitis.  Even more important — infection of the mice with HSV1 induced abeta production in their brains.

People have been claiming infections as the cause of just about every neurodegeneration since I’ve been a neurologist, and papers have been written about HSV1 and Alzheimer’s.

Which brings me to the second paper (ibid. pp. 64 – 82) that looked for the viral RNAs and DNAs in over 900 or so brains, some with and some without Alzheimer’s.  They didn’t find HSV but they found two other herpes viruses known to infect man (HHV6, HHV7 — which cause roseola infantum).  Humans are subject to infection with 8 different herpes virus (Epstein Barr — mononucleosis, H. Zoster — chickenpox etc. etc.).   Just about everyone of us has herpes virus in latent form in the trigeminal ganglion — which gets sensory information from our faces.

So could some sort of indolent herpesvirus infection be triggering abeta peptide production as a defense with the senile plaque as a byproduct?  That being the case, given the minimal benefits of any therapy we have for Alzheimer’s disease so far, why not try acyclovir (Zovirax) on Alzheimer’s.

I find it remarkable that neither paper mentioned this possibility, or even discussed any of the antivirals active against herpesviruses.

How many more metabolites like this are out there?

3′ deoxy 3′ 4′ didehydro cytidine triphosphate — doesn’t roll’ tripgingly on the tongue’ does it? (Hamlet Act 3, scene 2, 1–4).  Can you draw the structure?  It is the product of another euphoniously named enzyme — Viperin.  Abbreviated ddhCTP it is just cytidine triphosphate with a double bond between carbons 3 and 4 of the sugar.

Viperin is an enzyme induced by interferon which inhibits the replication of a variety of viruses. [ Nature vol. 558 pp. 610 – 614 ’18 ] describes a  beautiful sequence  of reactions for ddhCTP’s formation using S-Adenosyl Methionine (SAM).  ddhCTP acts as a chain terminator for the RNA dependent RNA polymerases of multiple members of Flaviviruses (including Zika).

However the paper totally blows it for not making the point that ddhCTP is extremely close to a drug (which has been used against AIDS for years — Zalcitabine (Hivid) — http://www.molbase.com/en/name-Zalcitibine.html which is just ddC.  ddhCTP is almost the same as ddC — except that there is no triphosphate on the 5′ hydroxyl (which enzymes in the body add), and instead of a double bond between carbons 3 and 4 of the sugar, both carbons are fully reduced (CH2 and CH2).  So ddhCTP is Nature’s own Zalcitabine.

It is worth reflecting on just how many other metabolites are out there acting as ‘natural’ drugs that we just haven’t found yet.

Remember entropy – take III

Pop quiz.  How would you make an enzyme in a cold dwelling organism (0 Centrigrade) as catalytically competent as its brothers living in us at 37 C?

We know that reactions go faster the hotter it is, because there is more kinetic energy of the reactants to play with.  So how do you make an enzyme move more when it’s cold and there is less kinetic energy to play with.

Well for most cold tolerance enzymes (psychrophilic enzymes — a great scrabble word), evolution mutates surface amino acids to glycine.  Why glycine?  Well it’s lighter, and there is no side chain to get in the way  when the backbone moves.  The mutations aren’t in the active site but far away.   This means more wiggling of the backbone — which means more entropy of the backbone.

The following papers [ Nature vol. 558 pp. 195 – 196, 324 – 218 ’81 ] studied adenylate kinase, an enzyme found in most eukaryotes  which catalyzes

ATP + AMP < — > 2 ADP.

They studied the enzyme from E. Coli which happily lives within us at 37 C, and mutated a few surface valines and isoleucines to glycine, lowered the temperature and found the enzyme works as well (the catalytic rate of the mutated enzyme at 0 C is the same as the rate of the unmutated enzyme at 37).

Chemists have been studying transition state theory since the days of Eyring, and reaction rates are inversely proportional the the amount of free energy (not enthalpy) to raise the enzyme to the transition state.

F = H – TS (Free energy = enthalpy – Temperature * Entropy).

So to increase speed decrease the enthalpy of activation (deltaH) or increase the amount of entropy.

It is possible to separately measure enthalpy and entropies of activation, and the authors did just that (figure 4 p. 326) and showed that the enthalpy of activation of the mutated enzyme (glycine added) was the same as the unmutated enzyme, but that the free energy of activation of the mutated enzyme was less because of an increase in entropy (due to unfolding of different parts of the enzyme).

Determining these two parameters takes an enormous amount of work (see the story from grad school at the end). You have to determine rate constants at various temperatures, plot the rate constant divided by temperature and then measure the slope of the line you get to obtain the enthalpy of activation.   Activation entropy is determined by the intercepts of the straight line (which hopefully IS straight) with the X axis.  Determining the various data points is incredibly tedious and uninteresting.

So enzymes  of cold tolerant organisms are using entropy to make their enzymes work.

Grad school story — back in the day, studies of organic reaction mechanisms were very involved with kinetic measurements (that’s where Sn1 and Sn2 actually come from).  I saw the following happen several times, and resolved never get sucked in to having to actually do kinetic measurements.  Some hapless wretch would present his kinetic data to a seminar, only to have Frank Westheimer think of something else and suggest another 6 months of kinetic measurements, so back he went to the lab for yet more drudgery.

 

 

Molecular biology’s oxymoron

Dear reader.  What does a gene do?  It codes for something.  What does a nonCoding Gene do?  It also codes for something, just RNA instead of protein. It’s molecular biology’s very own oxymoron, a throwback to the heroic protein-centric early days of molecular biology. The term has been enshrined by usage for so long that it’s impossible to get rid of.  Nonetheless, the the latest work found even more nonCoding genes than genes actually coding for  protein.

An amusing article from Nature (vol. 558 pp. 354 – 355 ’18) has the current state of play.   The latest estimate is from GTex which sequenced 900 billion RNAs found in various human tissues, matched them to the sequence(s) of the human genome and used computer algorithms to determine which  of them were the product of genes coding for proteins and genes coding for something else.

The report from GTex  (Genotype Tissue expression Project) found 21,306 protein-coding genes and 21,856 non-coding genes — amazingly there are more nonCoding genes than protein coding ones.  This  is many more genes than found in the two most widely used human gene databases. The GENCODE gene set, maintained by the EBI, includes 19,901 protein-coding genes and 15,779 non-coding genes. RefSeq, a database run by the US National Center for Biotechnology Information (NCBI), lists 20,203 protein-coding genes and 17,871 non-coding genes.

Stay tuned.  The fat lady hasn’t sung.

Chemistry and Biochemistry can’t answer the important questions but without them we are lost

The last two posts — one concerning the histone code and cerebral embryogenesis https://luysii.wordpress.com/2018/06/07/omar-khayyam-and-the-embryology-of-the-cerebral-cortex/ and the other concerning PVT1 enhancers promoters and cancer https://luysii.wordpress.com/2018/06/04/marshall-mcluhan-rides-again/ &#8212; would be impossible without chemical and biochemical knowledge and technology, but the results they produce and the answers they seek and lie totally outside both disciplines.

In fact they belong outside the physical realm in the space of logic, ideas, function — e.g. in the other half of the Cartesian dichotomy — the realm of ideas and spirit.  Certainly the biological issues are instantiated physically in molecules, just as computer memory used to be instantiated in magnetic cores, rather than transistors.

Back when I was starting out as a grad student in Chemistry in the early 60s, people were actually discovering the genetic code, poly U coded for phenylalanine etc. etc.  Our view was that all we had to do was determine the structure of things and understanding would follow.  The first xray structures of proteins (myoglobin) and Anfinsen’s result on ribonuclease showing that it could fold into its final compact form all by itself reinforced this. It also led us to think that all proteins had ‘a’ structure.

This led to people thinking that the only difference between us and a chimpanzee were a few amino acid differences in our proteins (remember the slogan that we were 98% chimpanzee).

So without chemistry and biochemistry we’d be lost, but the days of crude reductionism of the 60s and 70s are gone forever.  Here’s another example of chemical and biochemical impotence from an earlier post.

The limits of chemical reductionism

“Everything in chemistry turns blue or explodes”, so sayeth a philosophy major roommate years ago.  Chemists are used to being crapped on, because it starts so early and never lets up.  However, knowing a lot of organic chemistry and molecular biology allows you to see very clearly one answer to a serious philosophical question — when and where does scientific reductionism fail?

Early on, physicists said that quantum mechanics explains all of chemistry.  Well it does explain why atoms have orbitals, and it does give a few hints as to the nature of the chemical bond between simple atoms, but no one can solve the equations exactly for systems of chemical interest.  Approximate the solution, yes, but this his hardly a pure reduction of chemistry to physics.  So we’ve failed to reduce chemistry to physics because the equations of quantum mechanics are so hard to solve, but this is hardly a failure of reductionism.

The last post “The death of the synonymous codon – II” puts you exactly at the nidus of the failure of chemical reductionism to bag the biggest prey of all, an understanding of the living cell and with it of life itself.  We know the chemistry of nucleotides, Watson-Crick base pairing, and enzyme kinetics quite well.  We understand why less transfer RNA for a particular codon would mean slower protein synthesis.  Chemists understand what a protein conformation is, although we can’t predict it 100% of the time from the amino acid sequence.  So we do understand exactly why the same amino acid sequence using different codons would result in slower synthesis of gamma actin than beta actin, and why the slower synthesis would allow a more leisurely exploration of conformational space allowing gamma actin to find a conformation which would be modified by linking it to another protein (ubiquitin) leading to its destruction.  Not bad.  Not bad at all.

Now ask yourself, why the cell would want to have less gamma actin around than beta actin.  There is no conceivable explanation for this in terms of chemistry.  A better understanding of protein structure won’t give it to you.  Certainly, beta and gamma actin differ slightly in amino acid sequence (4/375) so their structure won’t be exactly the same.  Studying this till the cows come home won’t answer the question, as it’s on an entirely different level than chemistry.

Cellular and organismal molecular biology is full of questions like that, but gamma and beta actin are the closest chemists have come to explaining the disparity in the abundance of two closely related proteins on a purely chemical basis.

So there you have it.  Physicality has gone as far as it can go in explaining the mechanism of the effect, but has nothing to say whatsoever about why the effect is present.  It’s the Cartesian dualism between physicality and the realm of ideas, and you’ve just seen the junction between the two live and in color, happening right now in just about every cell inside you.  So the effect is not some trivial toy model someone made up.

Whether philosophers have the intellectual cojones to master all this chemistry and molecular biology is unclear.  Probably no one has tried (please correct me if I’m wrong).  They are certainly capable of mounting intellectual effort — they write book after book about Godel’s proof and the mathematical logic behind it. My guess is that they are attracted to such things because logic and math are so definitive, general and nonparticular.

Chemistry and molecular biology aren’t general this way.  We study a very arbitrary collection of molecules, which must simply be learned and dealt with. Amino acids are of one chirality. The alpha helix turns one way and not the other.  Our bodies use 20 particular amino acids not any of the zillions of possible amino acids chemists can make.  This sort of thing may turn off the philosophical mind which has a taste for the abstract and general (at least my roommates majoring in it were this way).

If you’re interested in how far reductionism can take us  have a look at http://wavefunction.fieldofscience.com/2011/04/dirac-bernstein-weinberg-and.html

Were my two philosopher roommates still alive, they might come up with something like “That’s how it works in practice, but how does it work in theory? 

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Omar Khayyam and the embryology of the cerebral cortex

“The moving finger writes; and, having writ, moves on”.  Did Omar Khayyam realize he was talking about the embryology of the human cerebral cortex?  Although apparently far removed from chemistry, embryology most certainly is not.  The moving finger in this case is an enzyme modifying histone proteins.

In the last post (https://luysii.wordpress.com/2018/06/04/marshall-mcluhan-rides-again/) I discussed how one site in the genome modified  the expression of a protein important in cancer (myc) even though it was 53,000 positions (nucleotides) away.  When stretched out into the usual B-form DNA shown in the text books this would stretch 1.7 microns or 17% of the way across the diameter of the usual spherical nucleus.  If our 3,200,000 nucleotide genome were chopped up into pieces this size some 60,000 segments would have to be crammed in.  Clearly DNA must be bent and wrapped around something, and that something is the nucleosome which is shaped like a fat disk.  Some 160 or so nucleotides are wrapped (twice) around the circumference of the nucleosome, giving a 10fold compaction in length.

The nucleosome is made of histone proteins, and here is where the moving finger comes in.  There are all sorts of chemical modifications of histones (some 130 different chemical modifications of histones are known).  Some are well known to most protein chemists, methylation of the amino groups of lysine, and the guanido groups of arginine, phosphorylation and acetylation  of serine and threonine.  Then there are the obscure small modifications –crotonylation, succinylation and malonylations.  Then there are the protein modifications, ubiquitination, sumoylation, rastafarination etc. etc.

What’s the point?  All these modifications determine what proteins and enzymes can and can’t react with a given stretch of DNA.  It goes by the name of histone code, and has little to do with the ordering of the nucleotides in DNA (the genetic code).  The particular set of histone modifications is heritable when cells divide.

Before going on, it’s worth considering just how miraculous our cerebral cortex is.  The latest estimate is that we have 80 billion neurons connected by 150 trillion synapses between them.  That’s far too much for 3.2 nucleotides to explicitly code for.

It turns out that almost all neurons in the cerebral cortex are born in a small area lining the ventricles.  They then migrate peripherally to form the 6 layered cerebral cortex.  The stem cell of the embryonic cortex is something called a radial glial cell which divides and divides each division producing 1 radial glial cell and 1 neuron which then goes on its merry way up to the cortex.

Which brings us (at last) to the moving finger, an enzyme called PRDM16 which puts a methyl group on two particular lysines  (#4 and #9) of histone H3.  PRDM16 is highly enriched in radial glia and nearly absent in mature neurons.  Knock PRDM16a out in radial glia, and the cortex is disorganized due to deficient neuronal migration.  Knock it out in newly formed neurons and the cortex is formed normally.  The moving finger having writ (in radial glia) moves on and is no longer needed (by mature neurons). “nor all thy Piety nor Wit shall lure it back to cancel half a line.  Nor all thy tears wash out a word of it”.

You may read more about this fascinating work in Neuron vol. 98 pp. 867 – 869, 945 – 962 ’18

Marshall McLuhan rides again

Marshall McLuhan famously said “the medium is the message”. Who knew he was talking about molecular biology?  But he was, if you think of the process of transcription of DNA into various forms of RNA as the medium and the products of transcription as the message.  That’s exactly what this paper [ Cell vol. 171 pp. 103 – 119 ’17 ] says.

T cells are a type of immune cell formed in the thymus.  One of the important transcription factors which turns on expression of the genes which make a T cell a Tell is called Bcl11b.  Early in T cell development it is sequestered away near the nuclear membrane in highly compacted DNA. Remember that you must compress your 1 meter of DNA down by 100,000fold to have it fit in the nucleus which is 1/100,000th of a meter (10 microns).

What turns it on?  Transcription of nonCoding (for protein) RNA calledThymoD.  From my reading of the paper, ThymoD doesn’t do anything, but just the act of opening up compacted DNA near the nuclear membrane produced by transcribing ThymoD is enough to cause this part of the genome to move into the center of the nucleus where the gene for Bcl11b can be transcribed into RNA.

There’s a lot more to the paper,  but that’s the message if you will.  It’s the act of transcription rather than what is being transcribed which is important.

Here’s more about McLuhan — https://en.wikipedia.org/wiki/Marshall_McLuhan

If some of the terms used here are unfamiliar — look at the following post and follow the links as far as you need to.  https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/

Well that was an old post.  Here’s another example [ Cell vol. 173 pp. 1318 – 1319, 1398 – 1412 ’18 ] It concerns a gene called PVT1 (Plasmacytoma Variant Translocation 1) found 25 years ago.  It was the first gene coding for a long nonCoding (for proteins RNA (lncRNA) found as a recurrent breakpoint in Burkitt’s lymphoma, which sadly took a friend (Nick Cozzarelli) far too young as (he edited PNAS for 10 years).

So PVT1 is involved in cancer.  The translocation turns on expression of the myc oncogene, something that has been studied out the gazoo and we’re still not sure of how it causes cancer. I’ve got 60,000 characters of notes on the damn thing, but as someone said 6 years ago “Whatever the latest trend in cancer biology — cell cycle, cell growth, apoptosis, metabolism, cancer stem cells, microRNAs, angiogenesis, inflammation — Myc is there regulating most of the key genes”

We do know that the lncRNA coded by PVT1 in some way stabilizes the myc protein [ Nature vol. 512 pp. 82 – 87 ’14 ].  However the cell experiments knocked out the lncRNA of PVT1 and myc expression was still turned on.

PVT1 resides 53 kiloBases away from myc on chromosome #8.  That’s about 17% of the diameter of the average nucleus (10 microns) if the DNA is stretched out into the B-DNA form seen in all the textbooks.  Since each base is 3.3 Angstroms thick that’s 175,000 Angstroms 17,500 nanoMeters 1.7 microns.  You can get an idea of how compacted DNA is in the nucleus when you realize that there are 3,200,000,000/53,000 = 60,000 such segments in the genome all packed into a sphere 10 microns in diameter.

To cut to the chase, within the PVT1 gene there are at least 4 enhancers (use the link above to find what all the terms to be used actually mean).  Briefly enhancers are what promoters bind to to help turn on the transcription of the genes in DNA into RNA (messenger and otherwise).  This means that the promoter of PVT1 binds one or more of the enhancers, preventing the promoter of the myc oncogene from binding.

Just how they know that there are 4 enhancers in PVT1 is a story in itself.  They cut various parts of the PVT1 gene (which itself has 306,721 basepairs) out, and place it in front of a reporter gene and see if transcription increases.

The actual repressor of myc is the promoter of PVT1 according to the paper (it binds to the enhancers present in the gene body preventing the myc promoter from doing so).  Things may be a bit more complicated as the PVT1 gene also codes for a cluster of 7 microRNAs and what they do isn’t explained in the paper.

So it’s as if the sardonic sense of humor of ‘nature’, ‘evolution’, ‘God’, (call it what you will) has set molecular biologists off on a wild goose chase, looking at the structure of the gene product (the lncRNA) to determine the function of the gene, when actually it’s the promoter in front of the gene and the enhancers within which are performing the function.

The mechanism may be more widespread, as 4/36 lncRNA promoters silenced by CRISPR techniques subsequently activated genes in a 1 megaBase window (possibly by the same mechanism as PVT1 and myc).

Where does McLuhan come in?  The cell paper also notes that lncRNA gene promoters are more evolutionarily conserved than their gene bodies.  So it’s the medium (promoter, enhancer) is the message once again (rather than what we thought the message was).