Force in physics is very different from the way we think of it

I’m very lucky (and honored) that a friend asked me to read and comment on the galleys of a his book. He’s trying to explain some very advanced physics to laypeople (e.g. me). So he starts with force fields, gravitational, magnetic etc. etc. The physicist’s idea of force is so far from the way we usually think of it. Exert enough force long enough and you get tired, but the gravitational force never does, despite moving planets stars and whole galaxies around.

Then there’s the idea that the force is there all the time whether or not it’s doing something a la Star Wars. Even worse is the fact that force can push things around despite going through empty space where there’s nothing to push on, action at a distance if you will.

You’ve in good company if the idea bothers you. It bothered Isaac Newton who basically invented action at a distance. Here he is in a letter to a friend.

“That gravity should be innate inherent & {essential} to matter so that one body may act upon another at a distance through a vacuum without the mediation of any thing else by & through which their action or force {may} be conveyed from one to another is to me so great an absurdity that I beleive no man who has in philosophical matters any competent faculty of thinking can ever fall into it. “

So physicists invented the ether which was physical, and allowed objects to push each other around by pushing on the ether between them. 

But action at a distance without one atom pushing on the next etc. etc. is exactly what an incredible paper found [ Proc. Natl. Acad. Sci. vol. 117 pp. 25445 – 25454 ’20 ].

Allostery is an abstract concept in protein chemistry, far removed from everyday life. Far removed except if you like to breathe, or have ever used a benzodiazepine (Valium, Librium, Halcion, Ativan, Klonopin, Xanax) for anything. Breathing? Really? Yes — Hemoglobin, the red in red blood cells is really 4 separate proteins bound to each other. Each of the four can bind one oxygen molecule. Binding of oxygen to one of the 4 proteins produces a subtle change in the structure of the other 3, making it easier for another oxygen to bind. This produces another subtle change in structure of the other making it easier for a third oxygen to bind. Etc. 

This is what allostery is, binding of molecule to one part of a protein causing changes in structure all over the protein. 

Neurologists are familiar with the benzodiazepines, using them to stop continuous seizure activity (status epilepticus), treat anxiety (Xanax), or seizures (Klonopin). They all work the same way, binding to a complex of 5 proteins called the GABA receptor, which when it binds Gamma Amino Butyric Acid (GABA) in one place causes negative ions to flow into the neuron, inhibiting it from firing. The benzodiazepines bind to a completely different site, making the receptor more likely to open when it binds GABA. 

The assumption about all allostery is that something binds in one place, pushing the atoms around, which push on other atoms which push on other atoms, until the desired effect is produced. This is the opposite of action at a distance, where an effect is produced without the necessity of physical contact.

The paper studied TetR, a protein containing 203 amino acids. If you’ve ever thought about it, almost all the antibiotics we have come from bacteria, which they use on other bacteria. Since we still have bacteria around, the survivors must have developed a way to resist antibiotics, and they’ve been doing this long before we appeared on the scene. 

TetR helps bacteria resist tetracycline, an antibiotic produced by bacteria. When tetracycline binds to TetR it causes other parts of the protein to change so it binds DNA causing the bacterium, among other things, to make a pump which moves tetracyline out of the cell. Notice that site where tetracycline binds on TetR is not the business end where TetR binds DNA, just as where the benzodiazepines bind the GABA receptor is not where the ion channel is. 

This post is long enough already without describing the cleverness which allowed the authors to do the following. They were able to make TetRs containing every possible mutation of all 203 positions. How many is that — 203 x 19 = 3838 different proteins. Why 19? Because we have 20 amino acids, so there are 19 possible distinct changes at each of the 203 positions in TetR.  

Some of the mutants didn’t bind to DNA, implying they were non-functional. The 3 dimensional structure of TetR is known, and they chose 5 of nonfunctional mutants. Interestingly these were distributed all over the protein. 

Then, for each of the 5 mutants they made another 3838 mutants, to see if a mutation in another position would make the mutant functional again. You can see what a tremendous amount of work this was. 

Here is where it gets really interesting. The restoring mutant (revertants if you want to get fancy) were all over the protein and up to 40 – 50 Angstroms away from the site of the dead mutation. Recall that 1 Angstrom is the size of a hydrogen atom, a turn of the alpha helix is 5.4 Angstroms and contains 3.5 amino acids per turn.The revertant mutants weren’t close to the part of the protein binding tetracycline or the part binding to DNA. 

Even worse the authors couldn’t find a contiguous path of atom pushing atom pushing atom, to explain why TetR was able to bind DNA again. So there you have it — allosteric action at a distance.

There is much more in the paper, but after all the work they did it’s time to let the authors speak for themselves. “Several important insights emerged from these results. First, TetR exhibits a high degree of allosteric plasticity evidenced by the ease of disrupting and restoring function through several mutational paths. This suggests the functional landscape of al- lostery is dense with fitness peaks, unlike binding or catalysis where fitness peaks are sparse. Second, allosterically coupled residues may not lie along the shortest path linking allosteric and active sites but can occur over long distances “

But there is still more to think about, particularly for drug development. Normally, in developing a drug for X, we have a particular site on a particular protein as a target, say the site on a neurotransmitter receptor where a neurotransmitter binds. But the work shows that sites far removed from the actual target might have the same effect

Action at a distance comes to chemistry

Allostery is an abstract concept in protein chemistry, far removed from everyday life. Far removed except if you like to breathe, or have ever used a benzodiazepine (Valium, Librium, Halcion, Ativan, Klonopin, Xanax) for anything. Breathing? Really? Yes — Hemoglobin, the red in red blood cells is really 4 separate proteins bound to each other. Each of the four can bind one oxygen molecule. Binding of oxygen to one of the 4 proteins produces a subtle change in the structure of the other 3, making it easier for another oxygen to bind. This produces another subtle change in structure of the other making it easier for a third oxygen to bind. Etc.

This is what allostery is, binding of molecule to one part of a protein causing changes in structure all over the protein.

Neurologists are familiar with the benzodiazepines, using them to stop continuous seizure activity (status epilepticus), treat anxiety (Xanax), or seizures (Klonopin). They all work the same way, binding to a complex of 5 proteins called the GABA receptor, which when it binds Gamma Amino Butyric Acid (GABA) in one place causes negative ions to flow into the neuron, inhibiting it from firing. The benzodiazepines bind to a completely different site, making the receptor more likely to open when it binds GABA.

The assumption about all allostery is that something binds in one place, pushing the atoms around, which push on other atoms which push on other atoms, until the desired effect is produced. This is the opposite of action at a distance, where an effect is produced without the necessity of physical contact.

Even though Newton invented a theory of gravity, which worked beautifully, he was disturbed by the fact that it acted through empty space. Here’s what he wrote in a letter to Bentley

“That gravity should be innate inherent & {essential} to matter so that one body may act upon another at a distance through a vacuum without the mediation of any thing else by & through which their action or force {may} be conveyed from one to another is to me so great an absurdity that I beleive no man who has in philosophical matters any competent faculty of thinking can ever fall into it. “

So physicists invented the ether which was physical, and allowed objects to push each other around by pushing on the ether between them.

But action at a distance without one atom pushing on the next etc. etc. is exactly what an incredible paper found [ Proc. Natl. Acad. Sci. vol. 117 pp. 25445 – 25454 ’20 ]. Here’s a link but it’s probably behind a paywall — https://www.pnas.org/content/pnas/117/41/25445.full.pdf

The paper studied TetR, a protein containing 203 amino acids. If you’ve ever thought about it, almost all the antibiotics we have come from bacteria, which they use on other bacteria. Since we still have bacteria around, the survivors must have developed a way to resist antibiotics, and they’ve been doing this long before we appeared on the scene.

TetR helps bacteria resist tetracycline, an antibiotic produced by bacteria. When tetracycline binds to TetR it causes other parts of the protein to change so it binds DNA causing the bacterium, among other things, to make a pump which moves tetracyline out of the cell. Notice that site where tetracycline binds on TetR is not the business end where TetR binds DNA, just as where the benzodiazepines bind the GABA receptor is not where the ion channel is.

This post is long enough already without describing the cleverness which allowed the authors to do the following. They were able to make TetRs containing every possible mutation of all 203 positions. How many is that — 203 x 19 = 3838 different proteins. Why 19? Because we have 20 amino acids, so there are 19 possible distinct changes at each of the 203 positions in TetR.

Some of the mutants didn’t bind to DNA, implying they were non-functional. The 3 dimensional structure of TetR is known, and they chose 5 of nonfunctional mutants. Interestingly these were distributed all over the protein.

Then, for each of the 5 mutants they made another 3838 mutants, to see if a mutation in another position would make the mutant functional again. You can see what a tremendous amount of work this was.

Here is where it gets really interesting. The restoring mutant (revertants if you want to get fancy) were all over the protein and up to 40 – 50 Angstroms away from the site of the dead mutation. Recall that 1 Angstrom is the size of a hydrogen atom, a turn of the alpha helix is 5.4 Angstroms and contains 3.5 amino acids per turn.The revertant mutants weren’t close to the part of the protein binding tetracycline or the part binding to DNA.

Even worse the authors couldn’t find a contiguous path of atom pushing atom pushing atom, to explain why TetR was able to bind DNA again. So there you have it — allosteric action at a distance.

There is much more in the paper, but after all the work they did it’s time to let the authors speak for themselves. “Several important insights emerged from these results. First, TetR exhibits a high degree of allosteric plasticity evidenced by the ease of disrupting and restoring function through several mutational paths. This suggests the functional landscape of al- lostery is dense with fitness peaks, unlike binding or catalysis where fitness peaks are sparse. Second, allosterically coupled residues may not lie along the shortest path linking allosteric and active sites but can occur over long distances “

But there is still more to think about, particularly for drug development. Normally, in developing a drug for X, we have a particular site on a particular protein as a target, say the site on a neurotransmitter receptor where a neurotransmitter binds. But the work shows that sites far removed from the actual target might have the same effect

How general anesthesia works

People have been theorizing how general anesthesia works since there has been general anesthesia.  The first useful one was diethyl ether (by definition what lipids dissolve in).  Since the brain has the one of the highest fat contents of any organ, the mechanism was obvious to all.  Anesthetics dissolve membranes.  Even the newer anesthetics look quite lipophilic — isoflurane CF3CHCL O CF2H screams (to the chemist) find me a lipid to swim in.  One can show effects of lipids on artificial membranes but the concentrations to do so are so high they would be lethal.

Attention shifted to the GABA[A] receptor, because anesthetics are effective in potentiating responses to GABA  — all the benzodiazepines (valium, librium) which bind to it are sedating.  Further evidence that a protein is involved, is that the optical isomers of enflurane vary in anesthetic potency (but not by very much — only 60%).  Lipids (except cholesterol) just aren’t optically active.  Interestingly, alfaxolone is a steroid and a general anesthetic as well.

Well GABA[A] is an ion channel, meaning that its amino acids form alpha helices which span the membrane (and create a channel for ion flow).  It would be devilishly hard to distinguish binding to the transmembrane part from binding to the membrane near it. [ Science vol. 322 pp. 876 – 880 2008 ] Studied 4 IV anesthetics (propofol, ketamine, etomidate, barbiturate) and 4 gasses (nitrous  oxide, isoflurance, devoflurane, desflurane) and their effects on 11 ion channels — unsurprisingly all sorts of effects were found — but which ones are the relevant.

All this sort of stuff could be irrelevant, if a new paper is actually correct [ Neuron vol. 102 pp. 1053 – 1065 ’19 ].  The following general anesthetics (isoflurane, propofol, ketamine and desmedtomidine) all activate cells in the hypothalamus (before this anesthetics were thought to work by ultimately inhibiting neurons).  They authors call these cells AANs (Anesthesia Activated Neurons).

They are found in the hypothalamus and contain ADH.  Time for some anatomy.  The pituitary gland is really two glands — the adenohypophysis which secretes things like ACTH, TSH, FSH, LH etc. etc, and the neurohypophysis which secretes oxytocin and vasopressin (ADH) directly into the blood (and also into the spinal fluid where it can reach other parts of the brain.  ADH release is actually from the axons of the hypothalamic neurons.  The AANs activated by the anesthetics release ADH.

Of course the workers didn’t stop there — they stimulated the neurons optogenetically and put the animals to sleep. Inhibition of these neurons shortened the duration of general anesthesia.

Fascinating (if true).  The next question is how such chemically disparate molecules can activate the AANs.  Is there a common receptor for them, and if so what is it?

Happy fiscal new year !