Category Archives: Chemistry (relatively pure)

Kuru continues to inform

Neurologists of my generation were fascinated with Kuru, a disease of the (formerly) obscure Fore tribe of New Guinea. Who would have thought they would tell us a good deal about protein structure and dynamics?

It is a fascinating story including a Nobelist pedophile (Carleton Gajdusek) https://en.wikipedia.org/wiki/Daniel_Carleton_Gajdusek and another (future) Nobelist who I probably ate lunch with when we were both medical students in the same Medical Fraternity but don’t remember –https://en.wikipedia.org/wiki/Stanley_B._Prusiner

Kuru is a horrible neurodegeneration starting with incoordination, followed by dementia and death in a vegetative state in 4 months to 2 years. For the cognoscenti — the pathology is neuronal loss, astrocytosis, microglial proliferation, loss of myelinated fibers and the kuru plaque.

It is estimated that it killed 3,000 members of the 30,000 member tribe. The mode of transmission turned out to be ritual cannibalism (flesh of the dead was eaten by the living before burial). Once that stopped the disease disappeared.

It is a prion disease, e.g. a disease due to a protein (called PrP) we all have but in an abnormal conformation (called PrpSc). Like Vonnegut’s Ice-9 (https://en.wikipedia.org/wiki/Ice-nine) PrPSc causes normal PrP to assume its conformation, causing it to aggregate and form an insoluble mess. We still don’t know the structure of PrPSc (because it’s an insoluble mess). Even now, “the detailed structure of PrPSc remains unresolved” but ‘it seems to be’ very similar to amyloid [ Nature vol. 512 pp. 32 – 34 ’14]. Not only that, but we don’t know what PrP actually does, and mice with no PrP at all are normal [ Nature vol. 365 p. 386 ’93 ]. For much more on prions please see https://luysii.wordpress.com/2014/03/30/a-primer-on-prions/

Prusiner’s idea that prion diseases were due to a protein, with no DNA or RNA involved met with incredible resistance for several reasons. This was the era of DNA makes RNA makes protein, and Prisoner was asking us to believe that a protein could essentially reproduce without any DNA or RNA. This was also the era in which X-ray crystallography was showing us ‘the’ structure of proteins, and it was hard to accept that there could be more than one.

There are several other prion diseases of humans (all horrible) — mad cow disease, Jakob Creutzfeldt disease, Familial fatal insomnia, etc. etc. and others in animals. All involve the same protein PrP.

One can take brain homogenates for an infected animal, inoculate it into a normal animal and watch progressive formation of PrPSc insoluble aggregates and neurodegeneration. A huge research effort has gone into purifying these homogenates so the possibility of any DNA or RNA causing the problem is very low. There still is one hold out — Laura Manuelidis who would have been a classmate had I gone to Yale Med instead of Penn. n

Enter [ Nature vol. 522 pp. 423 – 424, 478 – 481 ’15 ] which continued to study the genetic makeup of the Fore tribe. In an excellent example of natural selection in action, a new variant of PrP appeared in the tribe. At amino acid #127, valine is substituted for glycine (G127V is how this sort of thing is notated). Don’t be confused if you’re somewhat conversant with the literature — we all have a polymorphism at amino acid #129 of the protein, which can be either methionine or valine. It is thought that people with one methionine and one valine on each gene at 129 were somewhat protected against prion disease (presumably it affects the binding between identical prion proteins required for conformational change to PrPSc.

What’s the big deal? Well, this work shows that mice with one copy of V127 are protected against kuru prions. The really impressive point is that the mice are also protected against variant Creutzfedlt disease prions. Mice with two copies of V127 are completely protected against all forms of human prion disease . So something about V/V at #127 prevents the conformation change to PrPSc. We don’t know what it is as the normal structure of the variant hasn’t been determined as yet.

This is quite exciting, and work is certain to go on to find short peptide sequences mimicking the conformation around #127 to see if they’ll also work against prion diseases.

This won’t be a huge advance for the population at large, as prion diseases, as classically known, are quite rare. Creutzfeldt disease hits 1 person out of a million each year.

There are far bigger fish to fry however. There is some evidence that the neurofibrillary tangles (tau protein) of Alzheimer’s disease and the Lewy bodies (alpha-Synuclein) of Parkinsonism, spread cell to cell by a ‘prionlike’ mechanism [ Nature vol.485 pp. 651 – 655 ’12, Neuron vol. 73 pp. 1204 – 1215 ’12 ]. Could this sort of thing be blocked by a small amino acid change in one of them (or better a small drug like peptide?).

Stay tuned.

The uses of disorder

There was a lot of shock and awe about a report showing how seemingly minor changes in an aliphatic group on benzene led to markedly different conformations in its protein target (lysozyme from bacteriophage T4) http://pipeline.corante.com/archives/2015/06/18/tiny_and_not_so_tiny_changes.php.

Our noses are being rubbed in just how floppy proteins are, in contrast to the first glimpses of protein structure obtained by Xray crystallography. Back then we knew so little about proteins, that seeing all the atoms laid out in alpha helices and beta sheets was incredibly compelling. We talked about the structure of a protein rather than a structure. Even back then, with hemoglobin (one of the first solved proteins) it was obvious that proteins had to have more than one structure. The porphyrin ring in heme that oxygen binds to is buried deep in hemoglobin, and the initial structure had to move in some way to allow oxygen to find its way in (because the initial structure showed no obvious channel for oxygen). So hemoglobin had to breathe.

We now know that many proteins have intrinsically disordered segments. Amazingly, the most recent estimate I could find in my notes (or in Wikipedia) is this — It is estimated that over 30% of eukaryotic proteins have stretches of over 30 amino acids that are intrinsically disordered [ J. Mol. Biol. vol. 337 pp. 635 – 645 ’04 ]. Does anyone out there know of more recent data?

We’re a lot smarter now — here’s a comment on Derek’s post — “I have always thought crystal structures of proteins/enzymes are more a guide than actually useful. You are crystallizing a protein first-proteins don’t pack like that in vivo. Then you are settling on the conformation that freezes out- is this the lowest energy form? Then you are ignoring hte fact that these are highly dynamic structures that are constantly moving, sliding, shaking, adjusting. Then if you put a ligand in there you get the lowest energy form-which is what it would look like after reaction and before ligand dissociation- this is quite different from what it can look like at other stages of the reaction.”

Here is an interesting example of the uses of protein disorder going on right now in just about every neuron in your body. Most neurons have long processes, far too long for diffusion to move a needed protein to their ends. For that purpose we have microtubules (aka neurotubules in neurons) stretching the length of the processes, onto which two types of motors attach (dyneins which moves things to negative end of the microtubule and kinesins which move things to the positive end).

The microtubule is built from a heterodimer of two proteins (alpha and beta tubulin). Each contains about 450 amino acids and forms a globule 40 Angstroms (4 nanoMeters) in diameter. The heterodimers pack end to end to form a protofilament. 13 protofilaments line up side by side to form the microtubule, a hollow structure about 250 Angstroms in diameter. In cells microtubules are 1 to 10 microns long, but in nerve process they can be ‘up to’ 100 microns in length. Even at 1 micron (1,000 nanoMeters) that’s 13 * 250 heterodimers in a microtubule.

Any protein structure this important has a lot of modifications imposed on it to alter structure and function. Examples include phosphorylation and the addition of glutamic acid chains (polyglutamylation). The carboxy terminal tails of alpha and beta tubulin are flexible and stick out from the tubulin rod (which is why they aren’t seen on Xray crystallography). The carboxy terminal tail is the site of post-translational glutamylation. The enzyme polyglutamylating the carboxy terminal tail of beta tubular is TTLL7 (you don’t want to know what the acronym stands for). It binds to the alpha/beta tubular heterodimer by an intrinsically disordered region of its own (becoming structured in the process), then it binds to the intrinsically disordered carboxyl terminal tails, structuring them and modifying them. It’s basically a mating dance. There is a precedent for this — see https://luysii.wordpress.com/2013/12/29/the-mating-dance-of-a-promiscuous-protein/

So disordered regions of proteins although structureless are far from functionless

Not physical organic chemistry but organic physical chemistry

This post is about physical chemistry with organic characteristics in the sense that capitalism in China is called socialism with Chinese characteristics. A lot of cell biology is also involved.

I remember the first time I heard about Irving Langmuir and the two dimensional gas he created. It even followed a modified perfect gas law (PA = nRT where A is area). He did this by making a monolayer of long chain fatty acids on water, with the carboxyl groups binding to the water, and the hydrocarbon side chain sticking up into the air. I thought this was incredibly neat. It was the first example of organic physical chemistry. He published his work in 1917 and won the Nobel in Chemistry for it in 1932.

Fast forward to our understanding of the membrane encasing our cells (the technical term is plasma membrane to distinguish from the myriad other membranes inside our cells. To a first approximation it’s just two Langmuir films back to back with the hydrocarbon chains of the lipids dissolving in each other, and the hydrophilic parts of the membrane lipids binding to the water on either side. This is why it’s called a lipid bilayer.

Most of the signals going into our cells must pass through the plasma membrane, using proteins spanning it. As a neurologist I spent a lot of time throwing drugs at them — examples include every known receptor for neurotransmitters, reuptake proteins for them (think the dopamine transporter), ion channels. The list goes on and on and includes the over 800 G protein coupled receptors (GPCRs) with their 7 transmembrane segments we have in our genome [ Proc. Natl. Acad. Sci. vol. 111 pp. 1825 – 1830 ’14 ].

Glypiated proteins (you heard right) also known as PIGtailed proteins (you heard that right too) don’t follow this pattern. They are proteins anchored in the outer leaflet of the plasma membrane lipid bilayer by covalently linked phosphatidyl inositol. https://en.wikipedia.org/wiki/Phosphatidylinositol — the picture shows you why — inositol is a sugar, hence crawling with hydroxyl groups, while the phosphatidic acid part has two long hydrocarbon chains which can embed in the outer leaflet. We have 150 of them as of 2009 (probably more now). Examples of PIGtailed proteins include alkaline phosphatase, Thy-1 antigen, acetyl cholinesterase, lipoprotein lipase, and decay accelerating factor. So most of them are enzymes working on stuff outside the cell, so they don’t need to signal.

Enter the lipid raft. [ Cell vol. 161 pp. 433 – 434, 581 – 594 ’15 ] It’s been 18 years since rafts were first proposed, and their existence is still controversial (with zillions of papers saying they exist and more zillions saying they don’t). What are they — definitions vary (particularly about how large they are). Here’s what Molecular Biology of the Cell 4th edition p. 589 had to say about them — Rafts are small (700 Angstroms in diameter). Rafts are rich in sphingolipids, glycolipids and cholesterol. The hydrocarbon chains are longer and straighter than those of most membrane lipids, rafts are thicker than other parts of bilayer. This allows them to better accomodate ‘certain’ membrane proteins, which accumulate there. [ Proc. Natl. Acad. Sci. vol. 100 p. 8055 – 7’03 ] These include glycosylphosphatidylinositol anchored proteins (glypiated proteins), cholesterol linked and palmitoylated proteins such as Hedgehog, Src family kinases and the alpha subunits of G proteins, cytokine receptors and integrins.

Biochemical analysis shows that rafts consist of cholesterol and sphingolipids in the exoplasmic leaflet (outer layer of the plasma membrane) of the lipid bilayer and cholesterol and phospholipids with saturated fatty acids in the endoplasmic leaflet (layer facing the cytoplasm). The raft is less fluid than surrounding areas of the membrane. So if they in fact exist, rafts contain a lot of important cellular players.

The Cell paper introduced synthetic fluorescent glypiated proteins into the outer plasma membrane leaflet of Chinese hamster ovary cells and was able to demonstrate nanoClustering on scales under 1,000 Angstroms (way too small to see with visible light, accounting for a lot of the controversy concerning their existence).

How can the authors make such a statement? The evidence was a decrease in fluorescence anisotropy due to Forster resonance energy transfer effects. Forster energy transfer is interesting in that it doesn’t involve molecule #1 losing energy by emitting a photon which is absorbed by molecule #2 increasing its energy. It works by molecule #1 inducing a dipole in molecule #2 (by a Van der Waals effect). Obviously, to do this, the molecules must be fairly close, and transfer efficiency falls off as the inverse 6th power of the distance between the two molecules.

In Fluorescence Resonance Energy Transfer (FRET), one fluorophore (the donor) transfers its excited state energy to a different fluorophore (the acceptor) which emits fluorescence of a different color. For more details see — https://en.wikipedia.org/wiki/Förster_resonance_energy_transfer — its interesting stuff. Again an example of physical chemistry with organic characteristics (and pretty good evidence for the existence of lipid rafts to boot).

Now it gets even more interesting. Nanoclustering is dependent on the length of the acyl chain forming the GPI anchor (at least 18 carbons must be present). NanoClustering diminishes on cholesterol depletion in actin depleted cell blebs and mutant cell lines deficient in the inner leaflet lipid — phosphatidylserine (PS) — which has two long chain fatty acids hanging off the glycerol. So it looks as if the saturated acyl chains of the glypiated proteins of the outer leaflet interdigitate with those of PS in inner leaflet. The effect is also enhanced on expression of proteins specifically linking PS to the actin cytoskeleton of the cortex. Binding of PS to the cortical actin cytoskeleton determines where and when the clusters will be stabilized. The coupling can work both ways — if something immobilizes and stabilizes the glypiated proteins extracellularly, than PS lipids can form correlated patches.

This might be a mechanism for information transfer across the plasma membrane (and acrossother membranes to boot). This could also serve as a way to couple many outer leaflet membrane lipids such as gangliosides and other sphingolipids with events internal to the cell. Cholesterol can stabilize the local liquid ordered domain over a length scale that is large than the size of the immobilized cluster. A variety of membrane associated proteins inside the cell (spectrin, talin, caldesmon) are able to bind actin. “The formation of the contractile actin clusters then determine when and where the domains may be stabilized, bringing the generation of membrane domains in live cells under control of the actomyosin signaling network.’

So just like the integrins which can signal from outside the cell to inside and from inside the cell to outside, glypiated proteins and the actin cytoskeleton may form a two way network for signaling. No one should have to tell you how important the actomyosin cytoskeleton is in just about everything the cell does. Truly fascinating stuff. Stay tuned.

Should pregnant women smoke pot?

Well, maybe this is why college board scores have declined so much in recent decades that they’ve been normed upwards. Given sequential MRI studies on brain changes throughout adolescence (with more to come), we know that it is a time of synapse elimination. (this will be the subject of another post). We also know that endocannabinoids, the stuff in the brain that marihuana is mimicking, are retrograde messengers there, setting synaptic tone for information transmission between neurons.

But there’s something far scarier in a paper that just came out [ Proc. Natl. Acad. Sci. vol. 112 pp. 3415 – 3420 ’15 ]. Hedgehog is a protein so named because its absence in fruitflies (Drosophila) causes excessive bristles to form, making them look like hedgehogs. This gives you a clue that Hedgehog signaling is crucial in embryonic development. A huge amount is known about it with more being discovered all the time — for far more details than I can provide see http://en.wikipedia.org/wiki/Hedgehog_signaling_pathway.

Unsurprisingly, embryonic development of the brain involves hedgehog, e,g, [ Neuron vol. 39 pp. 937 – 950 ’03 ] Hedgehog (Shh) signaling is essential for the establishment of the ventral pattern along the whole neuraxis (including the telencephalon). It plays a mitogenic role in the expansion of granule cell precursors during CNS development. This work shows that absence of Shh decreases the number of neural progenitors in the postnatal subventricular zone and hippocampus. Similarly conditional inactivation of smoothened results in the formation of fewer neurospheres from progenitors in the subventricular zone. Stimulation of the hedgehog pathway in the mature brain results in elevated proliferation in telencephalic progenitors. It’s a lot of unfamiliar jargon, but you get the idea.

Of interest is the fact that the protein is extensively covalently modified by lipids (cholesterol at the carboxy terminal end and palmitic acid at the amino terminal end. These allow hedgehog to bind to its receptor (smoothened). It stands to reason that other lipids might block this interaction. The PNAS work shows this is exactly the case (in Drosophila at least). One or more lipids present in Drosophila lipoprotein particles are needed in vivo to keep Hedgehog signaling turned off in wing discs (when hedgehog ligand isn’t around). The lipids destabilize Smoothtened. This work identifies endocannabinoids as the inhibitory lipids from extracts of human very low density lipoprotein (VLDL).

It certainly is a valid reason for women not to smoke pot while pregnant. The other problem with the endocannabinoids and exocannabinoids (e.g. delta 9 tetrahydrocannabinol), is that they are so lipid soluble they stick around for a long time — see https://luysii.wordpress.com/2014/05/13/why-marihuana-scares-me/

It is amusing to see regulatory agencies wrestling with ‘medical marihuana’ when it never would have gotten through the FDA given the few solid studies we have in man.

When the active form of a protein is intrinsically disordered

Back in the day, biochemists talked about the shape of a protein, influenced by the spectacular pictures produced by Xray crystallography. Now, of course, we know that a protein has multiple conformations in the cell. I still find it miraculous that the proteins making us up have only relatively few. For details see — https://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/.

Presently, we also know that many proteins contain segments which are intrinsically disordered (e.g. no single shape).The pendulum has swung the other way — “estimations that contiguous regions longer than 50 amino acids ‘may be present” in ‘up to’ 50% of proteins coded in eukaryotic genomes [ Proc. Natl. Acad. Sci. vol. 102 pp. 17002 – 17007 ’05 ]

[ Science vol. 325 pp. 1635 – 1636 ’09 ] Compared to ordered regions, disordered regions of proteins have evolved rapidly, contain many short linear motifs that mediate protein/protein interactions, and have numerous phosphorylation sites compared to ordered regions. Disordered regions are enriched in serine and threonine residues, while ordered sequences are enriched in tyrosines — this highlights functional differences in the types of phosphorylation. Interestingly tyrosines have been lost during evolution.

What are unstructured protein segments good for? One theory is that the disordered segment can adopt different conformations to bind to different partners — this is the moonlighting effect. Then there is the fly casting mechanism — by being disordered (hence extended rather than compact) such proteins can flail about and find partners more easily.

Given what we know about enzyme function (and by inference protein function), it is logical to assume that the structured form of a protein which can be unstructured is the functional form.

Not so according to this recent example [ Nature vol. 519 pp. 106 – 109 ’15 ]. 4EBP2 is a protein involved in the control of protein synthesis. It binds to another protein also involved in synthesis (eIF4E) to suppress a form of translation of mRNA into protein (cap dependent translation if you must know). 4EBP2 is intrinsically disordered. When it binds to its target it undergoes a disorder to ordered transition. However eIF4E binding only occurs from the intrinsically disordered form.

Control of 4EBP2 activity is due, in part, to phosphorylation on multiple sites. This induces folding of amino acids #18 – #62 into a 4 stranded beta domain which sequesters the canonical YXXXLphi motif with which 4EBP2 binds eIF4E (Y stands for tyrosine, X for any amino acid, L for leucine and phi for any bulky hydrophobic amino acid). So here we have an inactive (e.g. nonbonding) form of a protein being the structured rather than the unstructured form. The unstructured form of 4EBP2 is therefore the physiologically active form of the protein.

The chemical ingenuity of the lake Ontario midge

Well we’re freezing our butts off here in sunny New England, so it’s time to discourse upon the chemical ingenuity of antifreeze proteins. They’ve long been known, with most found in fish living in arctic waters. A very unusual structure is found in a 79 amino acid protein from an insect living near Lake Ontario. It contains 79 amino acids with a set of 10 amino acid tandem repeats making up most of the protein. Here is the the repeat.

X X Cys X Gly X Tyr Cys X Gly ; X = any amino acid.

Can you as a computational chemistry expert figure out what it forms?

The 10 amino acids form a complete circle with the peptide backbone looking nothing like an alpha helix, a beta sheet or anything else I’ve seen. It just sort of wanders around for 360 degrees. In cross section the ‘circle’ resembles the Greek letter theta with a disulfide bond between the two cysteines forming a crossbar inside the circle. This puts all 7 tyrosines from the 7 repeats in a row on one side of the circle, where they form the presumed ice binding site. The solenoid is reinforced by intrachain hydrogen bonds, and side chain salt bridges. You can read about it and see some pictures in Proc. Natl. Acad. Sci. vol. 112 pp. 737 – 742 ’15 ].

The chemical ingenuity of some of these proteins is remarkable. None of them (except one) appear to have been figured out before their structures were determined.

[ Proc. Natl. Acad. Sci. vol. 108 pp. 7281 – 7282 ’11 ] Even now, the structural differences between the surface of ice nuclei and liquid water are poorly characterized (we don’t even know how many hydrogen bonds are involved), yet antifreeze proteins somehow recognize it. Some 12 different structural motifs have been found in antifreeze proteins. 3 are given — one is a small globular protein (sea pout) another is an alpha helix (winter flounder), and the third is a stack of left handed PolyProtein-II helices (snow flea). The present work gives a fourth example — a right handed parallel beta helix from (Marinomonas primoyensis). It is a 34 kiloDalton domain — it is a calcium bound parallel beta helix, with an extensive array of icelike surface waters that are anchored via hydrogen bonds directly to the protein backbone and adjacent side chains. The bound waters make an excellent 3 dimensional match to the primary prism and basal planes of ice.

Probably the most counterintuitive antifreeze protein is the following. It stands a lot of what we thought we knew about protein structure on its head.

[ Science vol. 343 pp. 743 – 744, 795 – 798 ’14 ] Almost all globular proteins reported to date have a dry protein core (e.g. water free). An antifreeze protein called Maxi from the winter flounder (Pseudopleuronectes americanus) has been found with a water filled core. It is a 3 kiloDalton alanine rich 4 helix bundle 145 Angstroms long. The periodicity of the alpha helices is 11 amino acids. A single turn of an alpha helix is 5.4 Angstroms high and 11 Angstroms wide. So 11 amino acids fairly neatly comes out to 16 Angstroms in length (because each helical turn is 3.7 residues (vs. the normal 3.6 in the classic alpha helix). The ice binding residues are Threonine at position i, Alanine at position i+4 and Alanine at position i + 8 (putting them along one face of the helix). The protein is a dimer of monomers each containing two helices. The core is comprised of 400 (yes 400 !) highly organized water molecules. The water is interleaved as a roughly two molecule thick layer between both intra and intermonomer helix interfaces, extending to the ice binding surfaces. Maxi must bind ice nuclei and inhibit their growth. The water molecules inside the bundle form pentagons ! ! ! Amazingly, this was predicted 50 years ago by Scheraga . The 5 membered water rings form cages around individual amino acid side chains, illustrating their semi-clathrate structure — rather than ice. Most of the carbonyls are involved in hydrogen bonding interactions with water — helping to keep the protein soluble. The protein denatures at low temperatures (16 C)

Ordered water can be found in most high resolution Xray crystallograpy protein structures, but they are usually between the proteins. Maxi retains the very structure of water.

Removal of water has been proposed as a potential rate limiting step in protein folding. Maxi folds to the point where water not in direct contact with the protein chain is removed from its core. It then arrests further folding to retain a beautifully ordered core of water interleaved between the protein helices.

Amazing! No one would ever have predicted something like Maxi (except Sheraga).

An interesting way to study the hydrophobic effect between protein surfaces

Protein interaction domains haven’t been studied to nearly the extent they need to be, and we know far less about them than we should. All the large molecular machines of the cell (ribosome, mediator, spliceosome, mitochondrial respiratory chain) involve large numbers of proteins interacting with each other not by the covalent bonds beloved by organic chemists, but by much weaker forces (van der Waals,charge attraction, hydrophobic entropic forces etc. etc.).

Designing drugs to interfere (or promote) such interactions will be tricky, yet they should have profound effects on cellular and organismal physiology. Off target effects are almost certain to occur (particularly since we know so little about the partners of a given motif). Showing how potentially useful such a drug can be, a small molecule inhibitor of the interaction of the AIDs virus capsid protein with two cellular proteins (CPSF6, TNPO3) the capsid protein must interact with to get into the nucleus has been developed. (Unfortunately I’ve lost the reference). For more about the host of new protein interaction domains (and potential durable targets) just discovered please see https://luysii.wordpress.com/2015/01/04/microexons-great-new-drugable-targets/

Hydrophobic ‘forces’ are certain to be important in protein protein interactions. A very interesting paper figured out a way to measure them using atomic force microscopy (AFM). [ Nature vol. 517 pp. 277 – 279, 347 – 350 ’15 ]. This is particularly interesting to me because entropy has nothing to do with the force as measured. I’ve always assumed that the the hydrophobic force was entropic, similar to the force exerted by rubber when you stretch it. It’s what pushes hydrophobic side chains into the interior of proteins (e.g water doesn’t have to decrease its entropy by organizing itself to solvate hydrophobic side chains). Not so in this case.

The authors prepared self-assembled monolayers using dodecyl thiol (CH3 (CH2) 10 CH2 SH) bound to gold. Every now and then an amino group or a guanido group was placed at the other end of the thiol. This allowed them to produce a mixture of hydrophobic groups (60%) and ionic species (NH4+ or guanidinium ions) within nanoMeters of the hydrophobic regions. The amine and the guanidino groups were the same distance as the hydrocarbon ends from the gold surface. A gold atomic force microscope (AFM) with a hydrophobic tip (the same C(12) moiety), was then used to measure the adhesive force between the tip and the surface in aqueous solution.

This is important because it is a measurement not a theoretical calculation (apologies Ashutosh). This is particularly useful since water is so complex that we don’t have a good understanding (potential function) for it.

Methanol was added (which eliminated most of the hydrophobic interactions). Sensitivity to methanol was taken as a signature of the hydrophobic component of the force. The pH could be manipulated, so the R – NH2 could be charged to R -NH3+, ditto for guanidinium to the uncharged species.

So guess what the effect of amino and guanidine groups were on the hydrophobic interaction. I was rather surprised.

The strength of hydrophobic interactions between the mixed monolayers and the tip doubled when neutral amino groups found within nanoMeters of hydrophobic regions are charged to form R -NH3+ ions by lowering the pH. A similarly placed guanidinium ion eliminates the hydrophobic interactions at all pHs. So the effect of the two side chains (NH2 for lysine, guanidinium for arginine) is opposite.

They note that the ammonium ion is well hydrated, but guanidinium is hydrated only at the edges of the plane (where the electrons are) but not above it. This allows guanidinium an amphipathic behavior, which is why it can be a denaturant (did you know this? I didn’t).

I’m sure that the effect of negative ions (e.g. carboxyl groups) and every other conceivable side chain will be studied in the future.

Thus hydrophobicity is not an intrinsic property of any given nonPolar domain. It can be changed by functional groups within 10 Angstroms.. So placing a charged group near a hydrophobic domain, should allow tuning of the hydrophobic driving force. I’d be amazed if this isn’t found to be the case evolutionarily.

They also studied some wierd looking stuff resembling proteins (beta peptides { e.g. the amino and carboxyl groups on adjacent carbons rather than the same one as with alpha amino acids) with weird side chains which are known to adopt an amphipathic helical conformation. THe nonpolar side chains were trans 2 aminocyclohexanecarboxylic acid (ACHC), and the cationic side chains were beta3 homolysine. Why didn’t they use something more natural. The peptide forms an ACHC rich nonPolar square domain 10 Angstroms on a side with a polar patch on the other side of the helix.

So it’s a fascinating piece of work with large implications for the design of drugs attacking protein protein interfaces.

Do enzymes chase their prey?

Do enzymes chase their prey? At first thought, this seems ridiculous. However people have been measuring diffusion of substances in water for over a century. Even Einstein worked on it (his paper on Brownian motion). So it’s fairly easy to measure the diffusion of an enzyme in water. Several enzymes (catalase — one of the most efficient enzymes known, and urease) diffuse faster when their substrate is present. [ Nature vol. 517 pp. 149 – 150, 227 – 230 ’15 ] The hydrolysis of urea by urease and the conversion of H2O2 to O2 and water by catalase enhances the molecular diffusion of the enzymes (this is called anomlous diffusion).If you inhibit catalase enzymatic activity using azide the anomalous diffusion disappears (even though there’s still plenty of H2O2 around). This work also showed that the rate of diffusion of catalase, urease and 2 more ezymes correlates with the heat produced by the reaction catalyzed.

Heating the catalytic center of catalase (using a short laser pulse) produces the same anomalous diffusion. Proteins exist in a world in which Brownian motion is governed by viscous forces rather than by inertia, so coasting (a la Galileo and Newton’s law of inertia) isn’t an option — continuous force generation is required.

Heat generated from each catalytic cycle could be transmitted through the enzyme as a pressure wave. For this to happen the catalytic center must be NOT at the center of mass of the enzyme, so the pressure wave will create differential stress at the enzyme solvent interface (which should propel the enzyme). They call this the chemoacoustic effect.

Molecular dynamics simulations suggest that the transmission of energy through a protein can be quite fast (5 Angstroms/picoSecond) and nonuniformly distributed.

Some enzymes have a near perfect catalytic efficiency. Every time a substrate hits them, the substrate is converted to product. Examples include catalase, acetyl cholinesterase, fumarase, and carbonic anhydrase. There are 100 million to a billion collisions per mole per second in solution.

Could this be a product of evolution (to make enzymes actively search out substrates?). Note, this won’t work if the catalytic center of the enzyme is in the center of mass.

I doubt that much catalytic efficiency is gained by having a huge protein molecule sluggishly move through the cytoplasm. Why? The molecular mass of H2O2 is 19 Daltons (vs. 18 for water), so it moves slightly more slowly but water moves at 20C in water at 590 meters/second. Of course it doesn’t get very far before it bumps into another water molecule and gets deflected.

Is there an ace physical chemist out there who can put numbers on this. I couldn’t believe that I couldn’t find a simple expression for the relation between the diffusion coefficient and the mass of the diffuser, ditto for the atomic volume of a water molecule, although I’m guessing that it’s pretty close to the length of the H – O bond (.95 Angstroms) giving a mass of 3.6 cubic Angstroms. I wanted this so I could see how much room to roam a water molecule has.

Paul Schleyer 1930 – 2014, A remembrance

Thanks Peter for your stories and thoughts about Dr. Schleyer (I never had the temerity to even think of him as Paul). Hopefully budding chemists will read it, so they realize that even department chairs and full profs were once cowed undergraduates.

He was a marvelous undergraduate advisor, only 7 years out from his own Princeton degree when we first came in contact with him and a formidable physical and intellectual presence even then. His favorite opera recording, which he somehow found a way to get into the lab, was don Giovanni’s scream as he realized he was to descend into Hell. I never had the courage to ask him if the scars on his face were from dueling.

We’d work late in the lab, then go out for pizza. In later years, I ran into a few Merck chemists who found him a marvelous consultant. However, back in the 50’s, we’d be working late, and he’d make some crack about industrial chemists being at home while we were working, the high point of their day being mowing their lawn.

I particularly enjoyed reading his papers when they came out in Science. To my mind he finally settled things about the nonclassical nature of the norbornyl cation — here it is, with the crusher being the very long C – C bond lengths

Science vol. 341 pp. 62 – 64 ’13 contains a truly definitive answer (hopefully) along with a lot of historical background should you be interested. An Xray crystallographic structure of a norbornyl cation (complexed with a Al2Br7- anion) at 40 Kelvin shows symmetrical disposition of the 3 carbons of the nonclassical cation. It was tricky, because the cation is so symmetric that it rotates within crystals at higher temperatures. The bond lengths between the 3 carbons are 1.78 to 1.83 Angstroms — far longer than the classic length of 1.54 Angstroms of a C – C single bond.

I earlier wrote a post on why I don’t read novels, the coincidences being so extreme that if you put them in a novel, no one would believe them and throw away the book — it involves the Princeton chemistry department and my later field of neurology — here’s the link https://luysii.wordpress.com/2014/11/13/its-why-i-dont-read-novels/

Here’s yet another. Who would have thought, that years later I’d be using a molecule Paul had synthesized to treat Parkinson’s disease as a neurologist. He did an incredibly elegant synthesis of adamantane using only the product of a Diels Alder reaction, hydrogenating it with a palladium catalyst and adding AlCl3. An amazing synthesis and an amazing coincidence.

As Peter noted, he was an extremely productive chemist and theoretician. He should have been elected to the National Academy of Sciences, but never was. It has been speculated that his wars with H. C. Brown made him some powerful enemies. I’ve heard through the grapevine that it rankled him greatly. But virtue is its own reward, and he had plenty of that.

R. I. P. Dr. Schleyer

Paul Schleyer (1930 – 2014) R. I. P.

This is a guest post by Peter J. Reilly, Anson Marston Distinguished Professor Emeritus, Department of Chemical and Biological Engineering, Iowa State University, fellow Schleyer undergraduate advisee Princeton 1958 – 1960, friend, and all around good guy.

I’ll follow with my own reminiscences in another post. Obits tend to be polished and bland, ‘speak no evil of the dead’ and all that, but Peter captures the flavor of what it was actually like to be Paul’s advisee and exposed to his formidable presence.

“Following are my thoughts on our undergraduate chemistry advisor at Princeton, Paul von Ragué Schleyer, who died on November 21 of this year at 84.

Paul was an amazingly prolific chemist. He started publishing in 1956, soon after he arrived at Princeton from receiving a Ph.D. at Harvard, where he studied from 1951 to 1954 after earning an A.B. from Princeton. He was still publishing at the time of his death. In fact, he had promised to deliver a book chapter over this Thanksgiving weekend. Over his latter years at Princeton, in the early 1970’s, his annual production of papers averaged the middle 20’s. He kept up the same pace at Universität Erlangen-Nürnberg in Germany from 1976 to 1992. From 1993 to 1997, when he had appointments at both Erlangen-Nürnberg and the University of Georgia, he was in the 40’s. When fully at Georgia, after 1997, he gradually slacked off, publishing only 16 papers this year. Altogether he had 1277 publications, when a really productive chemist with ready access to students and postdoctoral fellows hopes to have 200–250 in a full career.

Another way to consider Paul’s productivity is by how often his work had been cited (partly by his own later papers but mainly by the papers of others). A 1981–1997 survey reported that he was the third most cited chemist in the world. Althogether his works were cited over 75,000 times. His h-index is 126 in the Thomson Reuters Web of Science database, meaning that he had 126 publications that were cited at least 126 times, an astounding number.

I first met Paul in the fall of 1958, two years after I arrived at Princeton. I needed to find someone to supervise my junior paper, a ritual common to all Princeton undergraduates doing A.B. degrees. I had originally approached Edward Taylor, a somewhat older chemistry professor, but when I told him that I was somewhat interested in becoming a chemical engineer, he directed me to Paul. Paul was 28 at the time, but he seemed older to me (I supposed all professors did). He was tall, with dark black hair combed to the side over his forehead. He had a scar on his cheek and talked very precisely.

My father met him once and came away asking if he had been a German U-boat captain during WWII.

I must say that I spent a sizable part of the next two years being terrified of Paul. He had a laboratory in the second floor of the southwest corner of Frick Chemical Laboratory. The benches were full of glassware, to the point where it seemed hard to do any research. However, the item that spooked me the most was a cauldron full of boiling black liquid, supposedly mainly nitric acid, in which dirty glassware was submerged to be cleaned.

Paul gave me a project to research the incidence and properties of the benzyne intermediate, a short-lived benzene ring with a triple bond. This was my first exposure to research beyond short papers for classes, and I suppose that I did well enough for him to invite me to do a senior thesis with him. The topic was to determine the mechanism by which an obscure organic chemical rearranged itself. The title of the thesis that came from a year’s dogged effort was “A Study of the Cleavage Products of 2,5-Dimethyltetrahydropyran-2-Methanol”, but what I mainly made was black goop. Paul’s written comments to me started with the statement that he was sorry that the problem was so intractable, but at least he liked my writeup. I still have the thesis (and the junior paper). Back in 2007 I was contacted by the Princeton University Library, which had lost its copy. They asked if I could send them mine so that they could microfilm it, which of course I did.

I remember that at least four of us chemistry majors spent much of our senior years in a very large and empty laboratory working on our theses under Paul’s direction. I must say that the various chemicals that I worked on smelled a lot better than the ones that you dealt with. I used to take weekend dates up to the laboratory to show them where I worked, and I would open one of your very small tubules, I think containing butyl mercaptan. Its smell still permeated the room on Mondays. (Editor’s note — people used to look at their shoes when I walked into the eating club after working with n-Bu-SH or similar compounds).

Despite my lack of success on my thesis, I learned from it how to do research. My chemical engineering major professor at the University of Pennsylvania was hard to contact, so much of my doctoral dissertation was done without much supervision. Between the two experiences, I had a good foundation for my 46 years of being a chemical engineering professor, six at the University of Nebraska-Lincoln and 40 at Iowa State University after four years at DuPont in southern New Jersey.

I only saw Paul four times after leaving Princeton. The first was when I returned there for a short visit. The second time was at my 25th Princeton reunion, when one of his daughters was graduating. A third time was when he visited the Iowa State chemistry department to present a prestigious lecture. The fourth and last time was in 2005 when I visited the University of Georgia for a meeting. Paul spent about 30 minutes telling me about his latest research, of which I understood very little.

I will close with a little story. When I told Paul during my senior year that I wanted to go to graduate school in chemical engineering, he asked why I wanted to become a pipe-fitter. Probably because of my chemistry background at Princeton, my research was always chemistry- and biology-based, first in fermentations at Penn and Nebraska (with a detour to chloro- and fluorocarbons at DuPont), and then in enzymes and carbohydrates at Iowa State. I moved more and more into computation late in my career, and when Paul visited around 2002 I told him that I would be sending a manuscript to the Journal of Computational Chemistry, which he and Lou Allinger at Georgia had founded and were still editing. Being Paul, he immediately said in his deep voice that it had better be good. As it turned out, it sailed through the review process with hardly a blip, and I followed it up with a second manuscript a few years later.

So, we were fortunate to have Paul as a mentor during our formative years. He certainly wasn’t the sweetest guy, but he was brilliant, and hopefully a very small part of his brilliance rubbed off on us.”

Peter J. Reilly

Follow

Get every new post delivered to your Inbox.

Join 75 other followers