Prospects for Hydrogen

What are the prospects for hydrogen as part of a sustainable, green economy?

Hydrogen is perennially spoken of as a fuel of the future- clean, renewable, light. It is particularly appealing in an environment (like that of California) where solar energy is having a huge impact on the grid and causing rising portions of solar production to be "curtailed". That is, turned off. But even in California, solar power has hardly scratched the surface. Only few roofs have solar and the potential for more power production is prodigious. Over time, as more renewable sources of energy come on line, the availability of excess power at peak times will rise dramatically, prompting a huge need for storage, or other ancillary uses for excess power. Many storage schemes exist or are under development, from traditional water pumping to batteries, flywheels, gravitational weights, etc. Hydrogen is one of them, spoken of as a versatile storage and fuel medium, which can be burned, or even more efficiently put through fuel cells, to return electrical power.

A typical day on California's electrical grid. The top teal line is total demand, and the purple zone is power not supplied by renewables like wind, hydropower, and solar. During the mid-day, most power now comes from solar, an amazing accomplishment. Roughly 2 GW are even turned off at the highest peak time, due to oversupply, either locally or regionally. How could that energy be put to use?

Unfortunately, as a fuel, hydrogen leaves much to be desired. We have flirted with hydrogen-powered cars over the last couple of decades, and they have been a disaster. Hydrogen is such an awkward fuel to store that battery-powered electric vehicles have completely taken over the green vehicle market, despite their slowness in refueling. The difficulties begin with hydrogen's ultra-low density. The Sun has the gravitational wherewithal to compress hydrogen to useful proportions, at the equivalent of 100,000 earth atmospheres and up. But we on Earth do not, and struggle with getting hydrogen in small enough packages to be useful for applications such as transport. The prospect of Hinden-cars is also unappealing. Lastly, hydrogen is corrosive, working its way into metals and weakening them. Transforming our natural gas system to use green hydrogen would require replacing it, essentially.

The awkwardness, yet usefulness, of (reduced) hydrogen as an energy currency in an oxygenated atmosphere is incidentally what led life during its early evolution to devise more compact storage forms, i.e. hydro-carbons like fats, starches and sugars. And these are what we dug up again from the earth to fuel our industrial, technological, and population revolutions.

But how useful is hydrogen for strictly in-place storage applications, like load balancing and temporary grid storage? Unfortunately, the news there is not good either. Physical storage remains an enormous problem, so unless you have a handy sealed underground cavern, storage at large scales is impractical. Second, the round-trip efficiency of making hydrogen from water by electrolysis and then getting electricity back by fuel cell (both rather expensive technologies) is roughly 35 to 40%. This compares unfavorably to the ~95% efficiency of electrical batteries like Li ion, and the 80% efficiency of pumped water/gravity systems. Hydrogen here is simply not a leading option.

Does that mean we are out of luck? Not quite. It turns out that there already is a hydrogen economy, as feedstock for key chemical processes, especially ammonia and fertilizer production, and fossil fuel cracking, among much else. Global demand is 80 million tons per year, which in electrical terms is 3-4 tera watt hours. That is a lot of energy, on the order of total demand on the US electric grid, and could easily keep excess power generator's hands full for the foreseeable future. Virtually all current hydrogen is made from natural gas or coal, so the green implications of reforming this sector are obvious. It already has storage and pipeline systems in place, though not necessarily at locations where green energy is available. So that seems to be the true future of hydrogen, not as a practical fuel for the economy in general, but as a central green commodity for a more sustainable chemical industry.

• Unions
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Squeezing Those Electrons For All They've Got

How respiratory complex I harnesses electron transfer from NADH to quinone to power the mitochondrial battery.

Energy- we can't live without it, we can't make it ourselves, and we use all sorts of complex technologies to harvest and store it. Solar power is reaching a crisis as we realize that it isn't going to work without storage. Life faced similar crises billions of years ago, and came up with core solutions that we know now as the chemical transformations of photosynthesis and metabolism. Plants make storage compounds from sunlight, which we in turn eat for energy, transforming them into a series of currencies from short- to long-lived, such as NADH, protons, ATP, glucose, and finally, fat.

Within us, the mitochondrion is the engine, not making energy, but burning it from the food we eat. The core citric acid cycle disassembles the reduced carbon compounds that serve as our food and longer-term storage compounds into oxidized CO2 and energy carriers NADH, FADH. While used widely in the cell for specialized needs, these compounds are not our core energy stores, and are generally sent to the electron transport chain for transmutation into a proton gradient that serves as the battery of the mitochondrion, which is in turn used to synthesize our general energy currency, ATP. ATP is used all over the cell for general needs, including the synthesis of glucose, glycogen, and fat as needed for longer term storage.

The discovery of the proton battery was one of the signal achievements of 20th century biochemistry, explaining how mitochondria, and bacteria generally, (from which they evolved), handle the energy harvested via the electron transport chain from food oxidation in an organized and efficient way, without any direct coupling to the ATP synthesis machinery. The electron transport chain is a series of protein complexes embedded in the innermost mitochondrial membrane that receive high-energy electrons from NADH / FADH made in the matrix through the citric acid cycle and use them to pump protons outwards. Then the ATP synthetase enzyme, which is another highly specialized and interesting story, uses the energy of those protons, flowing back in through its rotary structure, to synthesize ATP. The proton gradient is short-lived, a bit like our lithium batteries, continually needing to be recharged- a key form of storage, but just one part of a larger energy transformation system.

A recent pair of papers from the same lab, capitalizing on the new technologies of atomic structure determination, describe in new detail the structure of respiratory complex I, which is a huge complex of 45 proteins that receives NADH, conducts its two electrons to ubiquinone, and uses that energy to pump out four protons from the mitochondrial matrix. Not all questions are answered in these papers, but it is a fascinating look into the maw of this engine. Ubiquinone (often abbreviated as Q) is then later taken up by another respiratory complex that squeezes out a few more protons, while transferring the electrons to cytochrome C, which goes to yet another respiratory complex that squeezes out a final few protons.  Like in our macroscopic world, a lot of complicated machinery is needed to keep a power system humming. 

The complex hinges literally on the Q binding site, which is at the elbow between the intracellular portion that binds NADH, and the series of proteins that all sit in the membrane. When Q binds, the bend is larger, (called the closed form), and when it leaves, the bend is smaller (called the open form). The electron path through the paddle is reasonably well understood, going through several iron-sulfur and flavin mononucleotide complexes that have special overlapping quantum tuning to allow extremely efficient electron transport. The key to the whole system is how the transfer of electrons from NADH through the paddle domain down to Q, which protonates it to QH2 and makes it leave to travel through the membrane to its other destinations, is coupled with a long-range physical and electrostatic shift through the rest of the complex to run the proton pump cycle. 

Structure of complex I, emphasizing the electron path in the paddle (upper right) and the many possible proton conduction paths in the membrane-resident part of the complex (bottom). The Q binding site is shown in brown at the elbow. Each protein subunit is named and given a distinct color. A conductive "wire" through the middle of the membrane components is isolated from the solvent, but connected to each membrane side with dynamically gated pathways. Whether these gates have more of a physical character or an electrostatic character, or both, remains uncertain.

The membrane domain, made up of several similar proteins all side-by-side, seems to have a sort of wire running through the middle, made up of charged amino acid side chains and water molecules, capable of conducting protons parallel to the membrane. It also has specific proton conduction paths within each subunit that provide the possible entry and exit paths for protons getting pumped from the interior outwards. The authors propose that there is a sort of hokey-pokey going on, where one bent form (the open form, with Q ejected) of the machine exposes the matrix-side proton channels, while the other bent form (closed form, with Q present) closes those channels and opens a corresponding set of four channels on the other side that let those same protons out to the cell. The internal wire, they propose, may possibly redistribute the protons to buffer the input channels. Or it might even allow all four to exit on the last, fourth pump complex. In any case, this in essence is the core of biological pump designs, opening channels in one direction to capture protons from one side, (by diffusion), and then executing a switch that closes those and opens ports to the other side, again using diffusion to let them go, but in a new direction. It is the physical cycle that translates energy into chemical directionality, aka pumping.

Proposed mechanism, with the insertion or ejection of Ubiquinone Q dictating the  proton channel accessibility along the membrane proton pump subunits of complex I. Protons enter from the mitochondrial matrix in the blue structures (closed), and exit via the other side in the green structures (open).

Closeup of one of the membrane proton pump segments, showing the dynamic formation of one proton conduction channel in the "open" state (left) vs the closed state (right, circled). The somewhat dramatic turning of the center protein helix carrying residues M64 and F68 opens the way

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• Table of the week.. Are we as free as China? Are we great yet? A comparison of the US and China in key Covid measures, taken Feb 9.

Rank	Country	Total Cases	New Cases	Total Deaths	New Deaths	Total Recovered	Active Cases	Serious, Critical	Tot Cases/1M	Deaths/1M
1	USA	27,798,163	+93,759	479,726	+3,219	17,631,858	9,686,579	21,446	83,682	1,444
83	China	89,720	+14	4,636	0	84,027	1,057	18	62	3

The Silicon Age

This magical element brings us the modern age- in computation, and in power.

In geologic terms many regard the current epoch as the Anthropocene, based on our various far-reaching (and often obscene) effects on earth's biosphere and geology. But where are we in the sequence of cultural epochs, starting from the stone age, and continuing through the bronze and iron ages? This somewhat antiquated system of material culture-based divisions seems to have petered out with the iron age, about 500 BC. What came after? There was certainly a technological hiatus in the West (and perhaps elsewhere) around the dark ages, where iron remained the most advanced material, though one might make a case for concrete (a Roman invention, with extensive use in antiquity), glass, or porcelain as competitor, though the latter never had the broad impact of iron.  The industrial age was perhaps founded on steel- the new material that brought us well into the twentieth century, until we hit the atomic age, an age that did not age well, sadly, and seems to be headed for the scap heap- one that will be radioactive for eons.

Now we are clearly indebted to a new element- silicon. That it is the magic ingredient in computers goes without saying. But now it is also providing the power for all those computers, in its incarnation as solar cells, as well as light for our lives, as efficient LEDs. It is incidentally intriguing that silicon resides just one row down, and in the same column, from the central element of life- carbon. They have the same valence properties, and each have unusual electronic properties. For silicon, its magic comes from being a semiconductor- able to be manipulated, and in switchable fashion, from conducting to insulating, and back again. A magic that is conjured by doping- the peppering-in of elements that have either too many valence electrons (phosphorous; n for negative) or too few (boron; p for positive). Too many, and there are extra electons that can conduct. Too few, and there are positive charges (holes) that can conduct similarly.

Charge and electrochemistry across the p-n junction.

At the interface between n and p doped zones something amazing happens- a trapped electrical charge that forms the heart of both transisters and solar cells. The difference in composition between the two sides sets up conflicting forces of diffusion versus charge. Electrons try to diffuse over to the p doped side, but once they do, they set up an excess of electrons there that pushes them away again, by their negative charge. Holes from the p doped side likewise want to migrate over to the n doped side, but set up a similar zone of positive charge. This zone has a built-in electric field, but is also insulating, until a voltage going from p to n, which squeezes this zone to smaller and smaller size, making it so narrow that charge can flow freely- the diode effect. The reverse does not work the same way. Voltage going from n to p makes this boundary zone larger, and increases its insulating power. This, and related properties, gives rise to the incredibly wide variety of uses of silicon in electronics, so amplified by the ability to do all this chemistry on precisely designed, microscopic scales.

Solar cells also use a p-n doping regime, where the bulk of the silicon exposed to the sun is p-doped, and a small surface layer is n-doped. When a photon from the sun hits the bulk silicon, the photoelectric effect lets loose an electron, which wanders about and meets one of two fates. Either it recombines with a local atom and releases its photon energy as infrared radiation and heat. Or it finds the p-n junction zone, where it is quickly whisked off by the local electric field towards the positive pole, which is all the little wires on the surface of solar panels, taking electrons from the n-doped surface layer. The p-n interface has a natural field of about 0.6 volt, which, when ganged together and scaled up, is the foundation for all the photovoltaic installations which are taking over the electric grid, as a cheaper and cleaner source of electricity than any other. Silicon even plays a role in some battery technologies, helping make silicon-based solar power into a full grid power system.

Solar power is scaling to provide clean energy.

Silicon gives us so much that is essential to, and characteristic of, the modern world. Like carbon, it is very abundant, not generally regarded as rare or precious. But that doesn't mean it lacks interest, let alone importance.

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• Meanwhile, an outstanding article describes the slow destruction of US pharmaceutical and public health capabilities.

Origin of Life- RNA Only, or Proteins Too?

Proteins as Participants in the Origin of Life

After going through some momentous epochs in the history of life in the last two weeks (the origin of eukaryotes, and the nature of the original metabolism), we are only part of the way to the origin of life itself. The last common ancestor of all life, (LUCA), rooted at the divergence between bacteria and archaea, was a fully fledged cell, with many genes, a replication system and translation system, membranes, and a robust metabolism based on a putative locale at hydrothermal vents. This is a stage long after the origination of life, about which our concepts remain far hazier, at the chemical interface of early Earth.

A recent paper (and prior) takes a few more speculative shots at this question, (invoking what it calls the initial Darwinian ancestor, or IDA), making the observation that proteins are probably as fundamental as RNA to this origination event. One popular model has been the "RNA world", based on the discovery that RNA has some catalytic capability, making it in principle capable of being the Ur-genetic code as well as the Ur-enzyme that replicated that same code into active, catalytic molecules, i.e., itself. But not only has such a polymathic molecule been impossible to construct in the lab, the theory is also problematic.

Firstly, RNA has some catalytic ability, but not nearly as much as it needs to make a running start at evolution. Second, there is a great symmetry in the mechanisms of life- proteins make RNA and other nucleic acids, as polymerases, while RNA makes proteins, via the great machine of the ribosome. This seems like a deep truth and reflection of our origins. It is probable that proteins would, in theory, be quite capable of forming the ribosomal core machinery- and much more efficiently- with the exception of the tRNA codon interpretation system that interacts closely with the mRNA template. But they haven't and don't. We have ended up with a byzantine and inefficient ribosome, which centers on an RNA-based catalytic mechanism and soaks up a huge proportion of cellular resources, due to what looks like a historical event of great significance. In a similar vein, the authors also find it hard to understand how, if RNA had managed to replicate itself in a fully RNA-based world, how it managed to hand off those functions to proteins later on, when the translation function never was. (It is worth noting that the spliceosome is another RNA-based machine that is large and inefficient.)

The basic pathways of information in biology. We are currently under siege by an organism that uses an RNA-dependent RNA polymerase to make, not nonsense RNA, but copies of itself and other messages by which it blows apart our lung cells. Reverse transcriptases, copying RNA into DNA, characterize retro-viruses like HIV, which burrow into our genomes.

This thinking leads to a modified version of the RNA world concept, suggesting that RNA is not sufficient by itself, though it was clearly central. It also leads to questions about nascent systems for making proteins. The ribosome has an active site that lines up three tRNAs in a production line over the mRNA template, so that the amino acids attached on their other ends can be lined up likewise and progressively linked into a protein chain. One can imagine this process originating in much simpler RNA-amino acid complexes that were lined up haphazardly on short RNA templates to make tiny proteins, given conducive chemical conditions. (Though conditions may not have been very conducive.) Even a slight bias in the coding for these peptides would have led to a selective cycle that increased fidelity, assuming that the products provided some function, however marginal. This is far from making a polymerase for RNA, however, so the origin and propagation mechanisms for the RNA remain open.

"The second important demonstration will be that a short peptide is able to act as an RNA-dependent RNA polymerase."
- The authors, making in passing what is a rather demanding statement.

The point is that at the inception of life, to have any hope of achieving the overall cycle of selection going between an information store and some function which it embodies or encodes, proteins, however short, need to participate as functional components, and products of encoding, founding a cycle that remains at the heart of life today. The fact that RNA has any catalytic ability at all is a testament to the general promiscuity of chemistry- that tinkering tends to be rewarded. Proteins, even in exceedingly short forms, provide a far greater, and code-able, chemical functionality that is not available from either RNA (poor chemical functionality) or ambient minerals (which have important, but also limited, chemical functionality, and are difficult to envision as useful in polymeric, coded form). Very little of the relevant early chemistries needed to be coded originally, however. The early setting of life seems to have been rich with chemical energy and diverse minerals and carbon compounds, so the trick was to unite a simplest possible code with simple coded functions. Unfortunately, despite decades of work and thought, the nature of this system, or even a firm idea of what would be minimally required, remains a work in progress.

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Turning Biochemistry on its Head in Search of the Origin of Life

Early earth was anoxic. That means that metabolic reactions ran backwards, compared to what we regard as normal.

Following up on last week's post on the origins of eukaryotes, I ran across a brilliant body of work by William Martin and colleagues, which explores both that and the related topic of the origin of life, all of which took place on an early earth very different from our own. Perhaps the most fundamental theme in any biochemistry course, especially when it comes to metabolism, is controlled oxidation. We in our bodies recapitulate the action of fire, by transforming (reduced) hydrogen-rich carbon compounds (carbo-hydrates, fats, etc.) to the most oxidized form of carbon, CO2, which we regard as a waste product and make- from our food, and now by proxy out of our ramified economic metabolism- in prodigious amounts. Our rich metabolic inheritance essentially slows down and harnesses this energy-liberating process that, uncontrolled, runs wild.

But early earth was anoxic. There was no free oxygen, and this metabolism simply could not exist. The great oxygenation event of roughly 2 to 3 billion years ago came about due to evolution of photosynthesis, which regards CO2 as its input, and O2 as its waste product. Yet plants metabolize the other way around as well, (often at night), respiring the reduced carbon that they painstakingly accumulate from CO2 fixation back to CO2 for their growth and maintenance. Plants are firmly part of this oxidized world, even as they, in net terms, fix carbon from CO2 and release oxygen.

An energy rich, but reducing, environment, full of sulfides and other hydrogen-rich compounds.

In a truly anoxic world, the natural biochemical destination is reduced compounds, not oxidized ones. The deep-ocean hydrothermal vent has been taken as a paradigmatic setting, at least as common on the very early earth as today. Here, reduction is the order of the day, with electrons rampant, and serpentinzation a driving mineral process, which liberates reducing power, and generates methane and hydrogen sulfide. This is one home of anaerobic life- an under-appreciated demimond of micro-organisms that today still permeate deep sediments, rocks, hydrothermal vents, and other geologic settings we regard as "inhospitable". An example is the methanogens- archaea that fix CO2 using the local reducing power of hydrogen, and emit methane. Methane! A compound we in our oxygen atmosphere regard as energy-rich and burn in vast amounts, these archaea regard as a waste product. The reason is that they live where reduction, not oxidation, is the order of the day, and they slow down and harness that ambient (chemical gradient) power just as we do in reverse. This division of aerobic vs anaerobic, which implies metabolisms that run in opposite directions, is fundamental, accounting for the hidden nature of these communities, and why oxygen is so toxic to their members.

By now it is quite well known that not only was the early earth, and thus early life, anoxic; but the broadest phylogenies of life that look for our most distant ancestors using molecular sequences also place anaerobes like methanogenic archaea and acetogenic bacteria at the earliest points. Whether archaea or bacteria came first is not clear- they branch very deeply, and perhaps earlier than any phylogenetic method using the molecular clues can ever tell. Thus the archaeal progenitor of the eukaryotic host appears to have been anaerobic, and may have entered into a dependence with a hydrogen-generating, methane-using bacterium which had already evolved an extensive metabolism compatible with oxygen, but not yet dependent on it. It was only later that the oxygen-using capacity of this partner come to such prominence, after oxygen came to dominate the biosphere so completely, and after the partner had replaced most of the host's metabolism with its own enzymes for heterotrophic use (i.e. fermentation) of complex carbon compounds.

This overturns the image that was originally fostered by Darwin, in a rare lapse of prophetic skill, who imagined life originating in a quiet sunlit pool, the primordial soup that has been sought like a holy grail. The Miller-Urey experiments were premised on having complex compounds available in such a broth, so that heterotrophic nascent cells just had to reach out an choose what they wanted. But these ideas above end up proposing that life did not begin in a soup, rather, it began in a chemical vortex, possibly a very hot one, where nascent cells built an autotrophic metabolism based on reducing/fixing carbon from CO2, (the dominant form of carbon on early earth), using the abundant ambient reducing power, and local minerals as catalysts. Thus the energetics and metabolism were established first, on a highly sustainable basis, after which complexities like cell formation, the transition from mineral to hybrid mineral/organic catalysts, and the elaboration of RNA for catalysis and replication, could happen.

Much of this remains speculative, but one tell-tale is the minerals that underpin much of metabolism. Iron-sulfur complexes still lie at the heart of many electron transfer catalysts, as do several other key metals. RNA is also prone to oxidation, so would have been more robust in an anoxic world. More generally, this theory may widen our opinions about life on other planets. Oxygen may be a sign of some forms of life, and essential for us, but is hardly necessary for the presence of life at all. Exotic places with complex chemistries, such as the gas giant planets, may have fostered life in forms we are unfamiliar with.

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Cellular IM by GPCR

Looking into the mechanism of action of one our primary internal communication devices.

Cells need to protect themselves from the outside world, but they also need to interact with it and know what is going on. Bacteria have a lot of sensing mechanisms, primarily for food and toxins, but eukaryotes took this project to a whole new level, especially with the advent of multicellularity. While a few of the things cells sense come right through the cell membrane, like steroid hormones or fatty vitamins A and D, most things are blocked. This leads to the need for a large collection of proteins (receptors) that sit in the membrane and face both sides, with a ligand-binding face outside, and an effector face inside, which typically interacts with a series of other proteins that transmit signals, by phosphorylating other proteins, or modifying them with lipids, or just binding with a series of other proteins to form new complexes and activities.

A couple of GPCRs (red and orange) portrayed in a schematic membrane (black lines), bound by a couple of their primary intracellular targets and signaling partners, a G-protein (left, teal) and an arrestin (right, purple).

Human DNA encodes upwards of 800 receptors of one class, the G-protein coupled receptor (GPCR), which arose early in eukaryotic evolution, and duplicated / diversified profusely due to their effectiveness as a platform for binding all sorts of different molecules on the exterior face. They dominate our sense of smell as olfactory receptors, respond to 1/3 of all drugs ever approved, such as the opioids, and also conduct our sense of vision. Rhodopsin, which detects the photon-induced conformational flip-flop of retinal, is a GPCR receptor in the photoreceptor cell membrane. The fact that photons, which could have been detected anywhere, by many sorts of mechanisms, are detected by a membrane-bound GPCR receptor illustrates just how successful and dominant this mechanism of sensing became during evolution. More GPCRs are still being found all the time, and even after receptor genes are deciphered from the genome, figuring out what they bind and respond to is another challenge. Thus over 150 of our GPCR receptors remain orphans, with unknown ligands and functions.

But how do they work? Due to their great importance in drug targeting, GPCRs have been studied intensely, with many crystal structures available. It is clear that they conduct their signal by way of a subtle shape change that is induced by the binding of their ligand to the external face/pocket, and conducted through the bundle of seven alpha helixes down to the other face. Here, the change of shape creates a binding site for the G-proteins with which the (active) receptor is coupled, so-named because they bind GTP in their active state and can cleave off one phosphate to form GDP. Binding to the activated receptor encourages an inactive, GDP-bound G-protein to alter its conformation to release GDP and bind a new GTP. The G-protein then runs off and do whatever signaling it can until its slow GTPase reaction takes place, turning it off. There are endless complexities to this story, such as the question of how cells can tell the difference between signals from the dozens of GPCRs they may be expressing on their surface at the same time, or how some ligands turn these receptors off instead of on, or the wide range of other participants such as kinases, GTP/GDP exchange factors, arrestins, etc., which have developed over the eons. But I will focus on the signaling mechanism within the GPCR receptor.

Rough schematic of GPCR activation. Ligands bind at the top, and a conformational shift happens that propagates a structural change to the intracellular face of the receptor, where effector signaling molecules, especially G-proteins, bind and are activated. TM refers to each trans-membrane alpha helix of the protein structure.

A recent paper purported to have condensed a large field of work and done some mutant studies to come up with a common mechanism for the activation of the main (A) class of GPCR. This extends structural concusions that many others had already drawn about this class of receptors. As shown above, the main consequence of ligand binding is that key helices, particularly helix 6, make a substantial movement to the side, allowing the G-protein (shown in the top diagram in blue) to dock and stick a finger into the receptor. This is quite idealized, however, since GPCR receptors exist in a roiling sea of motion, being at the molecular scale, and can have subtle and partial responses to their ligands- many of which have contradictory effects. Some ligands (sometimes useful as drugs) have opposite effects from the main ligand, turning the receptors off, and others can have distinct forms of "on", or partial on effects, only fleetingly allowing the activated state to occur. Also, structures from several different GPCRs have been solved, with generally similar mechanisms, but not always informative about the dynamics of action- a structure made with an activating ligand may even show the inactive conformation, since the fraction of time spent in an active state may be much less than 100%.

Closeup of one switching event during receptor activation. Orange is the inactive state, where phenylalanine 6x44 (#44 on helix 6) contacts leucine 3x40 (amino acid 40 on helix 3), but it butted out of the way, upon ligand binding and activation, by tryptophan 6x48.

These researchers analyzed 234 structures of GPCRs in various conformations to come up with an offset mechanism conducted by ~35 amino acids principally on helices 5, 6, and 7 as they conduct the tickle from the surface to the other face of the membrane. It is a classic meta-stable structure, where a small shove (by the ligand binding on the external face) causes a cascade of offsets of these amino acid side chains as they interact with each other that pushes the structure into the new, active, semi-stable conformation. A conformation that is additionally stabilized by a G-protein if one comes along, but only while in its GDP-bound state. An example of one of these individual atomic switches is shown above, where residues close to the ligand binding site undergo a dramatic shift that establishes a contact between amino acids 40 (leucine) and 48 (tryptophan), which were not close at all in the inactive state of the receptor. The larger scheme of detailed switches and shifts is shown below.

Detailed scheme of the authors for structural change propagation through the GPCR body. Each amino acid is referred to by a code, since this summarizes behavior of hundreds of different, though homologous, proteins. Contacts characteristic of the inactive state and broken or changed during activation are in orange, while those formed on activation are in green. For example, the "Na+ pocket", which contains a sodium ion in the inactive state, collapses in the active state.

So this is biology descending to the level of engineering to understand an individual protein machine. We have such machines at all points, from thousands of genes, expressed in billions of copies, all cooperating and toiling in the service of us as a larger organism, blissfully unaware, certainly until the advent of molecular biology, of the wonders at work within. GPCRs have been an amazingly successful, ever-diversifying molecular machine, alerting animal and other eukaryotic cells of phenomena happening outside. A sort of instant messaging system on the cellular and organismal scale.

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Cells Put Their Best Face Outward

Structure and function of the flippase enzyme.

This dates me a little, but when I was in grad school, the fluid bilayer hypothesis of membrane lipids was still new and exciting. Now canonical, it proposed that cellular membranes have no more structure than a soap bubble, being flat fluids of phospholipids that self-organize into a bilayer with two leaflets, each leaflet keeping its polar or charged head groups out towards aqueous solution, and their lipid tails on the inside, facing the complementary leaflet. At our scale, it seems shockingly fragile and structure-less. But at the micro scale, it is a pretty tough affair. Typical membranes are about 5 nm thick, which seems negligible, but it takes a protein at least 7 alpha helical turns, or 25 amino acids, to span it. Given that the fatty tail length is freely adjustable, as is the chemical nature and charge of the head groups, evolution has evidently optimized the thickness of membranes to provide an optimal tradeoff of structure and lightness. They are tougher than they look.

In this microscopic technique, cells are frozen and cleaved sideways, causing some of the membranes to split along their inner leaflet boundaries. This highlights the proteins and other material embedded within them. Note at the top that a small portion of the plasma membrane of this cell has a quasi-crystalline raft of proteins- a sign of active signalling taking place.

Membranes are also chemically tough, impervious to charged molecules due to their fatty interior. These features made membranes incredibly successful- one of the key foundations of life. Eukaryotes developed a whole second frontier of membranes, as internal organelles like the nucleus, endoplasmic reticulum, golgi, lysozome, and mitochondria. Mitochondria particularly use the imperviousness of membranes to set up complex charge and chemical asymmetries, to serve as batteries, storing up electromotive force from respiration of food and using it to synthesize ATP.

But it turns out that there are some forms of structure amid all the fluidity of the fluid bilayer. There are the proteins, of course, which can organize into crystalline rafts, or hook onto cell walls (in plants and bacteria) or cytoskeletal supports to enforce overall cell shape. There are features of composition that can make membranes more stiff, such as using more rigid, more saturated lipid tails, or having more cholesterol, which serves as a plate-like stiffener. And it also turns out that the two sides of membranes can have markedly different compositions, another indication of just how stable and tough these tiny structures are.

A recent paper revealed the structure of an enzyme (flippase) that helps to enforce the asymmetry of composition between the inner and outer leaflets of eukaryotic plasma membranes. Why would such asymmetry exist? The reasons are not all clear, really. One aspect is the charge imbalance, whereby the inner (cytoplasmic) leaflet has more heavily charged phospholipids. There could also be defense issues, particularly among bacterial, which might want to present certain lipid head groups externally, and use other ones internally. Another is signaling, where certain phospholipids are chemically modified to serve as protein attachments and other forms of signaling, and thus need to be on the correct side of the wall. One prominent example is phosphotidylserine, which is usually kept on the inner leaflet. During cell suicide, (apoptosis), however, the (flippase) enzymes that keep it there are cleaved and disabled, while other enzymes (scramblases) that degrade the membrane composition asymmetry are activated, causing phosphatidylserine to be shown on the cell's outside, which is in turn a signal to traveling macrophages to attack and eat that cell.

So flippases spend their lives scavanging phophotidylserine from the outer membrane leaflet and transferring it to the interior leaflet, constituting one sign to the outside that yes, I am still alive. The process violates the concentration gradient of phosphatidylserine, so needs energy, which comes in as ATP. We end up with a rather complex two protein system that itself has to be consistently oriented the right way in the plasma membrane, cleaves ATP, phosphorylates itself briefly, grabs phosphatidylserine specifically from the outer leaflet of the membrane, and then transports it across to the inner membrane.

This schematic illustrates the enzymatic cycle. The phosphatidylserine to be transported is at bottom, in green, on the external face of the membrane. A complex ATP=>ADP cycle dramatically alters the shape of the top of the enzyme on the cytoplasmic face, which at the E2P step is propagated down to a gap which opens between the two proteins- the portions colored purple and beige, which are situated in the membrane. This lets a phosphatidylserine to slip into a pocket that binds it selectively, after which the phosphate leaves the upper part, the enzyme recloses, and the phosphatidylserine is released to the other face of the membrane.

This structure was arrived at with the new techniques of electron microscopy that have allowed protein structures to be determined without crystallization, a development that has been particularly beneficial for membrane proteins that tend to be very hard to crystallize. The project also used a series of ATP and phosphatidylserine analogs that helped freeze the proteins in certain conformations through the reaction cycle, providing the data that informs the model above.

A closeup of the phosphatidylserine binding site, the lipid tails pointing upward. Ther are numerous amino acid side chains from the protein (such as asparagine (N) 353, serine (S) 358, etc. that coordinate the phosphatidylserine specifically, making this a transporter almost exclusively for this phospholipid alone. Other hydrophobic side chains such as phenylalanine (F) 107 and 368 form congenial interactions with the lipid tails.

Binding of phosphatidylserine is specific, but it can not be very strong, since the point of the reaction cycle is to release it again rapidly. Once binding has established specificity, it induces dephosphorylation, which then induces further conformation changes that lock the outward access of the phospholipid and destabilize its binding to the protein.

A cross-section of the full structure (right), and schematic showing (left) the series of structural elements of the two proteins of the transporter (CDC50A, now called TMEM30A in red, and ATPA1, the ATPase, in all the other colors.) The full structure (with no phospholipid or ATP present) has the ATPase on a large domain sticking out into the cytoplasm, and the key phosphatidylserine binding cleft (between the purple and beige sections, buried in the membrane.

It is wonderful to live in an age when such secrets of life, once utterly unsuspected, and then veiled in unreachable technical obscurity, are revealed in mechanistic detail.

• A voyage through the cytoplasm. Warning- extreme colors.
• What an anti-corruption platform looks like.
• Whither Afghanistan?
• The pending impeachment, cont.
• The birds are not happy.
• What is earth's carrying capacity?

New Phases in the Nucleus

Special molecular interactions generate new phases of matter in various globs within the nucleus... but why?

One of the great events, near or at the orgin of life, was the advent of membranes- stable, flexible, but also rather tough structures build from amphipathic molecules, with water-loving head groups at one end and water-repellent, oil-like chains elsewhere. They sandwich together spontaneously to make the membrane (bilayer) sheet, which constitutes a separate phase from both the outside and inside of the cell. Getting across it is impossible for many molecules, which is highly protective, but has also necessitated a large zoo of transporters, channels and other mechanisms for transactions cells need to make with the outside.

Typical membrane, with a hydrophobic, oily interior that keeps it structurally coherent and impermeable to most aqueous substances. Note that it is, on a molecular scale, quite thick- bigger than most proteins.

It has gradually become apparent that the nucleus (whose envelope is a double membrane and which was borne of another great event in life- the origin of eukaryotes) harbors quite a variety of other phases of macromolecules, constituting zones, globs, speckles, assemblies- organelle-like structures that make study of the nucleus rather interesting. The story begins with the nuclear pore, which is where any moderate to large size molecule, up to partially constructed ribosomes, has to go to enter or leave the nucleus. Such cargo typically has a short segment in its protein chain that serves as a "signal", either for nuclear export or import. These signals bind to specialized transporter proteins which themselves have an unusual decoration of hydrophobic protein segments (HEAT repeats). The nuclear pore is lined with proteins carrying another decoration, forming an unstructured hydrophobic and homophilic mesh or gel of FG-repeats (named for their composition of phenylalanine and glycine) inside the pore. The transporter HEAT repeats can bind weakly,  but specifically, to these FG-repeats, or perhaps better, melt into them, and thus pass easily through the pore. It is a very clever scheme for controlling transport tightly with a mechanism that costs virtually no energy, since the transport is passive, going down the various molecules's concentration gradients.

Diagram of one nuclear pore complex. showing especially the mesh of FG-repeat protein tails that compose its interior and fringes. These interact with compatible transporter molecules to let large proteins and complexes through by selective diffusion.

But that is not all. The nucleus has long been known to have a large zone, the nucleolus, where ribosomal RNA genes are transcribed and where much of the assembly of ribosomes takes place. It is a dense mass of DNA, RNA, and proteins specialized to these tasks, a veritable factory for making this most abundant and complex component of cells.

An electron micrograph of one ribosomal gene in the act of being transcribed. Each rRNA transcript is a separate "branch" on this Christmas tree, showing the conveyor belt/factory nature of the process. Image at top, tracing at the bottom. The field is about 2.5 micrometers. This is only one of many ribosomal generation processes taking place within the nucleolus.

More recently, several other structures have been discovered in the nucleus, including speckles of RNA splicing components, Cajal bodies, PML bodies, paraspeckles, and others. And researchers have now realized that some transcriptional activation machinery forms similar blobs, called "super-enhancers". These have particularly high gene expression activity and seem to comprise a critical mass of regulatory RNAs, DNA-binding transcription factors, and a mess of mediators, histone modifiers, and other regulatory proteins in a sort of molten glob that segregates from the rest of the already-dense nuclear milieu. These are regarded as distinct liquid phases. Since DNA and RNA can bend, particularly between long-range enhancer regions and the promoter and coding regions of genes, it is possible to pack a lot of activity into a small, furiously active glob. And the high cooperativity that is implicit in the formation of such a glob is modeled, by a recent paper, to cause a sharp rise in transcriptional activation as well.

Model of condensed super-enhancers, (C, bottom), compared with run-of-the-mill enhancers, (C, top).  Their transcriptional activity (red) is, due to their greater size and stability, likely to be higher and far more consistent than that of even strong enhancers.

Why? One reason is that physical stability helps to keep the machine going, in contrast to usual interactions in the nucleus and elsewhere that are more sporadic, and fall apart as soon as they come together. Transcriptional activation, to take one example, relies on the coalescing (collusion, if you will!) of dozens of different proteins and complexes, all of which have to be available for other interactions as well, if dynamic gene regulation is to take place all over the genome. Most of these interactions are thus weak, so there is a critical mass (and perhaps composition) that distinguishes enduring, high-activity enhancer complexes, which can then be termed super-enhancer globs, from the normal form of enhancer that comes together on a far more temporary, ad-hoc basis. It is yet one more way, based on, but emergent from, the detailed composition of an enhancer, to turn up the gain on the target process that they regulate- transcription.

Different phases of matter thus have very significant roles in the cell, especially in the nucleus, allowing the establishment of mini-organelles / factories for operations that can be more efficient via the time-honored route of separation and specialization. They add to the sense of a sort of convergent evolution, since we already knew that there are conveyor belts, (DNA and RNA templates), just-in-time material and metabolic logistics, transport networks (actin, microtubules), and extraordinarily complex management methods in play.

• The pathological tau proteins in Alzheimer's bind to the nuclear pore proteins and gum up the works.
• One reason why our tax filing system is insane.
• Even evangelicals are getting fed up.
• Krugman is has it sort of backwards- Medicare for all may be politically difficult, but other countries show it can be done. Accomplishing much via a Green New Deal, on the other hand, is, while critically important, also very difficult.
• We have a savings glut.
• Craven catering to the Taliban, cont.
• Religiosity and brain damage?
• Impeachment richly warranted, but unlikely due to craven corruption.
• Veblen and the rot of inequality.
• Another view of MMT.

Frankenplant

40% more efficient plants? Done!

What is the most common protein in the biosphere? It occurs in plants, right? Right- it is RuBisCO, the enzyme that fixes carbon dioxide from the atmosphere, is the workhorse of agriculture, and hero of the fight against global warming, should we choose to grow more plants instead of burning them down. Its full name is ribulose-1,5-bisphosphate carboxylase-oxygenase, meaning that its substrate is a five carbon sugar (ribulose) that has two phosphates attached, and the enzyme attaches a carboxyl group from CO2, but can also attach an oxygen instead (the oxygenase part). And therein lies the problem. RuBisCO is phenomenally inefficient (maybe ten reactions per second) and error-prone (using oxygen [O2] instead of CO2 roughly a quarter of the time), which is why it is made in such prodigious quantities, amounting to half the protein complement of leaves.

Plant researchers have been casting about for a long time for ways to make this core reaction more efficient. But have had no success. Indeed, an interesting paper came out a few years ago arguing that as far as this enzyme is concerned, the shape and chemical similarity between CO2 and O2 are so close that RuBisCO is perfectly optimized, exchanging speed for what specificity is possible given its substrates. It varies quite a bit in this tradeoff, depending on the specific environment, arguing that the optimization is quite dynamic over evolutionary time. One of the few innovative solutions that plants have developed is not a tweek to the enzyme, but a physiological compartment present in C4 plants (like corn), which concentrates CO2 and excludes O2, thus resolving the competitive constraint for some of their chloroplasts. Their RuBisCO enzymes are adapted to have a slightly more relaxed attitude- slightly less specific for CO2, while also almost 2-fold faster, gaining an significant advantage.

The error pathway, fixing oxygen instead of CO2, is called photorespiration, since it uses up oxygen like regular respiration, but now in a completely wasteful way. The product is phospho-glycolate instead of 3-phospho-glycerate, and the glycolate is both inhibitory to photosynthesis and difficult to dispose of. It is typically exported from the chloroplast, and bounced around between the peroxisome and mitochondrion in its way to being turned into the amino acid glycine, all at the cost of roughly twelve ATP. It is hard to believe that this waste goes on day in and day out across the biosphere, but it seems to be the case. One might note that this yet another case of the steep price of success, since RuBisCO evolved in a high CO2 environment. It was the success of the photosynthetic process that covered the earth with green and filled the atmosphere with what was to all existing life forms a poison- oxygen.

Now, a team of researchers have engineered a way around this conundrum, at least reducing the cost of glycolate recycling, if not resolving the fundamental problems of RuBisCO. They describe the import of a set of genes from other species- one from pumpkin, one from the alga Chlamydomonas, and five from the bacterium E. coli, plus a genetic suppressor of glycolate export from the chloroplast, all resulting in a far less costly recycling system for the waste product glycolate.

New pathways (red, blue) inserted into tobacco plants, plus inhibition of the glycolate transporter PLGG1. Some of the wild-type pathway for diposing of glycolate is sketched out on the right.

Firstly, glycolate export was suppressed by expressing a tiny RNA that uses the miRNA system to target and repress the gene (PLGG1) encoding the main glycolate transporter. Secondly, the researchers imported a whole metabolic system from E. coli (red part at top of diagram) that efficiently processes glycolate to glycerate, which, with a phosphorylation (one ATP) can be taken right up by the RuBisCO cycle. Lastly, they backstopped the bacterial enzymes with another pair that oxidize glycolate to glyoxylate (glycolate oxidase), and then (malate synthetase) combine two of them into malate, a normal intermediate in cellular metabolism. This was all done in tobacco plants, which, sadly, are one of the leading systems for molecular biology in plants.

Wild-type plant is on the far left, and a sample plant with all the engineered bells and whistles (AP3) is on the far right, showing noticeably more robust growth.

Combining all these technologies, they come up with plants that show biomass productivitiy 40% higher than the parent plants, as well as reducing plant stress under high light conditions. After 3 billion years of plant evolution, this is a shocking and impressive accomplishment, and can be extended to all sorts of C3 plants, like wheat and other grains (that is, non-C4 plants). Due to the number of genes involved, unintentional spread to other plants, such as weeds, is unlikely. But given the urgency of our CO2 waste problem, one wonders whether we might welcome such escapes into the wild.

• Some notes on Sweden.
• MMT is coming into the mainstream, despite kicking and screaming.
• Complete regulatory capture of the consumer financial protection bureau. Now protecting predatory lenders.
• For some countries, history is circular.
• The tribulations of absolute pitch.

Arthur Kornberg

Notes on a great biochemist.

One thing that has made America great is our biomedical research establishment. Over the second half of the 20th century, the US created a uniquely effective set of funding institutions, and grew a large cadre of scientists who have led the world in the adventure of figuring out what makes us tick. Biology, at the molecular level, is an alien technology, based on chemistry, yes, but otherwise utterly unlike to any technology we have developed or been previously familiar with. It has taken decades to get to our current incomplete level of knowledge, and it will take decades more to unravel such complex processes as the detailed genetics of early development, or of schizophrenia, or the nature of consciousness.

Yes, it has led to biotechnology and growing prospects for improved medicine. But the historical significance of this epoch lies in the knowledge gained, of finding and exploring a vast and ancient new world. One of the leading scientists of the early days of enzymology and molecular biology was Arthur Kornberg, whom I learned from through his textbook on DNA replication. It was a model of clarity and focus, filled with apt illustrations. It was the rare textbook that didn't try to cover everything, and thus could treat its proper subject with loving care and detail.

The cover depicts a micrograph of replicating viral DNA, the new duplex forming in a loop in the middle, and much of the DNA covered with proteins that help the process along.

Kornberg was the subject of both an autobiography and a biography / hagiography. The latter was written by a fellow scientist, but steers only gingerly into the science, sticking mostly to the story of Kornberg's life, times, and relationships. And what science there is is rather biased. For example, several years after the Watson-Crick model of DNA came out, Kornberg's lab developed a compositional assay for their short snippets of replicated DNA made in the test tube, and deduced that replication was anti-parallel. That is, one DNA strand of the duplex runs in one direction, chemically speaking, while the other strand runs in the opposite direction. This is portrayed as a discovery, for which Crick was very grateful in correspondence. But the Watson-Crick model had already posited the anti-parallel nature of DNA as an intrinsic property, and the model had been richly supported by that point, so Kornberg's work was at best confirmatory. Crick was just being polite.

An interesting side-light is that this epoch in biochemistry and molecular biology was substantially enabled by the scientific and technological breakthroughs of the Manhattan project and nuclear physics. It was isotopes like phosphorous-32 and sulphur-35 that allowed far more sensitive assays for nucleic acids than ever before, allowing tiny amounts of enzyme to be tracked down. DNA sequencing began with ladders of size-selected nucleic acids digested chemically from longer molecules and visualized by X-ray film thanks to radioactive P-32 enzymatically attached to the ends.

Early days, Arthur (right) and his wife Sylvie, who played a central, though unheralded, role in his laboratory and work.

One irony of the story is that Kornberg was so stuck in his system that he was resistant to the new fields it gave birth to, i.e. molecular biology. In his prime, he ran a factory of a lab, indeed a whole department, (first at Washington University, St Louis, then at Stanford), devoted to finding and characterizing the enzymes of nucleic acid synthesis and particularly DNA replication. These groups purified enzymes on a massive scale from E. coli cells, which were broken open, filtered, and then passed over various charge-selective and size-selective media, in extensive multi-step protocols to come out at the end with more or less pure single proteins or complexes of proteins. Some of these enzymes turned out to be extremely useful in biotechnology, for the cutting, copying, ligating, repairing of DNA, etc. As time went on, scientists realized that enzymology, while an important part of understanding how things work in cells, is usefully supplemented by the many methods of genetics and cell biology, which resulted in a hybrid field called molecular biology.

For example, Kornberg's Nobel prize was won, and name was made, on DNA polymerase I from E. coli. This enzyme replicates DNA, but not very well. It tends to fall off a lot. Some years later, another lab created a mutant E. coli strain that lacked the gene encoding this enzyme. And lo and behold, the cells were fine. They reproduced and replicated their DNA. It turned out that E. coli encodes five DNA polymerases, of which DNA polymerase I is among the least important- a repair enzyme that finishes gaps and other problems in the duplex, leaving the bulk of replication to other, far more ornate enzyme complexes. It was genetics that provided the critical clues in this story, showing how an integrated and diverse approach to research questions provides more productive answers.

Master of his realm, in later years, with fruit-themed computer.

Kornberg ran a family-style system in his departments. He had drawn most of its members (at Stanford) from among his own post-docs and students. It had communistic, but also authoritarian, elements. Space was shared, reagents were shared, even funding was shared- something unheard of today. At the same time, Kornberg had the last word on everything and was a ferocious micromanager. Researchers who wanted to make a name for themselves and build their own empires had to leave. But Kornberg picked well, and many of his colleagues had very influential careers, especially Paul Berg, a pioneer of recombinant DNA methods. Kornberg's strong dedication to his field and his system- his sense of meaning and purpose- was a precondition of success, communicating itself to all around him and fostering an unquestioned work ethic and community ethic. The Stanford department was legendary in its day, and inspired many other researchers to become enzymologists and use the laborious methods of protein purification to get their hands on the very gears and cogs of life.

• Steve McCarroll on contemporary biochemistry.
• Reformation and enlightenment as coincidences.
• Failure to deal with white collar crime may have led to a white collar criminal in the White House.
• Racism, yes.
• Who could have expected any better?
• The Chicago school, now for banking regulation?
• How much of military spending is waste, even on its own terms?
• Secular stagnation? Hogwash. Response, Wiki page. Response.
• MeToo and Kavanaugh. And perjury.