About this Author
College chemistry, 1983
The 2002 Model
After 10 years of blogging. . .
Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases.
To contact Derek email him directly: email@example.com
June 28, 2013
While writing up that eight-toxic-foods rebuttal the other day, I started reading up on Olestra, the "fake fat" that made the list. While it has to be considered a failure for its developers, I found the chemistry behind it interesting, and it got me to thinking.
First off, for those outside the chemical/biochemical field, a brief introduction to fat. We (and most other organisms) all store it in pretty much the same way: a backbone of glycerol (three carbons in a row, each with an alcohol), and each alcohol turned into an ester with a fatty acid. Those fatty acids are long carbon-chain compounds with a carboxylic acid group on the end, and when you've combined three of them onto a glycerol (fully loaded, as it were), you have a triglyceride. When the body wants to break that down for use as energy, it cleaves off the fatty acids one at a time (leading to diglycerides and monoglycerides), and the fatty acids are then chewed up two carbons at a time. (They're made two carbons at a time, too, so the ones found in living creatures are very heavily biased towards even-numbered carbon counts).
That intro is enough to make sense of some of the things you'll see in a blood test, like the tryglyceride levels and the free fatty acids. But there are a lot of details hidden in there. For one thing, there's a whole suite of different enzymes that do the work of assembling and breaking down the glycerides, and they're under all sorts of control mechanisms. And while glycerol is glycerol, the fatty acids themselves come in a huge variety - different lengths, presence of single or multiple double bonds up and down the chain (and keep in mind that double bonds in the middle of such a chain come in both cis and trans varieties), etc. So with this long list, glycerides get produced in all kinds of combinations, depending on diet, the tissue involved, and other factors. And beyond that, most all these components, up and down the list, are involved as signaling molecules on various proteins, substrates for other enzymes, starting materials for whole other chemical sequences, etc. Lipidology gets very complicated very quickly, and you may have noticed (via the changing dietary advice over the years) that we don't quite have it figured out yet. Nowhere near.
So what's Olestra? It's nothing more than table sugar (sucrose) with its alcohol groups given the fatty-acid-ester treatment. What you end up with is a molecule that acts very much like normal fats - both of them are polyhydroxy compounds decorated with fatty acids, after all. But the enzymes that cleave the various fatty ester groups don't recognize an esterified sucrose as anything they've seen before, and thus Olestra goes on its way uncleaved and unmetabolized. That, actually, is one of the things that seems to have sunk it in the market. A good-sized dose of Olestra has to go somewhere, namely, right through your digestive tract. The reports of the side effects this could bring on were not a good selling point, although there's a debate about how often they were observed in the real world.
Otherwise, though, it seems to have been a reasonably convincing substitute for actual fats. I've never had any Olestra myself - it would be interesting to see if I could distinguish potato chips made with it from the conventional ones. Procter and Gamble were of the opinion that there was no discernable difference in taste or texture, but I've heard from people who say that they can tell under blinded conditions
Another side effect is that the stuff would tend to dissolve greasier substances and carry them along. Thus the problems with fat-soluble vitamin absorption with Olestra, which was compensated for by adding more of these vitamins (such as A, D, and K) the to potato chips made with it. It should be remembered that potato chips are not a major source of vitamins - well, not for most consumers - but the concern was that a steady diet of Olestra-containing foods could interfere with nutrient absorption from the other foods eaten at the same time. This is purely a greasiness/water solubility issue (logP being the medicinal chemist's measuring scale), and Olestra has also shown an ability to sequester and remove things like ingested PCBs, for the same reasons. It doesn't know a vitamin from anything else; it just knows what it can dissolve.
Olestra spent a lot of time in human testing. Since lipid molecules (as mentioned above) are involved in a lot of different processes, these studies were done to see if there were any signs of Olestra participating in other pathways. Nothing was ever found; the stuff was too odd-looking to the body's enzymes to be digested, and too odd-looking to work its way into these other mechanisms as well. It just sort of made its way through.
But such cross-bred biomolecule hybrids are an interesting class. Just as you don't see fully-esterified sugar molecules in cells, there are many other things like this that don't show up - at least, as far as I know. The carboxylic acids at the C-terminals of amino acids, oligopeptides, and proteins don't get handled in living systems as esters much (or if they do, I've missed it). Imagine glycerol with peptides esterified off the OH groups, for example, in sort of a protein-fat hybrid. Now try it with glucose - I've never seen that, either. In the same way, the OH groups on amino acids like serine are available to be esterified, but that's another class of compounds I don't know much about. Phosphorylation, yes, but not plain esters. It's not like esters are somehow alien to biochemistry - you have the glycerides, for one, and esters of cholesterol are a well-known class of compound. Biochemistry as we know has just never gotten around to using these things.
It's easy to imagine a slightly alien life form using fatty acid esters of the higher sugars as its energy storage class rather than stopping at glycerol. These creatures would have enzymes that would take Olestra apart like a wooden puzzle, and might be baffled at our own molecules. Somewhere, some unusual-looking alien is perhaps proposing glycerol esters as an indigestible substitute for the diet - worried, perhaps, about the way everyone's tentacles are getting so swollen these days, what with the overabundance of cheap food and all, and sensing a market opportunity. Perhaps Zarkon & Yipslarg will succeed where Procter and Gamble failed.
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May 22, 2013
Just how many different small-molecule binding sites are there? That's the subject of this new paper in PNAS, from Jeffrey Skolnick and Mu Gao at Georgia Tech, which several people have sent along to me in the last couple of days.
This question has a lot of bearing on questions of protein evolution. The paper's intro brings up two competing hypotheses of how protein function evolved. One, the "inherent functionality model", assumes that primitive binding pockets are a necessary consequence of protein folding, and that the effects of small molecules on these (probably quite nonspecific) motifs has been honed by evolutionary pressures since then. (The wellspring of this idea is this paper from 1976, by Jensen, and this paper will give you an overview of the field). The other way it might have worked, the "acquired functionality model", would be the case if proteins tend, in their "unevolved" states, to be more spherical, in which case binding events must have been much more rare, but also much more significant. In that system, the very existence of binding pockets themselves is what's under the most evolutionary pressure.
The Skolnick paper references this work from the Hecht group at Princeton, which already provides evidence for the first model. In that paper, a set of near-random 4-helical-bundle proteins was produced in E. coli - the only patterning was a rough polar/nonpolar alternation in amino acid residues. Nonetheless, many members of this unplanned family showed real levels of binding to things like heme, and many even showed above-background levels of several types of enzymatic activity.
In this new work, Skolnick and Gao produce a computational set of artificial proteins (called the ART library in the text), made up of nothing but poly-leucine. These were modeled to the secondary structure of known proteins in the PDB, to produce natural-ish proteins (from a broad structural point of view) that have no functional side chain residues themselves. Nonetheless, they found that the small-molecule-sized pockets of the ART set actually match up quite well with those found in real proteins. But here's where my technical competence begins to run out, because I'm not sure that I understand what "match up quite well" really means here. (If you can read through this earlier paper of theirs at speed, you're doing better than I can). The current work says that "Given two input pockets, a template and a target, (our algorithm) evaluates their PS-score, which measures the similarity in their backbone geometries, side-chain orientations, and the chemical similarities between the aligned pocket-lining residues." And that's fine, but what I don't know is how well it does that. I can see poly-Leu giving you pretty standard backbone geometries and side-chain orientations (although isn't leucine a little more likely than average to form alpha-helices?), but when we start talking chemical similarities between the pocket-lining residues, well, how can that be?
But I'm even willing to go along with the main point of the paper, which is that there are not-so-many types of small-molecule binding pockets, even if I'm not so sure about their estimate of how many there are. For the record, they're guessing not many more than about 500. And while that seems low to me, it all depends on what we mean by "similar". I'm a medicinal chemist, someone who's used to seeing "magic methyl effects" where very small changes in ligand structure can make big differences in binding to a protein. And that makes me think that I could probably take a set of binding pockets that Skolnick's people would call so similar as to be basically identical, and still find small molecules that would differentiate them. In fact, that's a big part of my job.
But in general, I see the point they're making, but it's one that I've already internalized. There are a finite number of proteins in the human body. Fifty thousand? A couple of hundred thousand? Probably not a million. Not all of these have small-molecule binding sites, for sure, so there's a smaller set to deal with right there. Even if those binding sites were completely different from one another, we'd be looking at a set of binding pockets in the thousands/tens of thousands range, most likely. But they're not completely different, as any medicinal chemist knows: try to make a selective muscarinic agonist, or a really targeted serine hydrolase inhibitor, and you'll learn that lesson quickly. And anyone who's run their drug lead through a big selectivity panel has seen the sorts of off-target activities that come up: you hit someof the other members of your target's family to greater or lesser degree. You hit the flippin' sigma receptor, not that anyone knows what that means. You hit the hERG channel, and good luck to you then. Your compound is a substrate for one of the CYP enzymes, or it binds tightly to serum albumin. Who has even seen a compound that binds only to its putative target? And this is only with the counterscreens we have, which is a small subset of the things that are really out there in cells.
And that takes me to my main objection to this paper. As I say, I'm willing to stipulate, gladly, that there are only so many types of binding pockets in this world (although I think that it's more than 500). But this sort of thing is what I have a problem with:
". . .we conclude that ligand-binding promiscuity is likely an inherent feature resulting from the geometric and physical–chemical properties of proteins. This promiscuity implies that the notion of one molecule–one protein target that underlies many aspects of drug discovery is likely incorrect, a conclusion consistent with recent studies. Moreover, within a cell, a given endogenous ligand likely interacts at low levels with multiple proteins that may have different global structures.
"Many aspects of drug discovery" assume that we're only hitting one target? Come on down and try that line out in a drug company, and be prepared for rude comments. Believe me, we all know that our compounds hit other things, and we all know that we don't even know the tenth of it. This is a straw man; I don't know of anyone doing drug discovery that has ever believed anything else. Besides, there are whole fields (CNS) where polypharmacy is assumed, and even encouraged. But even when we're targeting single proteins, believe me, no one is naive enough to think that we're hitting those alone.
Other aspects of this paper, though, are fine by me. As the authors point out, this sort of thing has implications for drawing evolutionary family trees of proteins - we should not assume too much when we see similar binding pockets, since these may well have a better chance of being coincidence than we think. And there are also implications for origin-of-life studies: this work (and the other work in the field, cited above) imply that a random collection of proteins could still display a variety of functions. Whether these are good enough to start assembling a primitive living system is another question, but it may be that proteinaceous life has an easier time bootstrapping itself than we might imagine.
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November 21, 2012
We'll start off with a little extraterrestrial chemistry. As many will have heard, there are all sorts of hints being dropped that the sample analyzing equipment on the Mars Curiosity rover has detected something very interesting. We'll have to wait until the first week of December to find out what it is, but my money is on polycyclic aromatic hydrocarbons or something other complex abiotic organics.
Here's a detailed look at the issue. The Martian surface has a pretty vigorous amount of perchlorate in it, which was not realized for a long time (and rather complicates the interpretation of some of the past experiments on it). But Curiosity's analytical suite was designed to deal with this, and my guess is that these techniques have worked and that organic material has been detected.
I would very much bet against any sort of strong signature of life-as-we-know-it, though. For one thing, finding that in a random sand dune would seem pretty unlikely. Actually, finding good traces anywhere in the top layer of Martian rock and dust seems unlikely (as opposed to deeper underground, where I'm willing to speculate freely on the possible existence/persistence of bacteria and such). And I'm not sure the Curiosity would be well equipped to discriminate abiotic versus biotic compounds, anyway.
But organic compounds in general, absolutely. This brings up an interestingly false idea that underlies a lot of casual thinking about Mars (and space in general). Many people have this mental picture of everywhere outside Earth being sort of like the surface of our moon. It leads to a false dichotomy: here we have temperate air, liquid water, life and the byproducts of life (oil and coal, for example). Out there is all cold barren rock directly exposed to vacuum and hard radiation. We associate "space" with clean, barren, surfaces and knife-edge shadows, whereas "down here" it's all wet and messy. Not so.
There's plenty of irradiated rock, true, but there's water all over the outer solar system, in huge amounts. And while what we see out there is frozen, it's a near-certainty that there are massive oceans of the liquid stuff down under the various crusts of the larger outer-planet moons. All those alien-invasion movies, the ones with the extraterrestrials after our planet's water, are fun but ridiculous examples of that false dichotomy in action. There's plenty of organic chemistry, too - I've written before about how the colors of Jupiter's clouds remind me of reaction byproducts, and it's no coincidence that they do. The gas giant planets are absolutely full of organic chemicals of all varieties, and they're getting heated, pressurized, mixed, irradiated, and zapped by huge lightning storms all the hours of their days. What isn't in there?
Everything came that way. The solar system has plenty of hydrocarbons, plenty of small carbohydrates, and plenty of amines and other nitrogen-containing compounds in it. The carbonaceous chrondrites are physical evidence that's fallen to Earth - some of these have clearly never been heated since their formation (since they're full of water and volatile organics), so the universe would seem to be awash in small-molecule gorp. There's another false dichotomy, that the materials for life are very rare and precious and only found down here on Earth. But they're everywhere.
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October 8, 2012
The "arsenic life" bacterium has taken a number of blows in the scientific literature, and now it's taken another. A close look at its phosphate uptake system shows that these proteins in the GFAJ-1 bacteria not selective for arsenate (or at least tolerant of it, compared to normal lines). They are, in fact, extremely selective for phosphate.
All of the proteins can discriminate at least 500-fold over arsenate, but one of them from GFAJ-1 (highly expressed under the arsenate conditions) is 4,500-fold selective. The authors show, via X-ray crystallography, what sort of mutations hae occurred to give the binding site such high selectivity, which lead to the (slightly larger) arsenate disturbing a key hydrogen bond. This is what you'd expect if these bacteria were, in fact, still phosphate-dependent and needed to extract every bit of it they could from their arsenate-rich environment.
Here's a summary at Nature News. I believe that we can now declare this particular idea dead - everything is pointing the other way.
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June 5, 2012
Via Curious Wavefunction comes the news that Rosie Redfield and her lab have their paper coming out in Science refuting the "arsenic bacteria" results. It should be out on the journal's web site shortly, but is available at Arxiv beforehand.
I've been following Redfield's blogged results over the last few months, on and off, so the conclusions of this manuscript will not come as a surprise. She has been unable - completely unable - to substantiate the original claims of arsenate-driven growth and incorporation into biomolecules. Given the extraordinary nature of the original paper, the ball is now back in Wolfe-Simon et al.'s court. The default setting is that claims like those probably aren't real, and they need to be able to stand up to solid scrutiny.
I'll be very interested to see how this plays out. The authors of the original paper have been quite firm about only responding to criticism that's appeared in the official scientific literature, and have made remarks about how they're not going to deal with "website experiments" until they're published. Well, published they are, and in the same big journal as the original paper. What now?
I think their only hope is to advance specific, testable reasons why Redfield's results are incorrect. If it gets down to "Well, we get these results, and we don't see why you don't", and never advances from there, then the amazing results are almost certainly wrong. The world as we know it wins the tiebreakers in science.
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January 27, 2012
You may not have heard much about the arsenic-bacteria controversy recently, but you're about to hear quite a bit more. Rosie Redfield of UBC, one of the fastest and most vocal critics of the original paper, has been trying to reproduce it in her own group. There's a manuscript in preparation, but since she's been blogging on some of the progress, the import is clear: it hasn't been going well for the "bacteria can take up arsenic in their biomolecules" hypothesis. Scrolling back at that link will give you the story.
Here's a summary at Nature News (with a clarification from Redfield on one point). I look forward to seeing how this plays out - but remember, the startling results always have to prove themselves by happening again. Einmal ist keinmal.
Update: there's another story here, too. Redfield has been posting results as they come along, in a very prominent example of "open science". The first question is: will this affect journal publication? That is, will some editors look askance? The second point is to be found in that Nature News article, where Felisa Wolfe-Simon refers to those "website experiments", and how she basically can't discuss them until she sees them in a journal. Note that it's not "the UBC experiments" or "Redfield's experiments" - they're "website experiments", and thus (apparently) have more to prove.
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July 6, 2011
There's been a real advance in the field of engineered "unnatural life", but it hasn't produced one-hundredth the headlines that the arsenic bacteria story did. This work is a lot more solid, although it's hard to summarize in a snappy way.
Everyone knows about the four bases of DNA (A, T, C, G). What this team has done is force bacteria to use a substitute for the T, thymine - 5-chlorouracil, which has a chlorine atom where thymine's methyl group is. From a med-chem perspective, that's a good switch. The two groups are about the same size, but they're different enough that the resulting compounds can have varying properties. And thymine is a good candidate for a swap, since it's not used in RNA, thus limiting the number of systems that have to change to accommodate the new base. (RNA, of course, uses uracil instead, the unsubstituted parent compound of both thymine and the 5-chloro derivative used here).
Over the years, chlorouracil has been studied in DNA for just that reason, and it's been found to make the proper base-pair hydrogen bonds, among other things. So incorporating it into living bacteria looks like an experiment in just the right spot - different enough to be a real challenge, but similar enough to be (probably) doable. People have taken a crack at similar experiments before, with mixed success. In the 1970s, mutant hamster cells were grown in the presence of the bromo analog, and apparently generated DNA which was strongly enriched with that unnatural base. But there were a number of other variables that complicated the experiment, and molecular biology techniques were in their infancy at the time. Then in 1992, a group tried replacing the thymine in E. coli with uracil, with multiple mutations that shut down the T-handling pathways. They got up to about 90% uracil in the DNA, but this stopped the bacteria from growing - they just seemed to be hanging on under those T-deprived conditions, but couldn't do much else. (In general, withholding thymine from bacterial cultures and other cells is a good way to kill them off).
This time, things were done in a more controlled manner. The feat was accomplished by good old evolutionary selection pressure, using an ingenious automated system. An E. coli strain was produced with several mutations in its thymine pathways to allow it to survive under near-thymine-starvation conditions. These bacteria were then grown in a chamber where their population density was being constantly measured (by turbidity). Every ten minutes a nutrient pulse went in: if the population density was above a set limit, the cells were given a fixed amount of chlorouracil solution to use. If the population had falled below a set level, the cells received a dose of thymine-containing solution to keep them alive. A key feature of the device was the use of two culture chambers, with the bacteria being periodically swapped from one to the other (which the first chamber undergoes sterilization with 5M sodium hydroxide!) That's to keep biofilm formation from giving the bacteria an escape route from the selection pressure, which is apparently just what they'll do, given the chance. One "culture machine" was set for a generation time of about two hours, and another for a 4-hour cycle (by cutting in half the nutrient amounts). This cycle selected for mutations that allowed the use of chlorouracil throughout the bacteria's biochemistry.
And that's what happened - the proportion of the chlorouracil solution that went in went up with time. The bacterial population had plenty of dramatic rises and dips, but the trend was clear. After 23 days, the experimenters cranked up the pressure - now the "rescue" solution was a lower concentration of thymine, mixed 1:1 with chlorouracil, and the other solution was a lower concentration of chlorouracil only. The proportion of the latter solution used still kept going up under these conditions as well. Both groups (the 2-hour cycle and the 4-hour cycle ones) were consuming only chlorouracil solution by the time the experiment went past 140 days or so.
Analysis of their DNA showed that it had incorporated about 90% chlorouracil in the place of thymine. The group identified a previously unknown pathway (U54 tRNA methyltransferase) that was bringing thymine back into the pathway, and disrupting this gene knocked the thymine content down to just above detection level (1.5%). Mass spec analysis of the DNA from these strains clearly showed the chlorouracil present in DNA fractions.
The resulting bacteria from each group, it turned out, could still grow on thymine, albeit with a lag time in their culture. If they were switched to thymine media and grown there, though, they could immediately make the transition back to growing on chlorouracil, which shows that their ability to do so was now coded in their genomes. (The re-thymined bacteria, by the way, could be assayed by mass spec as well for the disappearance of their chlorouracil).
These re-thymined bacteria were sequenced (since the chloruracil mutants wouldn't have matched up too well with sequencing technology!) and they showed over 1500 base substitutions. Interestingly, there were twice as many in the A-T to G-C direction as the opposite, which suggests that chlorouracil tends to mispair a bit with guanine. The four-hour-cycle strain had not only these sorts of base swaps, but also some whole chromosome rearrangements. As the authors put it, and boy are they right, "It would have been impossible to predict the genetic alterations underlying these adaptations from current biological knowledge. . ."
These bacteria are already way over to the side of all the life on Earth. But the next step would be to produce bacteria that have to live on chlorouracil and just ignore thymine. If that can be realized, the resulting organisms will be the first representatives of a new biology - no cellular life form has ever been discovered that completely switches out one of the DNA bases. These sorts of experiments open the door to organisms with expanded genetic codes, new and unnatural proteins and enzymes, and who knows what else besides. And they'll be essentially firewalled from all other living creatures.
Postscript: and yes, it's occurred to me as well that this sort of system would be a good way to evolve arsenate-using bacteria, if they do really exist. The problem (as it is with the current work) is getting truly phosphate-free media. But if you had such, and ran the experiment, I'd suggest isolating small samples along the way and starting them fresh in new apparatus, in order to keep the culture from living off the phosphate from previous generations. Trying to get rid of one organic molecule is hard enough; trying to clear out a whole element is a much harder proposition).
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January 28, 2011
Here's the first response in the chemical literature to the arsenic-in-DNA controversy, from three authors in ACS Chemical Biology. They detail the argument, familiar to readers of the comment section here, that arsenate esters just would not be expected to have the hydrolytic stability needed for arseno-DNA to function usefully.
How far off is it? By, well, about
13 (make that 17) orders of magnitude, which is much worse than I'd thought. As the authors put it, "Overcoming such dramatic kinetic instability in its genetic material would present serious challenges to Halomonadacea GFAJ-1." Indeed it would.
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December 2, 2010
Update: a further look at the details of this paper is in a later post.
So: arsenic for phosphorus? That's the big news from NASA today. I listened to much of the press conference, and I've read the paper in Science. Is this real - and if it is, what does it tell us?
Let's do the second part first. Phosphorus is an extremely important element for every living thing on Earth. It's mostly found as phosphate, and phosphate groups are found all over the place: decorating proteins, carbohydrates, and lipids, as the invariable outside of DNA helices, and as the key part of the ultimate energy currency of every living cell, ATP. Phosphate's no bit player.
This is a good time to emphasize that (as far as we can tell) all life on Earth shares the same chemistry and the same kinds of biomolecules. Humans, frogs, fruit flies, fungi, tube worms on the ocean floor, lichens in Antarctica, and weirdo single-celled creatures living in boiling hot springs: we all have cells full of proteins, carbohydrates, lipids, and nucleic acids. We use DNA and RNA to pass on our genetic information, and the enzymes we use to manipulate them and to power our cells are all similar enough that we just have to share a common ancestor. (Either that, or life only gets going in a very specific way indeed).
One thought about today's press conference was that it might be announcing "alien life on Earth". That's been a subject of argument for quite a while. Even though everything we've ever found is of the same family tree, that doesn't rule out (logically or practically) the possibility that some other form of life, with different chemistry entirely, might be hanging out in its own environment. A good deal of searching has failed to turn it up, but (if it's such different stuff) we might be looking for it in the wrong ways, or might even have trouble recognizing it when we see it.
That's not what today's work has turned up, though - but it's probably the next best thing. What this group was looking for were hypothetical organisms that have learned to use arsenic instead of phosphorus. There are environments that are much richer in arsenic (and its corresponding arsenate salts) than they are in phosphorus. And arsenic is right under phosphorus in the periodic table, and forms similar sorts of compounds (albeit with rather different behavior), so. . .maybe it could substitute? Well, they didn't find any native arsenic-users - but they did force some into existence. They took a strain of bacteria from such an environment (Mono Lake sediments) and starved it of phosphate while providing it plenty of arsenate. The colonies that grew under these conditions were picked out and grown under even higher arsenate concentrations, and the process was continued stage after stage.
The end result appears to be bacteria that have incorporated arsenate into their metabolism. They still have phosphate in them, but not enough to keep everything running on a phosphate basis. Some parts have switched over to arsenate, without gumming up the works completely. That surprises me quite a bit - I really wouldn't have thought that things could be pushed that far. After all, in higher organisms, it's that arsenate-for-phosphate switch that's responsible for arsenic's reputation as a poison. Eventually, some key enzyme systems can't handle the switch and cease to function.
But not in these bacteria. They look different and grow more slowly than their phosphate-saturated brethren, and they'd clearly like ditch the arsenic at the first opportunity (add phosphate and they start growing more vigorously). But they're getting by, presumably with just enough phosphate to hold things together. (Have they hit the wall, one wonders?) A number of physical methods all point in the same direction, to arsenate being incorporated into their biomolecules. We still don't know where most of it goes, or how the various phosphate-manipulating enzymes manage to still work, but working out those details will keep a lot of people busy for quite a while. Personally, I'd love to see some X-ray structures of aresenate-containing proteins or nucleic acids, and I'm sure that the people who reported this are trying to get some.
So what does this mean? Well, you can apparently bend the most basic chemistry of life as we know it quite a bit before it breaks. As I said, I really would not have thought that this could be possible - we're all going to have to keep rather more open minds about what biochemical systems can handle. This makes the arsenic-from-the-ground-up idea look a lot more plausible, too, and you can be sure that the search for such organisms (using arsenate naturally, without having to be forced in the lab) will intensify.
It also makes you wonder about what other directions the biochemistry we know of can be stretched. Selenium for sulfur is my best guess - there, you have the advantage that selenium already has a small but real role in biochemistry as it is. I don't know of any environments that are higher in selenium than sulfur, but it would be worth trawling the closest candidates, culturing some bacteria, and giving them the same forcing treatment that was used here. If you really wanted to go wild, you could try pushing down to tellurium and down to antimony in the phosphorus column. Now, I really don't think those have much of a chance, but you never know. It's a lot more plausible to me than it was yesterday.
And the implications for extraterrestrial life are. . .what? Well, we keep finding the sorts of chemicals that we live with (amino acids, simple carbohydrates and the like) out in space. Our type of biochemistry might be fairly common - and if it is, it's good to know that it has a lot of wiggle room in it. It's hard for me to imagine a planet that's loaded down with arsenic and is short of phosphorus, but hey, it's a big universe. Big enough, it appears, for all kinds of weird things. It's great.
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September 23, 2010
I agree with many of the commenters around here that one of the most interesting and productive research frontiers in organic chemistry is where it runs into molecular biology. There are so many extraordinary tools that have been left lying around for us by billions of years of evolution; not picking them up and using them would be crazy.
Naturally enough, the first uses have been direct biological applications - mutating genes and their associated proteins (and then splicing them into living systems), techniques for purification, detection, and amplification of biomolecules. That's what these tools do, anyway, so applying them like this isn't much of a shift (which is one reason why so many of these have been able to work so well). But there's no reason not to push things further and find our own uses for the machinery.
Chemists have been working on that for quite a while. We look at enzymes and realize that these are the catalysts that we really want: fast, efficient, selective, working at room temperature under benign conditions. If you want molecular-level nanotechnology (not quite down to atomic!), then enzymes are it. The ways that they manipulate their substrates are the stuff of synthetic organic daydreams: hold down the damn molecule so it stays in one spot, activate that one functional group because you know right where it is and make it do what you want.
All sorts of synthetic enzyme attempts have been made over the years, with varying degrees of success. None of them have really approached the biological ideals, though. And in the "if you can't beat 'em, join 'em" category, a lot of work has gone into modifying existing enzymes to change their substrate preferences, product distributions, robustness, and turnover. This isn't easy. We know the broad features that make enzymes so powerful - or we think we do - but the real details of how they work, the whole story, often isn't easy to grasp. Right, that oxyanion hole is important: but just exactly how does it change the energy profile of the reaction? How much of the rate enhancement is due to entropic factors, and how much to enthalpic ones? Is lowering the energy of the transition state the key, or is it also a subtle raising of the energy of the starting material? What energetic prices are paid (and earned back) by the conformational changes the protein goes through during the catalytic cycle? There's a lot going on in there, and each enzyme avails itself of these effects differently. If it weren't such a versatile toolbox, the tools themselves wouldn't come out being so darn versatile.
There's a very interesting paper that's recently come on on this sort of thing, to which I'll devote a post by itself. But there are other biological frontiers beside enzymes. The machinery to manipulate DNA is exquisite stuff, for example. For quite a while, it wasn't clear how we organic chemists could hijack it for our own uses - after all, we don't spend a heck of a lot of time making DNA. But over the years, the technique of adding DNA segments onto small molecules and thus getting access to tools like PCR has been refined. There are a number of applications here, and I'd like to highlight some of those as well.
Then you have things like aptamers and other recognition technologies. These are, at heart, ways to try to recapitulate the selective binding that antibodies are capable of. All sorts of synthetic-antibody schemes have been proposed - from manipulating the native immune processes themselves, to making huge random libraries of biomolecules and zeroing in on the potent ones (aptamers) to completely synthetic polymer creations. There's a lot happening in this field, too, and the applications to analytical chemistry and purification technology are clear. This stuff starts to merge with the synthetic enzyme field after a point, too, and as we understand more about enzyme mechanisms that process looks to continue.
So those are three big areas where molecular biology and synthetic chemistry are starting to merge. There are others - I haven't even touched here on in vivo reactions and activity-based proteomics, for example, which is great stuff. I want to highlight these things in some upcoming posts, both because the research itself is fascinating, and because it helps to show that our field is nowhere near played out. There's a lot to know; there's a lot to do.
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April 8, 2010
A very weird news item: multicellular organisms that appear to be able to live without oxygen. They're part of the little-known (and only recently codified) phylum Loricifera, and these particular organisms were collected at the bottom of the Mediterranean, in a cold, anoxic, hypersaline environment.
They have no mitochondria - after all, they don't have any oxygen to work with. Instead, they have what look like hydrogenosome organelles, producing hydrogen gas and ATP from pyruvate. I'm not sure how large an organism you can run off that sort of power source, since it looks like you only get one ATP per pyruvate (as opposed to two via the Krebs cycle), but the upper limit has just been pushed past a significant point.
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March 5, 2010
Here's another outside the field - in fact, it's outside of a lot of people's fields. Where Is Everybody? presents fifty possible solutions to the Fermi Paradox: if there are a lot of planets in the galaxy, and if life is pretty easy to get going, and if it's possible to travel or just communicate between solar systems. . .why haven't we seen anything? Enrico Fermi, in his typically disconcerting way, ran the math on this question during a lunchtime conversation in 1950, and realized that at least one of the common assumptions behind it must be off, and by a great deal.
I was thinking about this last night, because this weekend I'll have swarms of fourth graders and their parents looking through my telescope (if the weather cooperates), under the auspices of the Amateur Telescope Makers of Boston. And it's impossible to look at the night sky without wondering what life might exist out there and what form it might take. That Wikipedia article is quite good, but if you find it interesting, this book goes into the question in greater detail. I should note that a new book, The Eerie Silence, has just come out on the same topic, but I haven't seen that one yet.
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July 7, 2009
While we're on the topic of hydrogen bonds and computations, there's a paper coming out in JACS that attempts to answer an old question. Why, exactly, does every living thing on earth use so much ribose? It's the absolute, unchanging carbohydrate backbone to all the RNA on Earth, and like the other things in this category (why L amino acids instead of D?), it's attracted a lot of speculation. If you subscribe to the RNA-first hypothesis of the origins of life, then the question becomes even more pressing.
A few years ago, it was found that ribose, all by itself, diffuses through membranes faster than the other pentose sugars. This results holds up for several kinds of lipid bilayers, suggesting that it's not some property of the membrane itself that's at work. So what about the ability of the sugar molecules to escape from water and into the lipid layers?
Well, they don't differ much in logP, that's for sure, as the original authors point out. This latest paper finds, though, by using molecular dynamic simulations that there is something odd about ribose. In nonpolar environments, its hydroxy groups form a chain of hydrogen-bond-like interactions, particularly notable when it's in the beta-pyranose form. These aren't a factor in aqueous solution, and the other pentoses don't seem to pick up as much stabilization under hydrophobic conditions, either.
So ribose is happier inside the lipid layer than the other sugars, and thus pays less of a price for leaving the aqueous environment, and (both in simulation and experimentally) diffuses across membranes ten times as quickly as its closely related carboyhydate kin. (Try saying that five times fast!) This, as both the original Salk paper and this latest one note, leads to an interesting speculation on why ribose was preferred in the origins of life: it got there firstest with the mostest. (That's a popular misquote of Nathan Bedford Forrest's doctrine of warfare, and if he's ever come up before in a discussion of ribose solvation, I'd like to hear about it).
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October 25, 2006
There's an interesting analytical chemistry paper in the preprint section of PNAS (open access if you want to read it) that may reopen an old controversy. It's from a large multinational team (Mexico, Spain, France, NASA-Ames) investigating the GC-mass spec instrumentation that was flown to Mars on the Viking landers in 1976. That's a key instrument in the life-on-Mars debate, so an attack on it is significant. First, though, some background - it's a tangled story.
The Viking landers each had three biology experiments to look for possible signs of Martian life, whose results were famously difficult to interpret. They produced both excitement and confusion at the time (scroll down in that NASA history page) and they've been fuel for arguments ever since.
There was the pyrolytic release experiment, which incubated Martian soil with 14C-labled carbon monoxide and carbon dioxide. After several days, the sample was purged, then heated to 650C and analyzed for the release of any labeled carbon compounds that might have been formed by living organisms. A control sample was heated before incubation, to kill off any such life forms. Seven out of the nine runs of this experiment seemed to produce positive results - that is, volatile labeled carbon was produced after pyrolysis.
The gas-exchange experiment used the same sort of apparatus, exposing the soil to either water vapor or nutrient solution under a mixed atmosphere of gases. The headspace was analyzed for changes in the concentrations of the various components, which could be due to biological uptake or release. This one showed a strong release of oxygen and carbon dioxide from the samples once moisture was added, but the amount decreased over time, leading to theories that this was the product of an inorganic reaction rather than a signature of life.
The labeled release experiment put Martian soil into a dilute nutrient broth, with several small organic compounds which were all labeled with 14C. After incubation, the headspace of the experimental cell was analyzed for any released labeled gases and again, a control experiment was done with pre-heated soil. This one produced exciting data, with release of labeled gas in the experimental samples well over those in the controls. One odd result, though, was that the subsequent injection(s) of nutrient solution did not produce a further spike of released gas. The final curves ended up looking neither like what you'd have expected from a classic bacterial positive, nor from a simple chemical reaction. This ambiguity has meant that the LR results have been re-analyzed and re-fought ever since the 1970s, with the experiment's designer, Gilbert Levin, leading the effort to rescue the data as a case for Martian life.
But then there were the GC-MS data, from an experiment considered to be the backstop test in case the biology experiments were difficult to interpret. Since they certainly were that, from beginning to end, this experiment became for many people the most important one on the landers. (It already had been for the people - a not insignificant group - who thought from the start that the biology tests were unlikely to provide a conclusive answer). This one heated soil samples directly and looked for volatile organics. Heating to 200C showed little or nothing in the way of carbon compounds, and very little water besides. By contrast, another sample taken up to 500 degrees released a comparative flood of water, but still showed no evidence of organic molecules.
And that, for most observers, was that. No organic molecules, no life. Explanations after the GC-MS results mainly turned to what sorts of inorganic chemistry might have given the behavior seen in the three other experiments. Martian soil was thus hypothesized to be a sterile mixture of interesting chemicals (iron peroxides? carbon suboxide polymers?) that had fooled the biology test packages, but couldn't fool the GC-MS.
There's always been an underground, though, that has held that the results were indeed the result of life. Gilbert Levin has never given up. In 1981, he pointed out that tests of a Viking-style GC-MS instrument had shown that it was insensitive to organics in a particular Antarctic soil sample, but that this same soil nonetheless gave a positive result in the LR experiment. And he really put his opinions out in the store window in 1997, with a paper that flatly concluded that the 1976 LR experiments had indeed detected Martian life.
In the last few years, others have joined the battle. Steven Benner at Florida, whose work I wrote about here, published a PNAS paper in 2000 which maintained that organic molecules on Mars would likely be retained as higher molecular weight carboxylates, which would not have been volatile enough for the Viking GC/MS instrument to detect. And now this latest group has weighed in.
They've also analyzed various Antarctic and temperate desert samples, and found that all of them contain organic matter that cannot be detected by thermal GC-MS analysis. And the ones that contain iron, including the NASA reference simulated Mars soil (a weathered basalt sample from near Mauna Kea), tend to oxidize their organics quickly under heating. The conclusion is that while much of the water and carbon dioxide produced in the Viking experiment from heating the Martian soil was surely inorganic, some of it could have been from the oxidation of organic material. The paper concludes that the Viking GC-MS results are. . .inconclusive, and should not be taken as evidence either way for the presence of organic molecules or life. The question, they feel, is still completely open.
The good news is that future missions are relying on other technologies. In addition to good ol' thermal volatilization/GC-MS, there are also plans for solvent extractions, laser desorption mass spec, short-path sublimation, and other nifty ideas. If these various US and European missions get off the ground (and on the Martian ground), we're going to have some very interesting data to look at. And argue about.
+ TrackBacks (0) | Category: Analytical Chemistry | Life As We (Don't) Know It
May 23, 2005
I haven't commented on the controversy about including "Intelligent Design" in school curricula, but I don't want that to be interpreted as any kind of approval. On the contrary - until it offers some testable predictions, which would seem an unlikely thing to hope for, I don't see how ID even rises to the level of a preliminary theory, much less one that can compete with the level of evidence backing up evolution. Many of ID's advocates, to a greater or lesser degree, strike me as intellectually dishonest.
Intelligent Design proponents are fond of arguing about "irreducible complexity", the idea that some structures are too complicated to have been generated through stepwise evolution. They argue this on the anatomical level, which I don't buy, but I'm not going to debate that one in this forum. (Allow me to refer the curious to my fellow Corantean Carl Zimmer, who's had plenty of run-ins with these folks, and his fine introduction to evolution. Those interested in the latest news on the ID/evolution battles should check out The Panda's Thumb. For sheer mockery, often irresistible in these cases, try this.)
But when they start making arguments at the chemical level, the what-are-the-odds stuff about proteins and DNA, well, that's when I come out of my lair. A paper in the latest issue of the journal ChemBioChem got me thinking about this today. (If you have access to Wiley journals, it's here as a PDF.) It's an update of the analytical work still being done on the Murchison meteorite (a href="http://www.publish.csiro.au/?act=view_file&file_id=AS03060.pdf">PDF), which fell in Australia in 1969. The more than 100 kg of recovered Murchison material have been attacked over the years with just about every instrument of the constantly shifting state of the art in analytical chemistry.
Why all the interest? Well, a short answer is that the pieces of this meteorite reek. Even now, they smell like low-grade gasoline, and they had a powerful odor indeed when they were freshly collected. The Murchison fall is a wonderful example of a rare class of meteorites called carbonaceous chondrites. Many people don't realize how much organic gunk is floating around out in space, but there are surely millions of tons of this stuff wandering around our solar system alone.
What's in the Murchison pieces? The list continues to lengthen. We're up to at least 500 different soluble compounds, but much more of the material is dark polymeric asphalty stuff that's hard to analyze. Most famously, the meteorite contains many amino acids. Save glycine, those come in left- and right-handed isomers, and a major find is that the Murchison material is slightly biased toward the left-handed ones, which happen to be the ones that life on Earth is built around. This is an important point: the chemicals that life as we know it is composed of are not at all odd or unlikely. They're all over our solar system, they're in interstellar clouds, and there's every reason to think that they're smeared and splatted all over the universe.
And more of the stuff is being made all the time. In 2002, several research groups took icy mixtures of water, methanol, ammonia, HCN, carbon dioxide and carbon monoxide (just the sort of mixtures that you see in cometary ices and the above-mentioned interstellar clouds.) They irradiated them with ultraviolet light - as would come from the Sun or untold billions of other stars - at cold outer-space temperatures, and obtained over a dozen of the most common amino acids - here are some more details.
So, here's another key point: the really big step is between making random chemical combinations and having carbohydrates and amino acids as inevitable products. Believe me, the molecules of life are an infinitesmal sliver of all the possible backbones of up to ten or twelve carbon, nitrogen, and oxygen atoms. But organic chemistry, with no active hand on the controls, turns out uncountable heaps of them. Compared to that, the gaps that need to be filled in on the way to living systems don't seem so large.
This, to me, is one of the major stories of the last few decades. Starting hundreds of years ago, astronomy gradually moved the Earth out of its supposed spot in the center of the universe and placed it in the huge (and hugely strange) context of the universe that we now know. Now chemistry is moving us away from the view of life as a strange and precious anomaly - granted, perhaps, by a divine being? - to something that could be everywhere and may well start of its own accord. The building blocks are ubiquitous, and if you give them half a chance they start to stack themselves up.
For better or worse, the presence of an active Designer does not suggest itself. That may not seem right to some people, for many different reasons. But if there's one thing that science has been showing us, it's the the universe doesn't much care what we think about it.
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February 23, 2005
Back in the early days of my pre-Corante blog, I wrote a piece about some other kinds of chemistry that might be used in living systems. There's now a wonderful one-stop review for all sorts of speculations on this topic, which incorporates everything I've ever thought of and plenty more. Steven Benner at the University of Florida, who my fellow Corantean Carl Zimmer has interviewed, and two co-workers (here's his research group) published "Is There a Common Chemical Model for Life in the Universe?" in Current Opinion in Chemical Biology late last year. (here's the abstract; I can't find the full text available yet on the Web.)
I can't say enough good things about this article. This is the sort of topic I've enjoyed thinking about for years, but there were still plenty of things in this review that had never occurred to me. Benner goes over the likely requirements for life as we know it, life as we'd probably recognize it, and life upon which we can barely speculate. As a chemist, he's particularly strong on discussions of the types of bonds that could best form the complex molecules that chemical-metabolism-based life needs. Energetic considerations - how much chemical bond energy is available, how soluble the materials are, how reactive they are at the various temperatures involved - are never far from his mind.
He devotes sections to ideas about living systems without chemical solvents (gas clouds, solid states) and the more familiar solvent-based chemistry. There's plenty of water out there in the universe - which is why bad movies about aliens coming to drain our oceans are so laughable - and it's natural enough that we should concentrate on water-based life. But there's plenty of ammonia out there, too, along with methane, sulfuric acid, and other potential solvents like the supercritical dihydrogen found in the lower layers of gas giant planets.
So, is all this stuff out there? Is life something that is just going to happen to susceptible chemical systems, given enough time? If so, which ones are susceptible? Benner's thoughts are, I think, best summed up by his take on Titan:
"Thus, as an environment, Titan certainly meets all of the stringent criteria outlined above for life. Titan is not at thermodynamic equilibrium. It has abundant carbon-containing molecules and heteroatoms. Titan's temperature is low enough to permit a wide range of bonding, covalent and non-covalent. Titan undoubtedly offers other resources believed to be useful for catalysts necessary for life, including metals and surfaces.
This makes inescapable the conclusion that if life is an intrinsic property of chemical reactivity, life should exist on Titan. Indeed, for life not to exist on Titan, we would have to argue that life is not an intrinsic property of the reactivity of carbon-containing molecules under conditions where they are stable. Rather, we would need to believe that either life is scarce in these conditions, or that there is something special, and better, about the environment that Earth presents (including its water)."
As for me, I can't wait to find out. I want Titan rovers, Jupiter and Saturn dirigibles, Venusian atmosphere sample return, instrument-laden miniature submarines melting down through the ice on Europa and Enceladus: the lot. How much of this will I ever get a chance to see in my lifetime? Current betting is running to "none of it, damn it", but things can change. Depends on how easily and cheaply we can get payloads up to (and out of) Earth orbit.
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August 13, 2002
As for phosphorylation, I've had some folks write to talk about the importance of phosphate cleavages for cellular energy production, and about the conformational effects of phosphorylation. All that's well taken - but I guess what I was getting at yesterday is that (for example) sulfation would seem to be a perfectly reasonable way to modify proteins. Why didn't life end up using it?
Perhaps the phosphate energy part is the key. That's such a basic mechanism that enzymes to handle phosphate groups must be archaic indeed. It could be that evolution just found a use for them, since they were there anyway, and that competing methods of post-transcriptional modification (like sulfation) never got off the ground. Of course, there's always glycosylation - wonder when that kicked in, evolutionarily?
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March 19, 2002
No longer can I say that the topic of allergenic extraterrestrial life hasn't been taken on in science fiction. Patrick Neilsen Hayden of Electrolite, who is certainly in a professional position to know, passes on the word that the 1999 novel BIOS (by Robert Charles Wilson, reviewed) includes this very idea.
On a related topic, a number of old sf stories made use of the chirality of amino acids and the resulting proteins, with a plot point usually being the possiblity of starvation when trapped in an environment full of the wrong-handed food source. Given that such enantiomeric compounds can have very different properties in the body, I'd think that such an environment would not only be non-nutritious, but extremely toxic.
But are there any such places (stipulating an abudance of life-as-we-know-it in the universe to make it more possible)? That gets right into the question of how we ended up with only L-amino acids (and only D-sugars, which get less attention, undeservedly.)
There are plenty of theories. One thing that most everyone agrees on is that what we see now is a founder's effect - life got started with the series that we know, and stuck with it ever since, across billions of years. (Reminds me of Microsoft.) But was it a pure 50/50 chance at the beginning, or was the deck stacked? For that to happen, you have to have a chiral environment somewhere for it to develop.
Explanations such as seeding by meteorites containing chiral amino acids (or planetary formation from a cloud containing a chiral mix of compounds) just push the question back a bit. Where did those excesses come from? I should note that a paper on the amino acid ratios in the Murchison meteorite was just presented at the same meeting that Jay Manifold has been reporting from over at "A Voyage to Arcturus." Maybe he can give us a report.
For some years, the explanation that was hauled out invoked the weak nuclear force, which is the first place you can find symmetry breaking down in the laws of physics. Trouble is, that's such a small effect on the thermodynamics (if there's an effect at all!) that it's really like sticking with the 50/50 chance.
Another interesting idea was that circularly polarized radiation, potentially from neutron stars, preferentially breaks down one enantiomer of simple molecules over another. This still doesn't give you much of an edge, but it's a lot more compelling explanation that the weak nuclear force.
Last year a theory was proposed that formation of simple biomolecules on rock surfaces (a hot topic in origin-of-life research) might have something to do with it. Calcite seems to absorb different enantiomers on different faces of its crystals, which could have led to local excesses - too local, some say - of one enantiomer. If one of these microenvironments was the first to start things on the road to life, though, that would be all you need. It's still another form of 50/50 chance, depending on what crystal face you pick, but at least (as with polarized radiation) you have a semi-plausible mechanism for generating an excess of one chiral form.
These ideas and others are discussed on this site, but note: that page also brings up an experiment from a few years ago that suggested that magnetic fields could induce chiral chemistry. This result has since been throroughly discredited. No one could ever reproduce it, and it turned out that one of the original graduate students faked the results. Not a smart career move, considering how much interest (and scepticism) the first report got.
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