Protip: Yankees can't drive in snow either

Any time it snows in the south, you can count on a few specific things happening. First, everyone goes to the grocery store to buy milk and bread (you know, for milk sandwiches). Second, your transplanted northern neighbor or friend laughs at that phenomenon (which is justified) and claims that because he's from the north, he "knows how to drive in snow." I tell you now, that man is lying. He lies without malice, to be sure—he honestly believes what he just told you. But he does not know how to drive in snow, and most especially he cannot drive safely in snow in the south.

As you probably know, there was a great big snowstorm in the northeast today. Doubtless you are imagining that plows rolled out like clockwork to clear the streets, but that's not what happened. Instead, as the snow fell, almost all the schools and businesses let out early simultaneously, resulting in what I always think of as the Raleigh Effect, a case of traffic paralysis resulting from an seemingly manageable winter weather event. The source of the problem in this case? Yankees who thought they could drive on snow. The result? Well, traffic was barely moving on South Street. Ralph's friend spent two hours going one block. And this is in Waltham MA.

The truth is that Yankees can't drive on snow because they almost never get a chance to learn how. The northeastern states all have very capable infrastructure that usually clears ice and snow off the roads fairly promptly. When that doesn't happen, in this case because plows can't clear roads that are full of cars, Yankee drivers are just as helpless as the rest of us. Your Yankee friend probably does know how to drive on roads that are a bit slushy, but he's almost certainly not equipped to drive on snowy roads in the South, where snowplows and salt trucks are about as common as an egg cream. Do not let him get in his car; he is in danger.

Now I know what you're thinking: "That may be true of citified Yankees, but what about those who come from the rural regions? They probably know how to drive on the powder." And you're right—New Englanders who don't come from the city are much more likely to be able to drive on actual snow. However, you must not let them drive on snow in the South! They are almost certain to wreck, though not out of any fault of their own. You see, these proud country men and women will get into their vehicles, drive confidently out onto the snowy streets, and promptly be hit by some jackass Southerner who thinks his 4 wheel drive means he knows how to drive in snow. Unless they're lucky, in which case they'll get hit by the southerner who thought his Honda was light enough to stay on top of the snow.

This is the painful truth your friend has not yet learned: Most likely, he actually doesn't know how to drive on a snowy road. Even if he does, this amounts only to overconfidence that will result in an accident due to the inevitable chaos of Southerners (who know even less about driving on snow than city Yankees) trying to get to the store because they've run out of fixin's for their milk sandwiches. Dissuade that man from getting in his car if you can... driving on snow, especially in the South, will only result in dents, dings, or a totaled car.

Posts the next couple of weeks will be sporadic as I get some travel in around the holidays. I shall return with more commentary in the new year.

Read the rest...

The Hierarchy of Motion in Adk

Well, the papers for which Ming produced his fantastic molecular artwork came out in Nature last week. There were two, as some of you might have noticed. Although the larger paper was probably more technically impressive, I'm going to skip over it and talk about the second Henzler-Wildman et al. In the interest of full disclosure, I will point out that although I did not work on this paper, those who did are in my lab. So, either I am a homer or this stuff is so exciting that I still want to write about it even after hearing it rehashed endlessly for more than a year. The question at hand is the nature of protein conformational changes. Specifically, the paper is meant to address whether motions on the timescale of nanoseconds to picoseconds are indicators of slower motions, and if so, what kind of indicators they are.

I don't mean to set up a false dichotomy here, but in order to set up this discussion it's most convenient to describe two extremes of an expected spectrum of behaviors. At one extreme of the spectrum we have a sort of "ball-and-string" model of protein behavior, in which slow motions generally reflect coherent motions of larger structural units with intervening regions of flexibility serving as "hinges". This would tend to produce the energy landscape shown at left, with two minima separated by a single large energy barrier, with perhaps a very low population of an intermediate state. Such a model predicts true two-state behavior in the slow time regime. On the fast timescale, dynamic behavior will be heterogeneous, with residues in the "ball" showing rigidity while residues in the "strings" are flexible.

As an alternative extreme, one could imagine a "jello" model of protein dynamics. In this model, domains or subdomains "wiggle" into new positions primarily through incoherent motions that occasionally produce a coherent shift. Thus, two well-populated minima might be separated by several intermediates with relatively low energy barriers (example energy diagram at right). If this is the case, one would expect the behavior in the slow time regime to be less clearly two-state (because the intermediates are all populated) and for the fast timescale dynamics to be fairly homogeneous and reflect significant flexibility. In the ball-and-string case the transition rate is governed by a monolithic energy barrier, while in the "jello" case the transition rate is limited by frustration.

I want to re-emphasize that this is not an either-or proposition. It is likely that both extremes are present in nature. Also, the ball-and-string extreme is really just a special case of the jello view. So what this paper cannot do is establish "how proteins move". However, it can establish how a particular protein moves and also indicate what techniques to apply to answer the same question in other cases.

The particular protein being studied in this paper is adenylate kinase, which I will refer to as Adk, a highly conserved protein that is present in nearly all forms of life. Its function is to convert one ATP and one AMP molecule into two ADP molecules and vice versa. Adk has two interesting properties. The first is that it is an equilibrium enzyme, which is to say that it catalyzes the phosphotransfer equally well in both directions. This is a useful feature to have in NMR studies. Additionally, although it is highly conserved, the slight differences in sequence between Adk from the intestinal bacterium E. coli (mesoAdk) and the hyperthermophile A. aeolicus (thermoAdk) produce enzymes that have very different thermal stabilities and activities.

Adk is a single fold with three subdomains: a "core" and two "lids", one of which typically binds AMP and the other ATP. It has previously been shown that the opening of these lids is the rate-limiting step of Adk catalysis, for both mesoAdk and thermoAdk. More recent research, published in a separate letter to Nature last week, indicates that these lids open and close even when the substrates are not present. The question then is whether the motion of the lids more closely resembles the ball-and-string model or the jello model. Henzler-Wildman et al. modeled thermoAdk and mesoAdk fast-timescale dynamics in order to resolve the question.

I have shamelessly stolen their first figure for your benefit. The order parameter S² is shown on the structure of mesoAdk (A) and thermoAdk (B). Both datasets were taken at 20 °C, a temperature at which mesoAdk is very active and thermoAdk is not. Just to orient the non-NMR people, for a structured protein one expects most of the bond vectors to have order parameters between 0.8 and 0.9, so even the red residues in these structures are not really all that unusually flexible. Also note that the AMP lid is located at the right end of these structures (folding over at hinges 1-4) while the ATP lid is at the top (hinges 5-8). It should be immediately apparent that thermoAdk is more rigid than mesoAdk. A more subtle point is that many of the "hinges" identified from crystal structures have increased flexibility relative to their surroundings. Also, although the lids (especially the ATP lid) are more dynamic than the core, they retain order parameters characteristic of folded proteins.

This is in contrast to earlier research from the Meirovitch lab, which indicated substantial nanosecond flexibility in the ATP and AMP lids (LID and AMPbD in their description). The supplementary information for Henzler-Wildman et al. is freely available and contains a good discussion of the discrepancy. For those who find it too technical, it can be summed up as follows: the Meirovitch group improperly applied an isotropic global rotational diffusion model, and their SRLS results produce order parameters that are inconsistent with the domains being folded. Moreover, the correlation times they derive with their method appear to be too close together to be reliably distinguished with the NMR data they collected. The Henzler-Wildman results are consistent with the previous relaxation dispersion data for Adk and also with molecular dynamics simulations (contained in the paper). If Katie reads this, perhaps she can elaborate on the reasons why her data is superior. Commentary from the Meirovitch group is also welcome.

Let us will continue on with the paper. As I mentioned, there is a bit of MD in there which supports the NMR findings. What I found to be more interesting, however, was the way that the dynamics of thermoAdk reacted to temperature. Because thermoAdk melts at a very high temperature, it was possible to measure its dynamics all the way up to 80 °C, although glycerol had to be added to the solution at this temperature in order to bring the tumbling rate back down to a point where internal and global correlation times could be reliably separated. The results, which you will have to read the paper to see because I forgot to email myself the figure from work, indicate that as the temperature increases the order parameters decrease (expected) and become very similar to those of mesoAdk at 20 °C. This matches up nicely with the results of activity studies which indicate that the activity of thermoAdk at the high temperature is similar to that of mesoAdk at the low temperature. Because it is known that the lid opening is rate-limiting, and because local deformations at the hinges are required for the lids to open, it is likely that the increased fast-timescale flexibility of thermoAdk hinges at high temperature is directly related to the increased activity of the enzyme at that temperature.

The dynamic results in this paper seem to support the ball-and-string view of motions in Adk (by contrast, the Meirovitch results would suggest the jello view). Although the lids are not as rigid generally as the core, their order parameters are still within a reasonable range for a folded protein, and are for the most part higher than the order parameters of residues located at reasonably well-defined hinges. In Adk, motions on the μs-ms timescale appear to be enabled by flexible hotspots that lie between well-defined elements of tertiary structure, with the overall ensemble dominated by the two discrete endpoints. For this case and others like it, a combination of relaxation dispersion measurements and model-free analysis suffice to characterize the motions.

For cases which more closely match the jello view, many NMR approaches may prove difficult to implement. Relaxation-dispersion analysis of such motions is likely to be frustrated by endpoints that constitute a slim majority or plurality of the ensemble, high rates of interconversion between similar conformers, and possibly a low Δδ for these conformers. Fast-timescale studies will be difficult because the motions involved will be complex and may violate some model-free assumptions. In such cases, high-field measurements of R₁ dispersion (yes, I said it) may prove especially valuable as the dominance of ω_N in those measurements at least gives us a chance of distinguishing motions in the high-ns regime. Of course, the major difficulty for NMR will be if the jello motions result in intermediate exchange and excessive signal averaging — in these cases paramagnetic spin labeling or EPR studies may prove to be the only practical method.

Henzler-Wildman, K.A., Lei, M., Thai, V., Kerns, S.J., Karplus, M., Kern, D. "A Hierarchy of Timescales in Protein Dynamics is Linked to Enzyme Catalysis" Nature 450 (2007) p913-916.

Read the rest...

Tin Man: recycled

As Tin Man, the Sci-Fi Channel's "re-imagining" of The Wonderful Wizard of Oz entered its fifth hour, I found myself wishing that I was watching The Wiz instead. I don't mean that The Wiz was a better piece of cinema than the new mini-series, though arguably it was more visually inventive. However, it shared an important virtue with the book and the original movie musical that the present adaptation lacks: it knew what it was. The Oz story is not a sophisticated adult story about contemporary morality, it is a child's story about wonder and adventure. It can be turned into something more sophisticated, with enough effort, but that's a task at which Tin Man manifestly failed.

The presentation looked more sophisticated, I grant. But dressing people up in black leather does not, in fact, make for a sophisticated story. All this dark, steampunk set-dressing looks pretty cool, but it doesn't really fit with the theme of magical little girls who save their world with the power of love. The more adult visuals simply didn't mesh with the story that was presented. As a result, I felt a certain dissonance, as if I were seeing Strawberry Shortcake depicted as a dominatrix.

And while the story was unsatisfying on its own merits—a tale of a counterproductive MacGuffin fetch quest that heavily involves amnesia, of all damn things—it was even less satisfying as a re-imagining of Baum's original. When you re-imagine something, you ought to say something new, otherwise you're just making fan fiction. But Tin Man is essentially childish in its presentation of good and evil, and the only adaptations it makes to Baum's world are to insert stock elements from other sci-fi worlds and movies, as well as importing Nurse Ratched's hair. You can read Wizard of Oz crossovers at fanfiction.net if that's what you want; there's no reason to make a movie out of them.

Don't misunderstand me, Tin Man isn't bad. It looks nice, and the actors largely do a good job (although the child who plays little DG was not very good). The music leaves something to be desired, but doesn't offend. But Tin Man just feels unnecessary, a superfluous bit of fluff trying to staple a grown-up look onto a childish and irrelevant story.

The Sci-Fi channel is a curious contradiction. Most of its original series (at present, anyway) are actually pretty good, and their re-imagining of Battlestar Galactica may be the best scripted show on television. Their original movies, however, are largely pathetic drivel starring actors that have washed up on this shore after fading from better careers. And then there are the mini-series, which are typically of very high quality, probably because many of them are adapted from books. In this case, it seems, the movie quality bled into the mini-series. Although the production values of Tin Man were high, the writing was poor, and in the end, you'd be better off watching the original, rather than this fanfic.

Read the rest...

Child's Play, again... plus, game reviews!

I went over to the Child's Play site this morning to check out how things were progressing. Apparently, they're up to $450k, with several weeks left to go. If you haven't already, please add your own 5 or 6 bucks to the pot. Readers in North Carolina will be pleased to know that Children's Hospital of North Carolina (in Chapel Hill) now has their own wish list available. Coverage of the 50 states seems to be increasing daily, so if you're concerned with giving locally, check it out. Most of the eastern states have at least one hospital you can give to. Canadians, Brits, and our friends down under in Australia and New Zealand have hospitals on the list, too. If you don't want to give games or gaming systems, remember that there's also a huge demand for (non-absorbent) toys, books, board games, and DVDs. Just click on a hospital you like, or the PayPal direct donation link.

You don't have to like the Penny Arcade guys (hey, plenty of people don't) to like what they're doing. The recent game-reviewing spat (exacerbated by Gerstmann's suspicious firing) is really relatively tame compared to their past feuds with, say Jack Thompson. Anyway, I think it comparatively obvious that Gabe is right—game reviews have adopted the approach of newspaper movie reviews and devolved to being dominated by their metrics. This is the same phenomenon that creates those movie print ads that heavily feature 5-star raves and "two thumbs up". Some gaming companies adopt the same approach, touting 10/10 scores and the like, to the exclusion of giving any idea what the gameplay is about. Consider Kane and Lynch, for example, the game that kicked off the present controversy. The advertisements feature a bunch of cutscene violence that looks decent, so I know the game is about killing dudes with reasonable graphical fidelity. But what's the story? How does it feel to play the game? Why is 'killing dudes with reasonable graphical fidelity'—a property shared by no less than 50% of extant games—their marketing focus? This is different from asking why it was their development focus; that's a question for its own post.

The length and breadth of most gameplay, paired with the diversity of personal goals associated with a person's approach to gaming, calls for a more measured and less numeric approach. Prince of Persia: The Sands of Time is a fantastic platformer wedded to a charming story and a dull combat system. For the platforming enthusiast it is a game sent from heaven, but someone who loves RPGs, fighting games, or 3rd person adventures might justifiably hate it. Game reviewers need to consider an alternative approach to reviewing games, one that pays less attention to a dubious and corrupt quantification scheme and puts more effort into opening a window for the reader onto the experience of playing.

Read the rest...

Purity is death

Perhaps you have already heard about the 14-year-old Washington boy who refused extended blood transfusion treatment and therefore died (some additional info can be found here and here). Obviously, this is a terrible tragedy for his family, and I certainly hope his aunt is a true believer because if she is not then she'll have no solace for the fact that her ignorant superstitious nonsense killed him. The judge in the case, who upheld the boy's right to refuse the transfusions, will come in for a great deal of much-warranted scrutiny, but the fact of the matter is that it is the Jehovah's Witnesses who deserve the scrutiny and the blame for what has happened.

I think anyone reading about this will have a knee-jerk reaction that the judge in the case made the wrong decision. The AP quotes judge Meyer as saying that the boy understood the consequences of his decision, which may well be true, in an analytical sense. Young Mr. Lindberg probably understood that he would die, but there are very few 14-year-olds, let alone 14-year-old boys, who have a good appreciation of what that means. Death isn't something you understand unless you've spent some time around it, brushed up against it. And think of the teenagers you know. Would you trust any of them with life-and-death decisions? Hell, we don't trust 14-year-olds with cars. It's also true that the boy's parents, who did not have custody of him, wanted him to take the transfusion—could his relationship with them have played a role in his decision?

So Meyer can be justly criticized on the grounds that Lindberg was not competent to make that judgment, or that it wasn't Lindberg's judgment to make. However, it should also be pointed out that this was not some one-off transfusion that would instantly cure the boy. The treatment under discussion was a long course of transfusions that would run alongside the chemotherapy. And according to the doctors the prognosis was that he had a 70% chance of surviving the ordeal, with all the discomfort and side effects to boot. Being forced to undergo the treatment against his will would certainly make this harder on the boy, and on his doctors.

That said, I personally feel that Meyer should have erred on the side of curing the boy. Lindberg's decision was dangerous and self-destructive, and this should have indicated the opposite ruling. However, I wasn't present for the hearing, and the decision Meyer did make wasn't groundless. Maybe there was something in Lindberg's demeanor suggesting greater maturity than his age would typically indicate.

You'll note that I didn't say anything about the religious sensibilities. That is because I give them no weight at all. Lots of people dislike the Jehovah's Witnesses for a variety of reasons, but I've never been bothered by them; certainly not to the degree that I am bothered by other odious "Christians" living a life of hatred at maximum volume. So this is not a statement emerging from a blanket dislike: their attitude towards transfusion reeks of ignorance, superstition, and flat-earthism. The soul, if it exists, is not bound up in any bodily organ or fluid. Certainly people who have received massive blood transfusions have not absorbed someone else's soul—or at least I'm sure that didn't happen to me.

There is a bright line with religious beliefs, especially laws of practice: they're fine as long as they don't hurt anyone. We in America do not allow cannibalism or polygamy, although these are both religious practices with long histories. Nor are we tolerant of female genital mutilation, stoning people to death for violations of the laws of Leviticus, or human sacrifice, religious practices all. This indoctrination against lifesaving medical procedures is just as dangerous and fatal, especially when the subject is a teenager lacking in perspective and a diversity of life experiences. Judge Meyer made a mistake by allowing Lindberg to finish the deed, but it was the Jehovah's Witnesses and their purity practices that killed that boy. That ought not be allowed.

Read the rest...

Paul Davies, supplemental

There's another point, one that I skirted around in my last post, that I feel is worth coming back to briefly. Remember that Paul Davies' argument asserts that science relies on an assumption, and that therefore science is based on faith. I admitted that I have faith in the assumption, but not the same sort of faith that a person has in a God. What I didn't address was the unspoken leg of Davies' argument, namely that operating on an assumption is equivalent to operating on faith. This is not true, and it's important to highlight this because it shines some light on the difference between science and religion.

Let's imagine for a moment that we are engaged in a debate on some topic that is neither religious nor scientific. For instance, we are debating whether Hitler's attack on the U.S.S.R. was a sound strategic move. I say, "Assuming Hitler hadn't attacked Russia, he would have had more capacity to defend Europe." You agree to this assumption, for the sake of argument, and we continue the discussion from there. Would you say you have faith in the proposition that Hitler did not attack the U.S.S.R.? Surely you know it to be counterfactual, so you do not even believe it. So assuming a proposition doesn't necessarily mean you have faith in that proposition.

Of course, this may not be perfectly analogous—after all, the proposition about Hitler is clearly unhistorical. So suppose we were dealing with an uncertain proposition. For instance, two detectives might be attempting to solve a murder with two suspects. One way to go about it would be to assume that one of the suspects is guilty and examine the evidence to see if any of it is inconsistent with that proposition. Again, nobody would agree that the detectives have faith in a particular proposition of guilt as they are reasoning. The assumption is merely a stipulation, a point from which to begin thinking.

All right, so it's pretty clear that making an assumption doesn't require an act of faith, not even the modest, empirical kind of faith I was writing about yesterday. Of course, it might seem strange for a scientist to perform experiments on the assumption that the universe can be accurately described by natural laws when he does not in fact believe that it is so. It is important to realize, however, that a scientist's actual state of belief towards the natural law assumption is irrelevant to the process of science. Science proceeds on the basis of that assumption, and I have faith in that assumption, but I do not design or perform experiments because I have faith in the assumption. Rather, I have faith in the assumption because the results of my research have always proven to be consistent with it. But even if I did not believe the assumption—for instance, if I believe that God occasionally usurps natural laws to make "miracles" occur—I could still perform science, so long as my experiments were predicated on the assumption of universal natural laws.

This is a sticky point, for religious believers and atheist scientists alike. A researcher must stipulate the truthfulness of the natural law assumption to interpret an experiment, but it is not necessary for the researcher to actually believe the natural law assumption is true, just as it is not necessary for our historical debaters to believe that Hitler did not attack the USSR, or our detectives to believe that a given suspect committed the murder. Making an assumption is not an act of belief, or of faith.

This is a significant difference between science and religion. Religious activities depend not only on the assumption of their legitimacy, but also an actual belief or faith in their central propositions. One can conduct meaningful scientific activities without believing the natural law assumption, but one cannot perform meaningful religious activities without believing the religious assumption. One can, of course, go through the motions of ritual without believing, but then the activity ceases to be religious in nature and becomes purely social or political. It is a problem in a religion, perhaps even a mortal sin, to be a hypocrite, but for science, hypocrisy (in the sense of using the natural law assumption without actually believing it) is just a personality quirk.

The only time that faith in the natural law assumption becomes relevant is when we move from the scientific process to the question of truth. Then we can object to Davies' argument on the grounds that have been exhaustively described by myself and others.

Not that I expect anyone to actually listen. After all, more Americans believe in the Devil than believe in evolution.

Read the rest...

Damn you, Paul Davies!

Initially, I didn't want to make any comment on Paul Davies' recent NYT column "Taking Science on Faith". In fact, I didn't even want to read it. News of its existence came to me through comments on scientific websites, and from these alone I knew that actually reading the damn thing would sadden and anger me. But, I was asked about it at work, and the internet clamor just kept rising, and I decided I might as well give it a read on the off chance that it would contain something interesting. This was a foolish gamble. Davies is a lesser man for having written such garbage, the New York Times is a lesser publication for having printed it, and I am less intelligent for having read it, because now that trash is in my brain. After the manner of Sherlock Holmes, I shall endeavor to forget it at once, but first I want to explain precisely why it is garbage so that you, at least, will have the benefit of the rebuttal.

In a strict sense, this is unnecessary: several blogs have already put up excellent posts knocking down Davies' arguments. My own commentary is hardly fresh, then, but I don't really write to impress the internet at large. All the same, there are a number of points that I feel haven't been made, or haven't been made clearly or forcefully enough. So I'll try and handle those.

The foundation of Davies' argument comes early on in the piece and, I'll grant, does have a certain appeal. Davies writes "All science proceeds on the assumption that nature is ordered in a rational and intelligible way." This is almost true, and I think it bears some elaboration. It breaks down to three ideas, which I will take up out of order.

Idea 1: Nature is ordered.

More expansively, one could say that the universe is described by laws. This is an assumption of science, but it's important to understand that this does not mean determinism is an assumption of science. The quantum universe is probabilistic, not absolute, and though this probabilism matters most on the microscale, events at that level can have profound effects at higher levels. The scientific acceptance of randomness matters later on.

Idea 2: The order of nature is intelligible.

This is also an assumption of science. Obviously, if there were no way to identify the order of nature then there would be no reason to perform experiments.

Idea 3: The order of nature is rational.

I saved this for last because it is where Davies fundamentally goes wrong. In the most limited sense, this may be accurate: scientists resist the idea that two contradictory laws are both true. But this does not seem to be what Davies means. In fact, he's never very clear on what this means or why it's important, but he does seem to feel that rationality is at odds with randomness. Yet, as I've already pointed out, science can be perfectly comfortable with randomness, even if Paul Davies can't. He's not exactly in bad company: Einstein famously felt a similar discomfort with the probabilistic descriptions emerging from quantum mechanics. But the personal predilections of particular researchers do not speak to the necessary assumptions for performing science. To assemble a scientific enterprise one needs not assume anything other than that there is something to learn (idea 1) and that there is a way to learn it (idea 2). Nothing about the nature of what is learned need be assumed. Thus, we can discard idea 3 as a necessary assumption. So that leaves us with with what I'll call for convenience the 'natural law assumption':

The universe can be described by natural laws, and there exists some method whereby those laws may be learned.

It's important to remember that the natural laws here aren't normative, as moral laws or political laws are. A natural law is a codification of empirical observation, not a prescription of outcome. Observation of a phenomenon that violates moral or political laws results in steps being taken to alter the phenomenon, while observation of a phenomenon that violates a natural law results in steps being taken to alter the law.

Davies goes on to say that making this assumption is an act of faith, which is at best misleading, and at worst outright dishonest. I can quite honestly say that I have faith in the natural law assumption, but what I mean by this is essentially the same thing I mean by saying I have faith that my car will start tomorrow, or that I have faith that the 'pause' button on my remote will cause the DVD to stop playing temporarily. This relates to a faith in something that is built on repeated success, not something that I preserve for its own sake. If my car fails to start tomorrow morning, I will not continue turning the key indefinitely, my faith in the inevitability of its starting intact. Rather, I will have it towed. Similarly for the remote. If natural laws failed to describe the universe, then I would give up on them, and why not? They're not helping my goal, which is to understand the universe. In short, my "faith" in the natural law assumption is an empirical expectation that it will be fulfilled.

This attitude would be utterly alien to a believer in a religion. Indeed, almost every religion contains admonishments not to give up on belief in hard times. The power of Job and similar works derives from the strength of a person's faith in the face of great troubles. The "Faith" of the religious is a metaphysical belief in the existence and intent of a supernatural entity.

Davies treats these meanings as equivalent, or at the very least expects his readers to conflate the two different meanings in this way. Yet Davies is clearly aware of the distinction, as the beginning of the column shows. If he has forgotten it by this point, then he is simply leading himself down the garden path with semantic sloppiness, and dragging us with him. If he remembers, then he's being dishonest.

Davies then goes on to describe his dissatisfaction with the idea that natural laws arose randomly. I've already pointed out that this is a personal problem of his, not a problem generally for science. But Davies is insistent, and points out that we really should be concerned about this because it just so happens that the natural laws were perfectly tuned for life, and it seems unlikely that this would just happen by chance. This is amazing stupidity.

The natural laws of the universe are not tuned perfectly for life, and we should suspect this, if for no other reason, because there is so little life and so much universe that is utterly, relentlessly hostile to it. If some transcendental entity was sitting at the control panel at Creation, with a mission to create a universe in which life would have a really good chance of existing, then he certainly did a terrible job. Vast, cold voids of space, impossibly hot fusion engines, a universe permeated with gamma-ray bursts sufficiently strong to burn a planet's atmosphere right off... these are not features hospitable to life. In fact, given the natural laws of our universe, life seems so cosmically unlikely that on that basis alone an entire cottage industry of science quacks makes a living arguing that life could only have arisen with the help of an omnipotent God.

"But but but," Davies and his ilk will argue, "if you tweaked any of the constants just a tiny bit, then life couldn't exist!" Such a statement is staggeringly unscientific—you couldn't possibly have any direct evidence for it. I will grant that, as a thought experiment, one might effectively argue that lifeforms such as ourselves might not survive in a universe where the gravitational constant were a little higher, or the vacuum permittivity constant were lower. Yet, to conclude from this that life is impossible is just evidence that you ignored what I said above. The universe we have is incredibly inhospitable to life, and yet here we've got a whole damn planet covered in it. Life, even life of our kind, might make its way in even less hospitable environments. And even if it couldn't, who knows what alternate kinds of life or intelligence might arise in vastly different universes? In such alternative existences, energy itself might be alive, and stars might recite poems to each other. We can say with confidence what existing structures of our universe would be abolished were a particular natural law to change, but how can we predict what new opportunities for life might emerge?

It's no surprise that we would have a hard time were the physical constants and natural laws of the universe to change. It is, however, idiotic to conclude that this is because the universe was tuned for us. We were tuned for it, because it is within this universe that we came to exist.

Davies then concisely explains why the question of where natural laws come from is not a scientific question, without realizing it. Shoved in there with the multiverse mumbo-jumbo is a brief moment of clarity when he realizes that the problem is turtles all the way down. Any explanation of the origin of natural laws necessarily requires a description of the natural laws governing that origin. Which then necessitates a description of the origin of those natural laws, and the natural laws governing them, and so on and so forth through turtle after turtle until we realize there is no bottom, or we reach the layer of elephants.

Of course, this is not a necessary consequence of assuming the existence of natural laws. Rather, it is a consequence of assuming causality. But quantum mechanics appears to be quite at home with uncaused events, and there is no special reason to believe that natural laws must be caused. Davies seems to believe this, but at this point I hope you are at least a little skeptical of his judgment in the matter.

Davies then concludes that religion and science are both founded on faith. I've already pointed out that the truth of this statement relies on the conflation of two totally different meanings of 'faith'. But even if Davies is correct, this is hardly a catastrophic failing. The world abounds in demonstrations that faith in a benevolent, all-powerful God is ill-placed—the faithful of all religions suffer death, disease, and horrible agony, even in their moments of worship. The wicked are everywhere rewarded, the virtuous everywhere trampled upon, and their prayers go unanswered. Oh sure, the jock on TV thanks God for the opportunity he was given, but what about those other 100 million kids who never made it to the NFL? Didn't they pray, too? Did God love those boys less than he loved Pacman Jones, or did he just have nothing to do with it?

The natural law assumption, by contrast, has proved to be phenomenally successful. In fact, it may be the single most successful idea in the entire history of mankind. Virtually everything that surrounds you as you read this—the clothes you wear, the treated air you breathe, the chair you sit in, the materials in your building, the computer you are looking at—is the fruit of this amazing idea. Even if we are dealing with equivalent kinds of faith, then it certainly seems you have substantially more reason to place your faith in the existence of natural laws than in the existence of God.

Davies then goes on to make much of the fact that the concept of natural law emerged from the concept of a deity. While this is quite true, it doesn't say anything essential about science. Science existed before the first codifications of "natural laws" and has advanced substantially since. The clockwork universe of the Enlightenment died with the Heisenberg Uncertainty Principle, and on the theological side, the watchmaker God has faded as well. So Davies is technically correct here, but he's babbling on about irrelevancies.

Until, that is, he comes to the end of the paragraph and says:

physicists think of their laws as inhabiting an abstract transcendent realm of perfect mathematical relationships.

I certainly hope he does not believe what he is writing here. Natural laws cannot 'inhabit' any 'realm' because they do not exist. They are not things. The representations of natural laws exist, as patterns of ink on paper, patterns of electrons on magnetic media, patterns of neural connections in human brains. But there is no thing you can point to and say "That is a natural law" any more than there is a thing you can point to and say "That is a 'That wall is brown'". Natural laws as we discuss them are ideas without physical existence—they are nothing more than shared descriptions of observations. Natural laws as they appear in nature are just properties of the universe—they are not separate from the matter and energy we presume them to govern. John Wilkins has a really good explication of this reification fallacy.

Once you see the mistake that Davies is making, the next paragraph evaporates, leaving a faintly unpleasant smell. His analogy is not appropriate because whereas religion imparts reality to its God, science does no such thing for its laws. Certainly the laws have no reality apart from the universe (as God supposedly does), and to claim that there is something analogous between the theological relation of God to the universe and the scientific relation of natural laws to the universe is to either completely misunderstand what natural laws are, or to engage in a willful deception.

Davies' closing to the article tumbles out of his assumptions, and follows them into the same black pit of shame. The whole article is an embarrassment—to Davies, to his "Beyond" institute, and to Arizona State University. What I find most infuriating about it is that I will now be forced to endure endless rehashes of this sloppy, misleading mess at the hands of creationists, flat-earthers, and alterna-quacks. "Science is faith!" they'll cry, "A scientist said so!" and then I'll have to remember the conflations and fallacies and patiently explain them, over and over again, until the day I die. So damn you, Paul Davies! Damn you to the abstract transcendent realm of perfect mathematical relationships!

Read the rest...

Lab courtesy haiku

In the bastardized American format...

Agar will gel up,
vile gray sink-lake develops.
Next time, use trashcan.

Technically, they're not
my Pipetmen either — that
does not make them yours.

Reducing agents,
like open sewers in lab —
put the cap back on.

Autoclaves melt agar;
now your waste is everywhere.
Remember, use trays!

It's called a Bunsen
burner for a reason — don't
leave unattended.

Foetid, abhorrent,
vile... your unbleached media,
or eldritch horror?

Seriously, man,
clean centrifuge after use;
it smells like a morgue.

Needles go in "Sharps
container", not underneath
the paper towels.

Floor grabs onto shoes;
try cleaning up spills as soon
as they happen, please.

Crystals belong in
screening trays, not pumps — wash with
water every time.

No, seriously,
I meant that about needles:
dispose properly.

Beware: grad students
will eat anything left out
in your lab's breakroom.

Water bottles and
computers — two great things that
ought not be combined.

This ain't no disco,
You ain't no DJ. This ain't
no foolin' around.

So I was a little bored today.
Read the rest...

One more thing to blame dad for...

Not all papers are revolutionary—some merely provide an entertaining test of common assumptions. A recent paper by Taylor, Wedell, and Hosken in Current Biology is a case in point. One assumption of evolutionary biology is that males who are attractive mates produce attractive offspring; this is supposed to underlie evolutionary developments such as the peacock's tail. Despite its intuitive appeal, this principle has not been put to the test very often. Taylor et al. made use of the fruit fly Drosophila simulans to investigate whether the assumption held up.

Their experiment was relatively simple. Isolated male flies were bred with 2-3 female flies, and the copulation latency, or the time between pairing and successful mounting, was recorded. This is a reasonable measure of male attractiveness because in D. simulans the female is in charge of the sex event—a male cannot copulate with an unwilling female. Granted, this was something of a desperation measure, the question being asked is, essentially, "If this was the last guy on earth, how long would it take for him to convince you to sleep with him?" A more convincing demonstration might have been to have females choose from different males, but as I am not a fly geneticist I'm not sure whether one could set up such an experiment. Additionally, it seems possible to me that the copulation latency might be affected by the attractiveness of the female, but I am not sure whether male flies are choosy. At any rate, the experiment might have been improved if repeated experiments with different females had been performed for each male to minimize bias arising from female attractiveness. This was done in the first part of the experiment, but not the second.

After maturing in isolation, so as to eliminate the possibility of learned behaviors affecting the results, the male progeny from these breeding events were isolated with females from a different population. The copulation latency was again recorded (no word on whether alcohol or dance music were supplied to help get things going). From these results, the authors concluded that the attractiveness of the father, as measured by the latency, contributes significantly to the attractiveness of the sons.

The authors did not provide much information on the nature of heritable features that give rise to attractiveness in their fruit flies, except to exclude body size as a factor. I myself don't really know what the Brad Pitt of the fly world would look like, though maybe cousin Kathy has some idea, but wingspan, pheromones, and maybe some kind of auditory factor could all contribute and conceivably be heritable and account for the observations. A knowledge of the specific means by which "attractiveness" is transmitted isn't relevant to the larger question of whether attractiveness is heritable, but matters when one wants to know whether the results in this model system can reasonably be extended to, say, birds or mammals.

Does this experiment tell us anything about the transmissibility of attractiveness in humans? Not necessarily. Mating behavior in humans is as much bound up in learned social behaviors as with unlearned biological ones. Many genes with the potential to result in male attractiveness are certainly heritable, but the diversity of lifestyles among humans means that the actual development of physical attractiveness from these potentials is uncertain. External non-heritable features (wealth, education, musical talent) also contribute significantly to human mating, which further complicates the issue. So I guess Dad is off the hook for this one, at least for the time being.

The article itself is relatively bare-bones and does not include any figures of raw data, which is something I never like to see. I don't mean to impugn the authors—the journal may have decided not to make the expenditure. I think it would have been beneficial to see the correlation, and I can't imagine any scientific reason not to include such a figure. Maybe it's some biology thing.

Taylor, ML, Wedell, N, and Hosken, DJ. "The heritability of attractiveness." Current Biology. 17 (2007) pp. R959-R960.

Read the rest...

Seven-score and four years ago...

...on November 19th 1863, one of America's greatest presidents gave one of America's most famous and influential speeches. Preceded by a masterful 2-hour oration—now practically forgotten—by Edward Everett, these remarks lasted a mere two minutes. The reaction at the time was muted, though some were surprised at the brevity of the speech. Of course, by the standards of the modern sound-bite, the Gettysburg Address is positively long-winded:

Four score and seven years ago our fathers brought forth on this continent a new nation, conceived in Liberty, and dedicated to the proposition that all men are created equal.

Now we are engaged in a great civil war, testing whether that nation, or any nation, so conceived and so dedicated, can long endure. We are met on a great battle-field of that war. We have come to dedicate a portion of that field, as a final resting place for those who here gave their lives that that nation might live. It is altogether fitting and proper that we should do this.

But, in a larger sense, we can not dedicate—we can not consecrate—we can not hallow—this ground. The brave men, living and dead, who struggled here, have consecrated it, far above our poor power to add or detract. The world will little note, nor long remember what we say here, but it can never forget what they did here. It is for us the living, rather, to be dedicated here to the unfinished work which they who fought here have thus far so nobly advanced. It is rather for us to be here dedicated to the great task remaining before us — that from these honored dead we take increased devotion to that cause for which they gave the last full measure of devotion — that we here highly resolve that these dead shall not have died in vain — that this nation, under God, shall have a new birth of freedom — and that government of the people, by the people, for the people, shall not perish from the earth.

This speech is justly famous. In a paltry two minutes Lincoln conveys the futility of the ceremony, the enormity of the preceding sacrifice, and the resolve to which that sacrifice ought to move the audience. The point is communicated clearly, forcefully, and with emotion. Importantly, something of substance is said.

Modern political communication, on the other hand, is almost stringently dedicated to not saying anything. Tired talking points and empty blandishments are repeatedly trotted out to an ever more disconnected and disinterested audience. Press agents and campaign materials label as "bold" and "visionary" candidates whose speeches inescapably depict them as timid and insipid. Why have so many reacted so strongly and positively to Ron Paul, even though he is undeniably one crazy motherfucker? Because he says something. Maybe it's crazy, but at least there's an underlying message aside from "I am trying to offend as few people as possible". Clinton, Obama, Giuliani, and Romney are people, not robots, so they must have some actual point of view. Why not express it?

It's fair tor criticize modern political discourse for its dedication to the 15-second sound bite, but that's not sufficient. Brevity in moderation is not injurious to discourse per se, but it is if nothing of substance is conveyed. In just two minutes you can say something, perhaps even something that's important and immortal. The problem with today's politics isn't the length of a sound-bite, it's the emptiness of both the words and of the ideas they are meant to express.

Read the rest...

Gamers 4 Health?

Gamasutra has reported that the Robert Wood Johnson foundation has launched a new initiative geared towards adapting video games for health purposes. Just two or three years ago this would be almost completely laughable—at that time, only games with large peripherals like Dance Dance Revolution offered a reasonable chance of improving health during play. In that era, a plan like this would either be geared towards developing purpose-built health gaming systems or creating health-education games. It's possible that some portion of this money will in fact go to the production of cheap flash games based on eating the right number of vegetables. We may even see the long-awaited Salad Master appear. However, the advent of the reasonably-affordable Wii, which has physical activity built into its control system, puts the use of games for health improvement into the home.

But why use games for this purpose? The press announcement includes a key rationale:

Computer and video games are an increasingly powerful medium, providing engaging ways for people to learn, be entertained, and connect and communicate with each other. The industry brings in more than $7.4 billion in annual revenue; nearly 70 percent of American heads of households play computer and video games, the average age of players is 33 and more than 40 percent are women. There is enormous opportunity to apply the power of this interactive medium to pressing health and health care challenges.

There's a lot of useful information in this little quote that suggests productive avenues. Now, games produced for the PC as yet cannot include a significant amount of real motion without costly purpose-built peripherals. "Health games" for that market will mainly consist of the flash-style games I mentioned earlier. Frankly, in my opinion there is no space in the adult market for health-education games. While I do not disagree that complex and useful information can be conveyed by a game, to effectively communicate health information to an adult audience without making them feel like they are being talked down to is a task to which most interested developers are probably unequal. Health-education messages would have to be sneaked into more mainstream games for this approach to be effective.

Moreover, PC gaming is a mostly sedate experience. You are not moving around, and to create games in this platform runs counter to the overall aim. A more focused and effective approach would be to create games that involve significant amounts of upper/lower body motion for the Wii, or for the XBox and PS3. Some of this program will hopefully go towards developing health-game peripherals for the latter consoles. However, in the near term the best bet is the Wii, for a variety of reasons. First, of course, is the built-in physicality of game control. Also, the lower price-point and the accessibility of this console make it a more attractive choice for gamers with families, which will include many of the gamers in the age bracket targeted above. Additionally the Wii (and the XBox) have a built-in way to distribute simple low-cost games directly (over their respective download systems) without the typical costs associated with game publication. Hopefully the PSN will equalize this factor.

Exercise via Wii should not be assumed to be automatic. Although I am reliably informed that doing so makes one a toolbox, one can play Wii games using only small wrist motions while sitting. This suggests that games intended to produce physical activity should also include compensation to decrease the sensitivity of the Wiimote, forcing more expansive motions.

Some fragment of this "health games" program should be dedicated to developing appropriate accessories. A weighted sheath for the Wiimote, for example, could increase the physicality of a game without interfering with control. Alternately, weighted, reinforced gaming gloves that minimize the coverage of the palm and restriction of the fingers could improve the workout and diminish the incidence of "Wii wrist". The one thing that's missing from the Wii is something to do with your feet. One could, however, develop peripherals to deal with this. For instance, one could make a motion-sensing ankle strap (weighted even) with a long cable to reach the Wiimote. I'm not sure there's any reason why one couldn't develop a game in which one person uses two Wiimotes with such peripherals to enforce full-body motions. Head-to-head would require 4 wiimotes in that case, however, which starts to increase the expense. Online play to the rescue?

As far as the design of workout games, aside from the typical quality issues, the time required to play should also be taken into account. Typical sports games typically require something like 10 minutes of continuous play. Based on the intensity of the activity, doctors and kinesiologists should calibrate gameplay so that it comes in the right duration—possibly something like 15 minutes for intense play, 30 minutes for light-to-medium intensity. The duration of a gameplay unit should also be calibrated for the intended audience: a 33-year-old father of two probably can't sit down to play for three hours every night, or even manage that 3 times a week. Games in which 30 minutes to an hour of play are enough to actually achieve something would be optimal; people who have more time can always continue.

A really clever person could also go beyond titles that are explicitly about the activity and exercise to create a title that tells a story and simultaneously achieves a physical end. The pace of the game could be modulated to encourage the right kind of exercise behaviors. For instance, an early part of each game segment could be devoted to motions that encourage stretching, build into motions that constitute the workout, pursue those motions for a calibrated workout period, and use cutscenes and puzzle segments to enforce breaks or a post-workout cooldown. Hack-and-slash adventures or RPGs could be easily adapted to such an approach, or one could use the classic shooter as a model. Using an episodic content download distribution system would keep it fresh for the player, and ensure a continuous revenue stream. Health education could even be incorporated into such a game.

The main point I'm making is that there should be a focus here that goes beyond creating a game that's "just" exercise. Game designers are some pretty creative people—don't come to "health gaming" with the idea that all of it has to be Wii Sports or even Rockstar's Table Tennis. Intense physical activity can also be built into games that tell a story. Doing so might even be an advantage, especially in reaching people who don't like the idea of working out, or who are very busy but don't want to have to choose between exercise and entertainment.

Again I'm talking about the Wii mostly, but there's no reason for the initiative to limit itself to that in the long run. Because it makes use of a two-hand grip, the sixaxis isn't as conducive to large motions as the Wiimote is, but the existing Playstation Eye could possibly be adapted to exercise use. And, of course, there are always purpose-built peripherals, though these on top of the ~$400 you need for the hi-def consoles may not be as much of a broad-based winner.

Also, of course, dedicated systems for creating exercise games could also work, but I feel they will be less effective. Creating an expensive, purpose-built whole-room video game system that pushes athletic activities may draw in a few people who otherwise wouldn't join a gym, but mostly such a system will only reach people who are willing to go to a gym anyway. That doesn't "grow the audience" for exercise. It's much more important to reach people in their homes, where they feel most comfortable. Also, you don't want to make people choose between outcomes if it's not necessary. If you create a conflict between entertainment and exercise there's a pretty good chance they'll choose the former. If you create synergy between these possibilities and mix exercise and entertainment in the same machine then your audience grows.

I think an initiative of this kind is a really good idea as long as the administrators don't tie themselves into archaic or stale game styles. A flash game about colon cancer might be informative, but it probably won't be enjoyable or entertaining, and besides it will encourage lethargy. A tennis game for Wii might give someone a workout, but hey, they already have one. The effort here needs to be dedicated to ways to make existing games into exercise (by desensitizing the Wiimote, adding weighted accessories) and to develop new modes to make future exercise games better by calibrating their durations and taking the gameplay into directions that aren't explicitly sports.

Read the rest...

Allosteric interactions of PDZ domains

Another article entering the preprint stage last week also has some history behind it, although it doesn't have quite the wayback machine mojo of the last articles I discussed. A collaboration between researchers at Radboud University Nijmegen and the University of Pisa produced a really interesting story about the protein tyrosine phosphatase PTP-BL (citation 1 below). Like many phosphatases, PTP-BL is a large, multi-domain protein; it is typical to view these domains as independent functional units and see their conjunction in a protein as just a form of scaffolding. In the case of PTP-BL, however, it appears that the domains interact with each other, and that this has consequences for their binding specificity. In addition, their results agree with previous work identifying allosteric networks in the PDZ domain.

However, the interesting part of this story starts almost a decade ago with a bioinformatics experiment carried out by Lockless and Ranganathan published in Science (citation 2 below). Standard bioinformatic analysis of homologous proteins involves aligning their sequences based on similarity and looking for residues which are conserved across evolutionary time and space. It is generally believed that invariant residues are essential to either the structural integrity of a given protein fold or its function. Lockless and Ranganathan decided to take this a step further and ask which residues were co-conserved with a particular amino acid position, that is, whether amino acid changes at some position X are correlated with changes at some other position Y. They performed this experiment using the PDZ domain, a very common ligand-binding domain that appears in multiple proteins (and often in multiple copies within the same protein) in nearly all eukaryotes as well as bacteria. As the target of their co-conservation analysis, they chose a histidine in the binding cleft of PDZ.

Naturally, one would expect other residues within the binding site to show co-conservation, and this is indeed the case. The surprise, however, comes from the fact that in addition to these expected residues, an additional patch of residues on the opposite side of the domain also appeared to be co-conserved with the histidine, as shown in their figure at right. The histidine in question is residue 76, and co-conserved residues are rendered with pinkish molecular surfaces. The peptide bound by this particular PDZ domain is shown as yellow sticks. What you can see here is that there appears to be some linkage in an evolutionary sense between residues in the active site and residues in the β-strand structure on the other side of the protein. Lockless and Ranganathan did some binding studies that seemed to support their findings, and attributed this apparent pattern to structural perturbations.

A few years later, NMR virtuoso Ernesto Fuentes performed an NMR dynamics study on the second PDZ domain of human protein tyrosine phosphatase 1e (citation 3 below), in which he compared side-chain dynamics of the free (isolated) domain to those of the protein in a ligand-bound state. The results of that study are remarkably similar to those of the Lockless and Ranganathan work, though not identical. The ligand is shown in green, while side-chains of dynamically-responding residues are shown in red, yellow, and blue. As expected, most of the dynamic changes upon ligand binding occur right next to the binding site. However, two distal surfaces of the domain also appear to feel dynamic effects from the binding of the peptide. This gives even more direct evidence of some kind of allosteric interaction between the binding site of a PDZ domain and parts of the protein that are further away.

The new paper by van den Berk et al. puts a kind of exclamation point on this story. Their effort began, essentially, as a fishing expedition to find what peptides exactly the various PDZ domains of PTP-BL—it has five total—bind. Among others, they found that PDZ2 would bind to peptides from APC (binding site -VTSV) and RIL (binding site -VELV). This was the case when the PDZ2 domain was tested alone. When a construct containing both the PDZ1 and PDZ2 domains was tested, however, the RIL peptide no longer bound to PDZ2. This was true whether or not the 200 amino-acid linker between them was included in the construct, indicating that a bona-fide interaction between PDZ1 and PDZ2 was responsible.

van den Berk et al. then used NMR chemical shift perturbation mapping to identify the binding site of PDZ1 on the PDZ2 domain. They found, in what should hardly be a surprise at this point, that the primary site of interaction is a distal surface of PDZ2. On the basis of modeling studies they suggest that the mobility of Ile 48 is critical to enabling the binding of the bulkier RIL peptide; they attribute the allosteric effect of PDZ1 to a restriction of PDZ2 Ile 48 so that it cannot move out of the way and allow RIL to bind. Thus we come to the model at right. Note that van den Berk et al. did not, as far as I can tell, determine what part of PDZ1 binds to PDZ2. It's possible that the normal binding cleft is used, but the binding site on PDZ2 looks like a broad hydrophobic surface rather than a narrow structure that could easily insert into a binding cleft. This suggests that the binding site on PDZ1 is still available, and thus that peptide binding to PDZ1 could fine tune the behavior of PDZ2.

The functional importance of this change in affinity is not yet clear. RIL is also bound by another PDZ domain in PTP-BL, so the PDZ1-PDZ2 interaction does not abrogate RIL binding. Additionally, even in the absence of PDZ1, APC has a higher affinity for PDZ2 than RIL, so the PDZ1-PDZ2 interaction is not really switching the target of PDZ2 or anything. However, if the local concentration of RIL is significantly higher than APC, improved specificity for APC may be necessary for kinetic reasons. Alternately, the improved APC specificity may be an incidental feature of an interaction that evolved for another reason, perhaps to bring APC into close proximity to some protein bound to PDZ1.

The possibility that allosteric communication pathways might exist in small protein modules like the PDZ domain was initially met with a great deal of resistance. The classic descriptions of allosteric and cooperative interactions all involved very large protein oligomers. Dynamics experiments like those carried out by Fuentes et al. and functional studies such as this one, however, have borne out the predictions of the bioinformatics studies. Allostery, or the potential for allostery, is a feature of small, isolated domains just as it for large protein assemblies.

Another point to keep in mind out of this paper is that it is a mistake to assume that domains within a protein are completely independent. It turns out that this is often the case, that, for instance, a binding domain and a catalytic domain exist together without really interacting, and when that happens it's not a problem to analyze each domain separately. However, domains within a protein can and do interact with one another, even when they are separated by sizable linking regions (the linker here is >200 amino acids). Often the easiest (and sometimes the only) way to investigate the structure of large proteins is to look at their domains individually. This paper, among others, is a reminder that this strategy is not always appropriate and may fail to capture important features of the domains' functions.

(1) van den Berk, LCJ, Landi, E, Walma, T, Vuister, GW, Dente, L, and Hendriks, WJAJ. "An Allosteric Intramolecular PDZ-PDZ Interaction Modulates PTP-BL PDZ2 Binding Specificity." Biochemistry ASAP (2007)

(2) Lockless, SW and Ranganathan, R. "Evolutionarily Conserved Pathways of Energetic Connectivity in Proteins." Science 286 (1999) p. 295-299.

(3) Fuentes E.J., Der C.J., and Lee, A.L. "Ligand-Dependent Dynamics and intramolecular signaling in a PDZ domain." J. Mol. Biol. 335 (2004) pp. 1105-1115.

Read the rest...

Fit H5N1 with oseltamivir resistance

I noted in a previous post on the persistence of oseltamivir in the environment that most known mutations conferring oseltamivir resistance also diminished the infectivity of H5N1 influenza. Unfortunately, new research seems to have proven me wrong on that point. The good folks at Effect Measure have a post up about a recent paper from researchers at St. Jude's indicating that at least some of the mutations that confer resistance to tamiflu preserve neuraminidase activity. Check it out.

For those of you at academic or industrial institutions that have online journal access, read the article:
Yen HL, Ilyushina NA, Salomon R, et al. "Neuraminidase inhibitor-resistant recombinant a/vietnam/1203/04 (H5N1) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo." J Vir 2007:81(22); 12418-12426.

Read the rest...

Can MD match model-free?

As I mentioned in my last post, a major challenge in the interpretation of protein motion has been the poor correlation between dynamics information arising from computer molecular dynamics simulations and NMR relaxation experiments. MD simulations complement the order parameters of the model-free formalism by providing detailed descriptions of motions that model-free parameters describe in a general way. However, the output of an MD simulation is only to be trusted if its specific model matches the experimental observations from NMR. Historically this has not been the case, especially when it comes to the description of side-chain motions.

Rafael Brüschweiller's lab has for several years been engaged, with some success, in an effort to improve MD simulations to the point where they can predict S² values that match NMR results for side chains. While a cold-eyed analysis of the correlations they've obtained so far (r values near 0.6) might not be very favorable, the comparisons aren't that bad. Their most recent communication to JACS (citation at the end of the post), appearing online last week, displays a marked improvement in the correlations between simulation and experiment. The r values are still not that close to 1, but the current results appear to be a significant step forward.

So, what was done differently? Showalter et al. use an altered version of the AMBER99 forcefield that has a modified dihedral angle potential. This had good results in simulating the dynamics of backbone amide moieties, although MD has historically done a reasonably good job with these anyway. In this communication they simulated the side-chain motions of calbindin and compared them to experimental values. They calculated spectral densities J(ω) from their simulated correlation functions, though they are required in this case to make use of experimentally determined molecular correlation times. They do a strikingly good job of predicting the J(ω) at the Larmor frequency of deuterium and also at twice that frequency (figure filched from paper):
This really is an amazingly good job. Yet, as you can see from their figure 2 (a part of it is at right), the S² values they obtain aren't very close to those that are derived from NMR experiments. This is also reflected in the relatively poor agreement at J(0) (r=0.86). This seems to be very odd, because the magnitude of the spectral density at J(0) is very strongly dependent on τ_m, which they took from an NMR experiment. As is evident from the model-free expression for J(ω), the order parameter scales this term. Keep in mind that τ_m is typically on the order of 10^-9 seconds while τ_e is on the order of 10^-11 seconds—this means that the second term in the spectral density expression is negligible at J(0). Because they took their τ_m from experimental data, the decreased correlation at J(0) indicates that their correlation functions converged to inaccurate values.

Giving the data a once-over, it appears that their fitted order parameters were mostly high. It's not clear to me why this should be so, except that over-constraint of the backbone may be affecting the side chains. From the supplementary information it appears that fits of backbone order parameters were also slightly higher in MD than experiment.

The most notable failure is not much help because it missed low. The significant outlier in the J(0) plot is threonine 45, shown in orange at right—this figure is made from PDB structure 3ICB, which was used in this simulation. The correlation function for this residue fails to converge, largely due to sampling of an alternate ψ angle. This behavior is consistent with the observations of low order parameters in that particular loop of the protein. From the crystal structure it appears that the hydroxyl moiety of Thr 45 is capping a helix (blue). It's possible that the misbehavior of this particular residue is due to some miscalibration of the force-field that doesn't accurately capture this capping interaction. The altered backbone dihedral angle potential may be overwhelming the hydrogen bonding interaction, resulting in the aberrantly low J(0) fit for this methyl group.

In general, the simulations for threonines and valines were not as accurate as those for other types of residues, which seems a little strange. These were also unusual in that they missed low, while, as I mentioned, on average residues tended to miss high. The branched nature of these amino acids causes some steric interactions with the backbone, so one would expect an improved potential to help these residues the most. However, if the altered potential is causing unwarranted excursions from the equilibrium structure, as seems to be the case with Thr 45, then valines and threonines, whose motions are at least partially controlled by steric interactions with the backbone, might be the most strongly affected.

I should stress that it's not necessary for the simulation to produce too much backbone motion to get this result. If the backbone dynamics are the wrong kind of motion that could have this effect even if the model-free parameters for the backbone derived from the simulation appear to be accurate.

It isn't terribly clear why an improved backbone potential should increase the correlation of side-chain order parameters between MD and NMR. Showalter et al. venture no explanation, and my own research and that of others hasn't shown any particular linkage between backbone dynamics and side-chain dynamics, except in the case of alanine residues. It may be that a more accurate depiction of backbone motions contributes to a more accurate dynamic environment generally. Or, the motions of the backbone and side chains could be related in unexpected ways. An in-depth analysis of the simulation probing for these correlations could be very instructive. Regardless, these results are a significant, encouraging step towards using MD simulations to interpret the findings of NMR dynamics experiments.

Showalter, S. A.; Johnson, E.; Rance, M.; Bruschweiler, R. "Toward Quantitative Interpretation of Methyl Side-Chain Dynamics from NMR by Molecular Dynamics Simulations" J. Am. Chem. Soc. (Communication); 2007;ASAP Article.

Read the rest...

The model-free dynamics formalism of Lipari and Szabo

I've seen a lot of really interesting articles in the past several days, though as always I admit that many things I find fascinating are sleep aids to others. I was going to write about the most esoteric article first, but as I started writing out an explanation I realized I needed to split things into two posts. The next post will be about an article I read last week in JACS ASAP pre-publication. But this post is about an article that appeared in JACS when I was about four years old. In it, Giovanni Lipari and Attila Szabo introduced a way of describing the dynamic information obtained from NMR so useful and successful that it has not been supplanted even after a quarter of a century. In addition, the article is really wonderfully written, and if you are a professional then I highly recommend reading it (the citation is at the bottom of this post). If you are not a professional, then read this post instead.

Regrettably, it is impossible to talk about this without using mathematics, but I promise you will not be forced to endure anything taxing, just exponents and multiplication. The Lipari-Szabo model-free formalism derives much of its power from its relative simplicity, so I promise this will not be too painful, and for those of my family who are interested, you will learn what all that junk I talk about means. Practicing NMR spectroscopists will notice a few differences in terminology and some (hopefully slight) conceptual fudging in what follows, but hey, you should be reading the original article anyway.

So. Let's imagine we have a really simple chemical system, like a single bond between two atoms, which we will call A and X (see right). Now, unless our AX compound is very, very cold it will be moving around all the time. Exactly how fast it moves will depend on what is around it (AX moves quicker through water than through honey) and what temperature it is (AX moves faster when warm), but unless we are at absolute zero, it will move. We can imagine two kinds of motion. AX can move forward or sideways: this is called translational motion, and having mentioned it, we won't worry about it anymore. In fact, we will temporarily pretend that it does not even happen. Also, AX can tumble—for instance, it could rotate around some point in the center of the bond, or swing around like a clock hand, or wobble at the ends (as shown). This is the motion we will consider.

Suppose you had a system like this and you wanted to come up with a way to show how it was moving. Well, one thing you could do is draw a picture or make a movie showing how quickly it tumbled. However, this would be a pretty awkward way to convey the data and might not be very informative. It might be better to try to come up with a single number that tells us how much AX has tumbled. Well, one way to do that would be to monitor AX over time, starting at some random time i, and compare the bond vector at any time i+t to the bond vector at time i. We could assign to any time t a number representing how likely it is to find AX in the same orientation at t that it had at i. Obviously, early on, this probability would be very high, and the larger t got, the smaller the probability would get. An example graph is over there on the left. This is called a correlation function, because it represents the correlation between the AX orientation at time i and the orientation at time i+t.

Obviously, for any real system the correlation function will be bumpy and difficult to represent mathematically. However, it turns out that we can approximate a correlation function reasonably well using an exponential decay, that is, a function based on a negative exponent of Euler's number. Thus we have:
where C(t) is the correlation function, t is the time (our x axis), and τ_m is a time constant, the value of which will depend on the speed with which AX tumbles. And now we have a single number that tells us about the tumbling. This number, τ_m, expresses the speed at which AX tumbles, and is called the rotational correlation time of the molecule. This particular number has been given an ungodly different number of subscripts, but I will use the original notation of Lipari and Szabo and call it τ_m—this means it is the molecular correlation time.

Well, so far so good, but a piddly system that just consists of two atoms isn't really that interesting. Your typical masochistic biomolecular NMR researcher is after molecules (proteins) that have hundreds or thousands of atoms, and even more bond vectors. Now, the picture would still be very simple if these very large molecules were perfectly rigid, but in fact proteins are very flexible and each bond vector is capable of moving on its own, independent of the overall molecular motion. So if we imagine that AX is part of a protein (so that X is bonded to other atoms and A is free) we have to figure out how to represent two motions. This might seem like a major problem, but we still have some hope.

First of all, let's pretend for a moment that the protein doesn't tumble. Well, in that case we could just use the same trick we did earlier, and draw up a correlation function to describe how AX moves inside the protein. Now, we have one difference from the earlier case. Because AX is now part of a much larger system, the correlation function does not decay to zero. Instead, it will decay to some static value, depending on how tightly constrained the bond motion is. If the bond is absolutely rigid, then that value is 1, meaning you have a 100% chance of finding the bond in the same position no matter when you look. If AX is completely flexible, then the ultimate value is 0. So, we'll call this number, which can take values from 0 to 1 and describes the rigidity of the bond, the generalized order parameter and denote it with S². Now, aside from the little wrinkle we just introduced, this is still an exponential decay. So, we need a correlation time, which we will denote with τ_e (an "extra" correlation time). So now we have a second correlation function:

We'll call our first function C_M(t) (for molecular motion) and the second one C_E(t) (for the extra motion). Obviously, these two correlation functions together will completely describe the motion of the bond. But how do we stick them together? If the two motions are correlated (i.e. the probabilities are not independent) this is really quite difficult to do in any general way. But, if the extra motion (the bond moving on its own) really is completely independent of the molecular motion (tumbling), then we can just multiply the functions to get C_T(t), the total correlation function:

where
So now we have an expression to describe the correlation function (i.e. the motion) of the bond, in terms of three parameters (τ_T is just a combination of two others so it doesn't count):

τ_m - the MOLECULAR correlation time
S² - the ORDER PARAMETER
τ_e - the EXTRA correlation time

That's great, but if we could actually measure the correlation function directly we'd hardly need all these parameters, right? We could just look at the correlation functions and compare them. But, it turns out that it is extremely difficult to measure the correlation function of a bond like this directly. Using NMR, however, we can measure something that is related, called the spectral density and denoted by J(ω). What the spectral density is and how NMR can be used to measure it is something for another time (this post is already really freakin' long). The upshot of all of it is, though, that our model of the correlation function C_T(t) translates into this model of the spectral density:
This should be familiar to NMR spectroscopists, though I have omitted scaling factors for clarity. S², τ_m, and τ_T mean exactly the same thing they did in equations 3 and 4 above. The ω represents a frequency, which should give you a little hint about what's going on here: the spectral density is the correlation function represented in terms of frequency, rather than time. This information can be extracted from various relaxation rates measured in NMR spectrometers.

So, now we have a lovely little model of bond motion and a way to get at the parameters experimentally. But it isn't really a model, because we haven't made any sort of assumptions about the nature of the movement involved. We've merely stipulated that there is a global motion, that there is an internal motion, and that these motions are independent. Hence this is called a model-free formalism for describing dynamics. The Lipari-Szabo model-free formalism is extremely powerful because it is very general—the main advantage of this approach is that it can be used to describe almost any motion of a bond. But, the main disadvantage of this approach is that it can be used to describe almost any motion of a bond. Using S², τ_m, and τ_e you can only find out how much a bond is moving, and how fast. The direction and distribution of motions is unknown. So if you want to know, for instance, whether two bonds move in a synchronous or asynchronous manner, the model-free approach can't help you.

Unless you have extremely good luck, you need computer simulations of protein motions to get those kind of answers. All-atom molecular dynamics simulations of proteins have the advantage of explicitly telling you exactly where every bond is moving at every moment. They have some disadvantages too, but the major one of importance for this discussion is that they have historically been very bad at reproducing the dynamics information that comes from NMR, especially when it comes to the motions of side chains. The resolution of that problem will be the subject of my next post.

Lipari, G. and Szabo, A. "Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity" J. Am. Chem. Soc. 1982: 104(17); pp. 4546-4559.

Read the rest...