Characterization of the mimivirus cyclophilin

An interesting article showed up today on the Articles in Press page at the Journal of Molecular Biology. Vu Thai, the most talented young crystallographer I have never wanted to punch in the face, and Elan Eisenmesser, a man of sharp wits and sharper dress, along with several collaborators, have performed a really extensive characterization of the cyclophilin encoded in the mimivirus genome. This is an intriguing study because the role of cyclophilins in viral life cycles is still very poorly understood, even though they are known to aid the infectivity of HIV and SARS.

Typically, these viruses do not encode their own cyclophilins -- this would be a pretty costly thing for such a small and limited organism -- but rather hijack them from the local cellular machinery. What still hasn't been cleared up is exactly why that happens. The catalytic activity of cyclophilins is to convert proline residues from a cis conformation, in which the C alpha atoms of sequential residues lie on the same side of the C'-N bond, to a trans conformation in which they are on the opposite side. It's been demonstrated that human cyclophilin A (hCypA) catalyzes this conversion on the HIV capsid. However, it's not known whether this is the key role and reason for its incorporation. hCypA is also known to mediate some cell-binding events for HIV, and it's hypothesized that this is the only reason for inclusion, and the cis-trans isomerization is incidental.

The mimivirus cyclophilin (mimicyp) has the potential to answer some of these questions. One of the keys here is that it has been carried along in the viral genome. Because viruses are such efficient parasites operating under strong selective pressure, it stands to reason that anything the virus considers important enough to carry along with it is critical to its survival. Granted, mimivirus is a bit of an odd duck, one of the largest viruses known. Additionally, its primary targets are amoebae, though mimivirus has also been known to cause pneumonia by direct attack of human cells. And indeed, Thai et al. find that mimicyp most closely resembles an amoeboid cyclophilin.

The results start to get progressively weirder from there. Firstly, mimicyp has a vanishingly low affinity for cyclosporin, the molecule that gave cyclophilins their name. Moreover, it doesn't appear to have catalytic activity towards a standard proline isomerase substrate at all. The sequence of the protein is missing key residues that typically interact with isomerization substrates and contribute to catalysis, and its putative active site is uncharacteristic of active cyclophilins. Mimicyp crystallizes with an unusual trimeric arrangement, and Elan has demonstrated that it also forms multimers in solution that can be dissociated by adding arginine. Nonetheless, mimicyp localizes to the outer capsid of the mimivirus and is thought to be critical to infectivity, though Thai et al. were unable to demonstrate this for infection of Acanthamoebae polyphaga.

So what we have here is a cyclophilin that is catalytically dead, forms strange multimer arrays, and can't yet be shown to be important for infectivity, that nonetheless is always carried along by mature virions, and is so critical to the success of the virus that it is carried along in the genome and expressed despite significant selective pressure in favor of exclusion, mutation, or silenced expression. Granted some of these pressures are alleviated by the sheer size of the genome and virus, but others (particularly expression) are not. Thai et al. suggest a number of reasons why mimicyp might be important, including charge neutralization of the capsid, mediation of entry and viral disassembly, or capsid-masking similar to that performed by hCypA for HIV. And, of course, the possibility exists that mimicyp has some other, as-yet-unidentified catalytic activity that is essential for mimivirus infectivity, or is only critical for infecting certain amoebae or cells.

One might also surmise that the ability of mimivirus to infect vastly different hosts to some degree relies on its carrying along this particular cyclophilin, which is probably quite dissimilar to any protein abundantly found in human cells. This only brings us back to the question of why mimicyp is so important in the first place.

Ultimately this paper does not reveal the precise reason why cyclophilins are used by viruses, and indeed it will probably do more to spur debate than end it. Nonetheless, Thai et al.'s research can be taken as adding weight to the proposition that the catalytic activity of cyclophilins on virions is incidental, or at least that its importance is an idiosyncratic feature of particular viruses. Mimicyp may also be our first introduction to a new class of cyclophilins, catalytically inactive, cyclosporin-free (cyclophobins? cyclomehs?), and multimeric. What it is that these cyclophilins do and why they are important will be an intriguing inquiry to follow.

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A bittersweet day in the Kern Lab

Yesterday Peter, one of our MD guys, left the lab after two years. He and his wife are returning to Sweden now that she has completed her study here. All of us will miss his insanely delicate orange-peeling procedure and his absurdly neat notebooks, though I believe we bid adieu to his segment of mini-meeting without any regret. According to Doro, Peter really "stepped it up" in these last two months and should have two papers when we lazy experimentalists get off our butts and finish those projects. So I guess Peter will be hounding Annette and I for those valuable papers.

Dorothee was especially nice and thoughtful, telling Peter how much she appreciated all his hard work. She had come back to town from the cape just to see him off, and even arranged a little party. Before he left, she gave him a little gift, and I really felt the emotion of the occasion.

All of us wish Peter the best, and hope he finds a good job in Stockholm. I suppose Annette and I will have to "step it up" in order to put some good publications in the pipeline for him.

Disclaimer: One of the above paragraphs consists only of lies.

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Automation for your nanodevice assembly

An interesting article appeared in JACS ASAP today from C.S. Hartley and J.S. Moore at UIUC on a clever way to direct the assembly of an asymmetric macrocycle. The approach relies on a simple idea to address a fundamental question about nanostructures, namely, how to make sure they assemble themselves as we would like. Given that protein design still isn't quite far enough along to create any product we desire, we have to rely on more conventional chemistry. The basics of the approach should be obvious from the image on the right (shamelessly stolen from the paper). Note the reactive groups on the lower side of these compounds; even without knowing the relevant chemistry it should be clear what mechanism for directed assembly is implied. Given that the smallest geometrically stable assembly consists of three units, and that entropy favors the creation of small assemblies and enthalpy dislikes dangling functional groups, it should be obvious that dumping all these into a reactive pot should mostly produce units that have a composition of 1-1-1 or 1-2-3.

This is in fact what the authors observe when they perform the experiment, though they find (perhaps surprisingly) that in mixtures of all three components the 1-2-3 macrocycle predominates. Glancing at the product it's clear that the macrocycles themselves will assemble into stacked arrays given the right conditions. If covalent chemistry is used to control this assembly, one has a rather simple method that could be used to construct fairly complex structures. Also, the yields from these reactions were encouragingly rich in the desired molecules, suggesting that only a little further optimization is necessary to produce effective scale-up.

Nothing in the paper is particularly earth-shattering, but then again, the cleverest answers often seem blazingly simple in hindsight. Hartley and Brown have come up with just such an answer here, in a commendably clear and readable paper.

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Bioscience is murder

While reading Jane Stemwedel's musings on the missing gray zone in animal-research discussions, it occurred to me that when taken as a philosophical position, rather than an emotional appeal, the animal-rights people are really demanding that all bioscience be ended. I don't mean to make the old slippery-slope argument here; rather, I mean to say that when viewed in the light of cold reason, rather than sloppy anthropomorphism, the demand to eliminate animal suffering boils down to a demand to end bioscience research. Although we biochemists investigate life, our tool is death, and we use it constantly.

My objection to the animal-rights crowd hinges on their attribution of equivalent values to human and animal life, or human and animal pain and suffering. If you are willing to accept degrees, to say that a certain amount of animal suffering is tolerable in order to alleviate human suffering, then I am not arguing directly against you. I run into trouble when the crowd says that no outcome justifies animal research, because this argument implies that no research should be performed at all.

Consider the case of the laboratory mouse. On the one hand, there is an intuitive appeal to the animal-rights argument in their case. "How would you feel," they might well ask, "if you were locked up in a cage all day and subjected to experiments?" And of course, most of us would not like it at all, but that isn't relevant because our likes and dislikes are psychological constructs for which it is not clear mice have any equivalent. We're relatively closely related to mice, and they have behavioral characteristics similar to our own, which leads us to empathize with them, or so it seems. But this is a false empathy; we empathize with the mouse as if it is a human being with human traits and psychology, but this is manifestly not the case. The human mother, even a fecund one, mourns over her dead infant; the mouse mother eats hers. A human behaving as the mouse does would be called depraved, but we do not call the mouse depraved because we recognize that it does not think or feel as a human does. The similarity of responses to stressful stimuli owes more to a coincidence of behavioral evolution than matched psyches. Arguments against research on the basis of perceived empathy are therefore largely empty.

Given that anthropomorphic empathy is a dead end, the next line on animals is that they have intrinsic rights, especially that no animal individual should be used in research that does not help that individual. This implies something else, namely that an animal must consent to be researched upon. As it is impossible for the animal to give consent, we must imagine conditions under which it might give consent and stipulate those as a precondition to research. But on what basis to we attribute these rights? What virtue of a living creature endows it with the right to self-determination?

The most natural response to this question is that a creature gains the right for self-determination by having the ability to weigh the consequences of its actions and choose wisely. Indeed, this is the attribution by which we typically operate, and why we allow children and the mentally infirm only limited degrees of self-determination. However, this is clearly not the attribution the animal-rights activists are using -- a mouse does not even have the ability to conceive of the possible consequences of research, much less weigh them. For the animal-rights activist, the simple fact of being alive gives rise to the right to self-determination.

But why then should we stop with mice? Untold millions of drosophila have been bred and destroyed in the name of genetic research, not to mention everything that's been done with nematodes. Sure, it's more difficult to relate their behavior to ours, but these creatures live and die in captivity, and exhibit stress responses during certain experiments. What property of a mouse means that it has self-determination and the flies and worms don't?

And we're still being biased even if we let them in. After all, what's so special about multicellular organisms? Why should they be the only ones with a right to self-determination? If all it takes to require consent is being alive, then E. coli, of which I have personally raised and destroyed billions in the pursuit of NMR dynamics data, qualify too. In fact, their tale is really gruesome when you think about it. They're subjected to extreme temperatures and such rapid changes in them that they ingest large chunks of foreign DNA (themselves the product of enormous bacterial slaughter). I feed them a starvation diet loaded with strange chemicals, get them so high on IPTG they start to produce one protein almost exclusively, and then once they're done I murder them by repeatedly freezing and thawing them before I shatter their bodies with sound waves or crush them to death.

So there we are -- bacteria have rights, too, at least if you accept the reasoning of the most extreme (philosophically) animal rights supporters. I could go on to make a case for cultured cell lines as an independent life form, but really I've already gutted molecular biology, any cell biology involving DNA manipulation or foreign proteins (think how many bacteria died to bring you that Pfu turbo), and all of biochemistry and structural biology. If we grant that animals have the right to self-determination, none of the biosciences can possibly survive the scrutiny. That's what animal-rights activists are demanding, whether they know it or not.

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The disingenue

The ingenue in fiction is a sweet young girl who because of her naïveté is often taken advantage of by more worldly members of the cast. Sometimes she is even duped into committing some crime or other offense and made a scapegoat by dastardly villains. It's an enduring theme in literature, enough so that it still has traction in modern society, though in an altered form. I call the many who make use of it disingenues, a word that has been used occasionally by others without definition. By it I mean someone who pretends to be a naïve innocent led astray by people who pretended to be her friends, with the intention of deflecting blame for some misdeed. More broadly, it could be taken to mean someone who is disingenuous generally (like, say, Alberto Gonzales).

The narrow meaning could be applied to any number of misbehaving celebrities. They blame "bad influences" around them and pledge to improve the company they keep so as not to wander down the garden path again. It's a cheap dodge, and one that devoted fans always buy. Thinking seriously, when Britney Spears or Lindsay Lohan go out to party, who's in charge of that entourage? They are. They wield the power and authority in their circles, and they make their own decisions. Having the wrong people around them enhanced their opportunity to make bad choices, but those "bad influences" didn't choose for them.

But let's not limit the definition to women; this is the 21st century, and gender balance is at work here in epic proportions. The greatest recent disingenue is in fact a man, Michael Vick, who until this very day made a dedicated effort to lay blame for his own failures and shortcomings on the people surrounding him. From the beginning of this dog-fighting debacle, his position was that he had placed ill-deserved trust in his relatives and acquaintances. He had given them a house and they had betrayed him by using it for these evil purposes. Of course this fanciful tale of Mike Vick, ingenue, eventually unraveled, leading to today's guilty plea. Encouragingly, today he also said, "Not for one second will I sit right here and point the finger and try to blame anybody else for my actions or what I've done." Maybe he has turned the page, but consider how his statement of fact went to great lengths to indicate that the most gruesome act -- the violent killing earlier this year of several dogs that were not "game enough" -- was a "collective effort" involving Vick but not solely carried out by him. This is not materially different from the statements of fact accompanying his cohorts' pleas, but the difference in language is telling; he is still trying to deflect blame.

Literary or not, the disingenue is here to stay. This line of defense is too successful to fade anytime soon. We always want to pity a poor celebrity led astray by the bad influences around him or her. But let us not forget that these people chose to associate with those influences, and moreover, that when those influences offered a turn onto the wrong road, Mike Vick and others like him chose to take it.

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The brass ring is tarnished

News this week from the world of science: impact factor doesn't really match up with research quality. A study by Brown and Ramaswamy of the University of Iowa in Acta Crystallographica D analyzed crystallographic structures that had been deposited in the PDB and came up with a generalized metric to describe several features of a structure's quality. They normalized it to the average quality of the PDB, and then set about to see what extraneous factors were the best predictors of quality.

There were several interesting results here. The first was that the quality of crystal structures has not generally improved over time, despite advances in technique and equipment. The authors attribute this to the increasing use of crystallography as merely part of an experiment, so that the focus is less on the quality of the structure as a whole and more on making the structure good enough to answer a specific question. One can also imagine that the democratization of crystallography has contributed to this effect. Early structures solved with more primitive techniques only led to a solution if the researcher was qualified and careful. As the tools have become better, more widely available and black-boxed, more people can do crystallography, but these tools have encouraged less-qualified individuals to model structures, and perhaps also encouraged sloppiness among the better-trained researchers. There is also the vacuum-cleaner effect to consider. As the tools for modeling structures have become more robust, individual crystallographers are expected to produce more structures. As a result, the time spent on each individual structure has declined, perhaps more so than the improvements in technology have justified.

Another important result is that structural genomics consortia generally produce slightly better structures than the PDB average. This is certainly reassuring to the government entities that have dumped millions of dollars into these initiatives, but it also bears thinking about why it is so. Certainly one reason for the improved quality is formalized screening and the highly trained people that are doing the structural genomics work. Moreover, these efforts are really focused on getting a good structure, without any direct emphasis on experimental utility. It stands to reason that if your goal is to get a good structure, then it is more likely that those structures you personally consider a success and therefore deposit in the PDB will be good structures. This assumes a certain competence on the part of the researchers, but this is a property that structural genomics researchers manifestly possess. An additional consideration, however, is that these consortia are explicitly oriented towards solving the structures of well-behaved proteins. The high degree of automation used by the consortia is not generally compatible with poorly-behaved proteins or any need to squeeze a model out of troublesome data. A willingness to leave troubled proteins alone is probably part of the success of the consortia in this regard.

The most troubling result from the paper came when structures were analyzed based on the journal of primary reference. Ramaswamy and Brown discovered that more crystal structures initially reported in high-impact journals like Nature, Cell, and Science had below-average quality than structures reported in lower-impact journals like Biochemistry, Proteins, and Eur. J. Biochem. Because the metrics used by Brown and Ramaswamy are not intrinsically sensitive to novelty, this cannot be blamed on the simple "newness" of structures reported to these high-impact journals. The authors of this study attribute their problematic finding primarily to the fact that the structures reported in these journals tend to be (as above) part of a paper, not the whole thing. Moreover, the reviewers for these papers may more often be something other than expert crystallographers, and even when they are, the extreme space constraints may prevent reporting in the paper of the relevant structure factors and raw data.


That the reviewers are important might also be supported by the data. Consider the figure above (my own creation from Table 5 of the paper). This graphs the aggregate quality score for a journal against the number of structures therein - remember that a lower aggregate score is better. There's clearly no linear correlation between quality (lower numbers are better) and the number of structures reported. But it is interesting to see that with two exceptions the 'bad' journals (positive quality score) have a number of structures between 100 and 1000. Inside this range the quality varies significantly but outside of it the quality is almost always good. Maybe this means nothing, but it also may mean that journals who have a significant stock in trade in protein crystal structures also have experienced editors and reviewers who know how to properly vet them. It's interesting to note that the only journal containing more than a thousand structures that has a positive score is Proceedings of the National Academy of Sciences, a journal which has a strange and inconsistent review policy. Similarly, journals where crystal structures are a rare event may have editors who react to them with caution and seek out expert reviewers. By contrast, in the middle range, no trend is discernible except a roughly inverse relationship between impact factor and structure quality.

The more disturbing implication, and one that the authors do not deeply address, is that an external property of novel structures causes them to be published with lower quality: namely, their very novelty makes quality more of an afterthought. This is not just the idea of rushing papers to publication causing trouble. Rather, I mean to say that the editors and reviewers of these papers are flat-out willing to accept lower quality of data in exchange for novelty and impact. In, for example, the recent high-profile pentaretraction by Geoffrey Chang, the reported features of the structures alone should have raised serious questions about publishing them, even without considering that they were contradicted by biochemical data. That these errors somehow did not rise to the level of alerting Chang to find the elementary error in his own software indicates that the reviewers and editors were as sloppy as Chang himself. And why? Because these structures were novel and potentially revolutionary. Good copy outweighed bad modeling.

Crystal structures have a useful feature in that their quality can to some degree be assessed quantitatively, without needing to ask subjective questions (say, whether a structure is consistent with some mechanistic model). That is, this kind of large-scale analysis of research quality is possible. The quality of structures in these journals isn't disastrous, but it is cause for concern. And it raises serious questions about other research published in these journals, data for which the quality is less quantifiable and objective. I won't say that Science and Nature are not to be trusted, but in light of this large-scale trend and recent data-falsification woes in other areas, it would be naive in the extreme to approach reports in these journals without a healthy skepticism.

Brown E.N., Ramaswamy S. "Quality of protein crystal structures" Acta. Cryst. D. 63 (2007) p. 941-950

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Biological data filters

I had a conversation this week with Annette about the structural ensemble versus individual structures that I'm still trying to coalesce into a fully-formed idea. The kernel of it is this: there is a dichotomy between the way we know that proteins act and the way we talk about their action. Proteins give rise to phenomenological effects as ensembles, but we discuss their states as individuals.

Consider a signaling molecule, say a member of a MAP kinase cascade (picture at right). A given protein can exist in either an inactive (A) or active (B) form. When active, the kinase phosphorylates some downstream target, otherwise it just sits in your cytoplasm taking a nap. Typically in this kind of system the active form of the kinase is also the phosphorylated form (red B). It's typical to say something like, "The kinase is activated by phosphorylation." At the same time we know from some of Dorothee's work with Dave Wemmer that certain bacterial proteins that get phosphorylated already sample their active conformations even before they are modified (blue B).

Even for systems where this kind of sampling hasn't been directly demonstrated it's reasonable to assume it takes place. After all, the active and inactive structures have the same amino acids to work with. Unless the phosphate group itself is a lynchpin of the new structure (perhaps by bridging two structural elements), then the active structure must be one the unmodified kinase can adopt. Naturally, we expect this structure to be higher in energy than the inactive state (so blue B is higher on our energy diagram than blue A), and that phosphorylation decreases the energy of the active state so that it is subsequently preferred (so red B is lower than blue A).

The implication of this is that, unless the unmodified active structure is much higher in energy than the inactive structure, some proportion of our kinase is active even when not phosphorylated. Perhaps this is as low as 1-2%, a fraction that's difficult to detect directly. Still, because enzymes are so efficient, this quantity may be significant. Or, for a single protein, we could say that it adopts an active form without phosphorylation 1-2% of the time. But we tend to talk about phosphorylation and other post-translational modifications as if they were switches, with phrases like "protein X is turned on by phosphorylation". The reality, though, is that the switch is less a matter of turning a protein on than of turning it on more.

This points to a reality far less clean and orderly than typically depicted in block schematics. Inappropriately active (i.e. active without modification) members of the various protein ensembles must give rise to a considerable amount of noise in biological information processes. The system must therefore have some way to distinguish the signal from the noise that's more than just the binary on/off typically depicted and discussed. These filters could take several forms -- for instance, the kinase of our kinase may mediate the interaction between our kinase and its target, though in this case inappropriate activation of the MAPKK could still give rise to signaling noise. Alternately, the phosphate could mediate the kinase - target interaction. Or the cell could simply have an inefficient signaling system, so that multiple nearly-simultaneous signaling events are necessary to activate a response.

Is this point important? Maybe and maybe not. Most of our experiments can only access the behavior of ensembles, so the ensemble nature of protein action is not likely to lead us astray. But as single-molecule studies become more popular it may be important to keep the ensemble perspective in mind so as not to be confused by their results. Moreover, a conceptually accurate picture of cellular signaling and regulation will require us to keep this feature in mind.

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To learn NMR...

I picked up James Keeler's Understanding NMR Spectroscopy because of a teaching dilemma. Students who come to an NMR lab often want to "learn NMR", though of course this is not really possible in a 2-3 month rotation. They can at least get started in NMR, but in order to do this effectively they need a resource to study and discuss with whomever has charge of them in the lab. I've had trouble finding an appropriate book for this task. High-Resolution NMR Techniques in Organic Chemistry, originally by Derome and now reincarnated by Claridge, is a fine introduction for general NMR study but is not at all oriented towards biomolecules and relies heavily on the vector representation that doesn't always help a student understand techniques such as HMQC or HSQC. Protein NMR Spectroscopy, by Cavanagh, Fairbrother, Palmer, and Skelton is an excellent resource for the advanced student, and has just come out with the long-awaited new edition, but the pages of mathematics and occasionally obscure language are really too intimidating for beginners.

Understanding NMR Spectroscopy is, I think, the resolution of this dilemma. Keeler's text is clear, describing the physical basis of NMR in a straightforward way that should work for just about any student. He handles the necessary quantum mechanics and operator representations with a deft touch that makes their mathematical derivations clear without producing an intimidating morass of equations. Naturally, some detail and rigor is swept under the rug in this approach, and an advancing student will want the Cavanagh book or Levitt's Spin Dynamics to get a firmer grasp of the nuts and bolts, but as an introduction to the theoretical underpinnings Understanding NMR Spectroscopy is superb. Keeler's explanation of relaxation processes is also excellent, and includes perhaps the best physical description of T2 relaxation I have ever read. The book also includes a useful little chapter on the workings of an NMR spectrometer that, while nothing special on its own, is also a good resource for an early-career grad student or rotator. Exercises at the end of each chapter can also be a good teaching tool (although, since the answers are available at spectroscopyNOW, not appropriate for a course).

Although the book is not explicitly oriented towards biomolecular NMR, it has a strong focus on heteronuclear experiments that ensures the information presented is appropriate for students interested in biomolecules.

I can't praise this book without reservation, however. Some topics that might be considered important are glossed over or skipped entirely -- chemical exchange, for example is barely mentioned, and REX not at all. Residual dipolar couplings are not discussed, and the angular dependence of the dipolar interaction is only skimmed. Chapters 10 and 11 are poorly structured and include inadequate and possibly confusing discussions of raising and lowering operators, coherence order, and coherence transfer pathways and diagrams. The mentor will need to take an active hand in explaining just what is going on in these sections.

That said, I think that Understanding NMR Spectroscopy will be an excellent book for grad students just starting out in biomolecular NMR or possibly rotating students who want a glimpse of the nuts and bolts of NMR theory. The gap between Derome/Claridge and Cavanagh has been pretty neatly filled by this affordable little volume ($40 at Amazon).

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"Jealousy among men is a terrible thing..."

Last night I rented and watched Taboo, or Gohatto if you use the original Japanese title. I don't think I would have found it on my own, but the Netflix recommendation algorithm spat it out and I gave it a try. Taboo is a fascinating, well-acted gay samurai mystery art film, and you cannot be any more surprised by reading that than I was by writing it.

The conceit of the film is that an exceptionally pretty boy named Kano Sozaburo joins a samurai military unit towards the end of the Shogunate (the politics of this era play a minor, but substantive role in the story). Many of the men become infatuated with him, and their jealousy starts to tear the unit apart until a bloody episode that ends the film. Beat Takeshi stars as the unit's second-in-command Hijikata Toshizo, who is the one that has to deal with the problems Kano creates. These are many, as Kano seems to catch absolutely everyone's eye, including the commander, at least one lieutenant, and even perhaps Hijikata himself.

This is not the best movie I've ever seen by any standard. The plot seems to come and go at times, and the tension builds only unevenly towards the final confrontation. While the movie centers on Kano little is done to give the viewer a real handle on his character. He blows hot and cold, and Matsuda Ryuhei at times just doesn't seem equal to his task. Takeshi does more with less; Hijikata is much easier to grasp, even though he has his own set of unresolved questions. In the end the viewer is left to resolve most of the movie's central mysteries.

The directing is very interesting. Oshima Nagisa's camera tries to let action speak for itself, which works splendidly in some scenes (Kano's embarrassing kendo bout against his lover) and not so well in others (an extended entrance by a geisha). As is often the case, color features prominently in the film (Kano's frequent wearing of white against the black of the militia's livery), but it bears thinking about what the colors mean in the cultural context.

Some cultural context may also be needed for westerners watching the film. An American might find it strange that all these men are attracted to Kano, who seems by our standards pale and rather pinched. The seeming lack of romance in the love scenes (there is one sex scene, but barely anything is shown) may also seem a little odd. These aren't flaws but rather important components of the historical and cultural setting, ultimately helping to build a fairly accurate picture of this world.

Taboo isn't easy to understand and just isn't for some people. I don't mean this pejoratively; you aren't a better person if it is for you. It's the strangest case ever made against gays in the military -- beware, all the straight men might become infatuated! And yet, though some of the plot seems totally alien, it works, even the slightly strained ending. Beat Takeshi is to be credited for this, as he grounds a film that could have easily flown off into space. Sakagami Jiro, Takeda Shinji, and a few others in the supporting cast also do great work, propping up (in my opinion) Matsuda, who just doesn't seem to have quite enough poison in his eyes for this role. If you think a gay samurai mystery art film might be for you, then try to get your hands on it, at least for a rental.

I also watched Hot Fuzz; all you need to know about this film is that it is very funny and you should rent it.

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Leverage

Man, there is a lot of chatter about Mike Vick these days. I used to think he wasn't quite as big a punk as his brother, but apparently family ties run deep. The talking heads all seem to expect a 1-year sentence at minimum, but I'm not so sure. Yeah, the Feds could give Vick all that jail time and make an example of him, but to me that seems like squandering their advantage. The sports guys have it right when they say that Vick's career can't really take even a year off -- he's an athletic freak, not a football mind, and without the constant exposure to field conditions his already-deficient technique could take an irreparable hit. If he spends a year in jail, that means he'll miss at least two seasons, and if that happens he may have no choice other than to come back as a running back. So basically the Feds have him over a barrel. If they hit him with even a fairly light sentence, his career might well be over.

But all that gets the Feds is Bad Newz Kennels. Wouldn't it make more sense to slap his wrist in exchange for having him roll over on all those guys his dogs fought against? There was a lot of talk early on in the investigation about how Mike Vick was a major player in the dogfighting world. The Feds can use that knowledge -- after all, the world of dogfighting has been notoriously difficult to breach -- and I think they might dangle a big fat carrot in front of Vick to get it. I think that maybe his plea deal will involve a 6-month sentence at Club Fed, with another 3-month commitment in front of a grand jury. Now if only somebody would throw Pacman Jones in jail...

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The Persistence of Memory

There's a neat article in today's Science that could creep you out. Shema, Sacktor, and Dudai demonstrate that an inhibitor of protein kinase M zeta, called ZIP, apparently erases the long-term memories of rats. Rats presented with a novel taste that makes them sick subsequently avoid water with that taste (this is called Conditioned Taste Aversion). If the rats are treated with ZIP after the initial conditioning, they lose this aversion, even if treated with ZIP up to 25 days after the initial conditioning. ZIP treatment prior to conditioning has no effect. The implication is that ZIP has erased the memory that gives rise to aversion. Moreover, this effect depends on where in the brain the ZIP is infused (hippocampal infusion has no effect), suggesting that there is some possibility of controlling which memories are removed.

For people suffering from post-traumatic stress disorder or similar problems this could be the beginning of a promising therapeutic avenue. Of course, for those of a more imaginative bent this holds the promise of being a frightening brainwashing tool.

Check out the research article and commentary in the August 17 issue of Science.

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Small-molecule allosteric catalyst

An interesting pair of articles showed up this week, both from Chad Mirkin's lab at the Nanotechnology Center of Northwestern University. You can find them both in the August 22 edition of JACS. The essence of these articles is that Mirkin's group has created a group of macrocyclic catalysts that can be allosterically regulated by small ligands. The basic principle is shown below (figure taken from the second paper):

The catalytic molecule has a small intrinsic ability to catalyze the chemical conversion of the small molecules, as shown on the left. The addition of the "Analytes" in this case Cl- and CO, displaces the sulfur coordinating groups that are weakly bound to the rhodium or copper metal centers. Removing the sulfur coordination allows the molecule to undergo a conformational change, opening up to adopt a new structure that is vastly more effective as a catalyst. This mimics the allosteric regulation of protein catalysts and binding proteins by ligands.

The approach is a pretty clever bit of molecular engineering, and an important step forward for nanotechnology. Though it remains to be seen how well these molecules survive under field conditions (reducing/oxidizing environments, complex solutions), they have a feature that will be absolutely essential for functional nanomachines, namely that they can be regulated. The ability to turn our microscopic tools on and off will be an important part of producing useful nanomachines, to say nothing of avoiding the "gray goo" of sci-fi nightmares.

If you're interested, check out these articles:
Kuwabara, J. Stern, C.L., and Mirkin, C.A. "A Coordination Chemistry Approach to a Multieffector Enzyme Mimic", J. Am. Chem. Soc. 129 (2007) p. 10074-10075.

Masar III, M.S., Gianneschi, N.C., Oliveri, C.G., Stern, C.L., Nguyen, S.T., and Mirkin, C.A. "Allosterically Regulated Supramolecular Catalysis of Acyl Transfer Reactions for Signal Amplification and Detection of Small Molecules" J. Am. Chem. Soc. 129 (2007) p. 10149-10158.

I recommend the first article for lay readers, and the second for the hardcore.

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HDTV

This story begins back in March, when I got an amazingly crappy hetNOE spectrum from our 500 MHz spectrometer. The hetNOE is always a low-sensitivity spectrum, but for a 1 mM sample the signal I got was simply unacceptable. At first, I didn't think much of this, for the simple reason that I thought the HX probe was in, and I'm used to getting poor signal-to-noise from that thing.

Except... except that everyone else was getting low-quality spectra too, no matter what probe was in. Finally one day Chris and Janice got almost no signal at all from an HSQC of a 1 mM monomeric protein, and that was the end. We had Sara test the magnet, and she found we had a four-fold reduction in signal to noise. To put things into perspective, we would have to take 16 times as many scans as before in order to achieve the same signal under these conditions. For a 1-hour HSQC, this is marginally acceptable. For a 2-day triple-resonance sequence, it is not. The new noise, strangely enough, came and went at odd times.

Al, because he knows all, immediately suspected a television signal, which he attributed to a rogue broadcaster somewhere out on Bear Hill. You see, an NMR spectrometer is really two things. It is a giant magnet, yes. But it is also a radiofrequency transmitter and receiver. The RF pulses induce a signal that the transmitter/receiver coil picks up. For our 500 MHz spectrometer, the primary signal we pick up is the proton signal at 499.75 MHz. This frequency is in a TV band.

Al was deflated, however, when Wlad tried to check the spectral band on his TV and found nothing. The search for the cause continued, with suspects ranging from the Brandeis student radio station to the construction crew radios to various parts of the magnet to secret government transmissions.

In the end, Al turned out to be right, and Wlad turned out to have a cheap TV. There was a channel broadcasting in this frequency range, but it was HDTV channel 18, a signal Wlad's cheap TV cannot decode. Our spectrometer can't decode it either, but we can sure see it. It knocks our signal down to an unrecoverable level. You can lose 75% of your signal for a small molecule and still be OK, but with proteins it's a different story. The spectrometer is essentially useless to us in this condition.

And the worst part, the very worst part about this is that it's a terrible channel! WMFP digital channel 18, broadcasting in the 494-500 MHz band, airs infomercials and "Gems TV"! Yes, that's right, our scientific research has been derailed by a display of cubic zirconium jewelry in glorious high definition. Their antenna is located just south of us and we're right in one of their strongest broadcast regions. Natural cures and cash-at-closing real estate ads are screwing us badly.

Hopefully, we'll be able to boost the spectrometer's frequency out of the channel 18 broadcast band to something like 500.13 MHz. However, there's a Channel 19 (WGBH) broadcasting in the 500-506 MHz band as well; the spectrum analyzer shows a little gap between them that we can hopefully hit, and the WGBH signal is only half as strong as WMFP. Still, we may be screwed even if we boost the frequency. And there's no way to shield the magnet, except maybe in the basement, but we'll need to renovate the basement area in order to do that. So basically, we are screwed.

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It came from the Protein Society! (Part 2)

Another feature of the protein society was a continued emphasis on trying to understand natively-disordered proteins, and by extension, the denatured state of natively ordered proteins. Because these two fields are highly related and use the same techniques, it seems to me best to lump them together for now. A couple of interesting points came up that I wanted to get down here for my own memory's sake.

One point, and one that became a recurring theme in several talks at the symposium, was averaging bias. The first real discussion of this came from a really good talk by Michele Vendruscolo on the study of the natively-disordered 131-deletion mutant of staphylococcal nuclease. Some models that Dave Shortle had produced of the disordered state on the basis of paramagnetic relaxation enhancement had predicted ensembles that were too small with respect to the known radius of gyration. Michele pointed out that the PRE is an ensemble measurement, and many different ensembles can give rise to the same PRE. Additionally, the PRE is biased because below a certain threshold the effect is invisible. This means that the measurement ends up being biased towards closer approaches. Essentially his point was that the normal distribution cannot be assumed for the ensemble average of distance measurements in the denatured state (and it's probably a questionable assumption in the native state as well).

Kevin Plaxco gave a talk later on that really hammered this point home. He did a series of SAXS experiments to determine the radius of gyration for a ton of proteins, including several that had shown residual structure in NMR experiments. His results indicated that the experimentally determined radius of gyration matched that predicted for a random coil for all these proteins. As he pointed out, though, the Rg is totally insensitive to local structure, whereas because of anomalous averaging much of the NMR data is hypersensitive to local structure. This means that both results can be right -- any given protein can have some percentage of its structure intact and as long as it's a different piece for each protein and not too much, the ensemble can retain a random-coil-like Rg. If tertiary interactions are preserved this becomes a slightly more difficult proposition to swallow, though. Still, his work, and several other talks and posters presented during the symposium, made an excellent point. We simply cannot rely on the assumption of a normal distribution when we are analyzing NMR data from systems with so many degrees of freedom.

Another thread that showed up repeatedly was the ongoing attempt to understand exactly how disordered states interact and are regulated, especially by post-translational modifications such as phosphorylation. Most disordered regions have multiple binding partners, with affinity enhanced for a particular partner by a particular modification. In the simplest model for these interactions, the modification itself and some of the surrounding primary sequence is recognized. However, there's an increasing amount of data, including a nice talk by a postdoc from Julie Forman-Kay's group, that the post-translational modifications alter the structural characteristics of the disordered state itself. The Forman-Kay talk suggested that phosphorylation induced a condensation of the protein by attenuating a surplus of positive charge.

This could conceivably be taken further. Consider a bit of sequence like DKRSDKA, which could conceivably take the form of a β-strand if it weren't for that concentration of positive charge on one side. A phosphate group on the serine could conceivably stabilize this structure and preorganize it for binding to a ligand, thus increasing affinity by reducing the energetic cost of binding.

It might even be possible to tune things more specifically. Take a sequence like GRDSSKAKSR. If you put this on a helix wheel you'll see a huge blast of positive charge on one side, but also a pair of serines. Phosphorylate S5 and S9 and you could stabilize the helix. At the same time, this would make a β-strand conformation less likely because such a strand would have negative charges on one side and positive charges on the other. By contrast, if you phosphorylate S4 you'd do nothing to stabilize the unfavorable charge concentration on the helix, but the positive charge concentration on the strand would be attenuated (see cartoon). In this way phosphorylation might be used as a kind of conformational switch to preorganize the same sequence in different ways and thus reach different downstream effectors. We know that conformational rearrangements of the kind that lymphotactin undergoes give rise to different signals and protein behaviors. The role of differential preorganization in disordered proteins hasn't been extensively studied yet, but may be equally important.

It's increasingly clear that disordered regions are a major factor in cellular signaling. I'm not having much luck with the one I'm working on now, but I'm excited to see where the next few years lead this field.

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It came from the Protein Society! (Part One)

I went to the annual Protein Society symposium a few weeks ago and have finally had some time to organize my thoughts about it, so I figured I'd put some of them up here. Expect a couple of these to show up.

One of the most interesting presentations at the Protein Society (besides my own scintillating poster on field-cycling, haha) was Brian Volkman's poster on lymphotactin. This is a really interesting story that somehow seems to keep flying beneath the radar of most people, but intellectually it represents a giant challenge.

In a nutshell, the story is this: lymphotactin is a small signaling protein of the chemokine family, a group of proteins that are important for various kinds of regulation, including in inflammation and disease. Under fairly standard experimental conditions (200 mM NaCl, 10 °C) the protein adopts a normal chemokine fold, but at 45 °C (for reference, body temperature is 37 °C) and low salt, it takes on a totally different fold. You can read the original paper on this here. Of course, when you see something like this it's natural to ask what the physiological relevance of the finding is. Brian's poster at Protein Society basically answered this question by illustrating different biological roles for the two forms. The short version is that the conformational change appears to be some sort of regulatory switch. I'll have more on that in the next episode.

What I want to talk about here was what wasn't said about this at the meeting. After all, we were forced to witness the usual ninny-argument over whether folding was a linear pathway or a funnel of some kind. While it was refreshing to hear a lot more people pointing out that this distinction is more or less meaningless, it's odd that nobody is tackling the question through a protein like lymphotactin. Consider the following experiment: perform phi-value analysis or GdHCl-dependent HX experiments on lymphotactin to find what portions of the protein are structured in the folding transition state. We can imagine two outcomes.

In the first, the transition state is found to be completely different; that is, residues with high phi-values or the last residues to lose protection in the HX experiment are completely different for the two conformations. This would suggest that the latest common intermediate is the random coil (RC), and that the very first move towards a folded state dictates the state one finally arrives at (N1 or N2). Any intermediates (I1 and I2) along the pathway are unique to the end state, rather than shared between the two conformations (see right). This would fit most closely with the pathway view promulgated by Englander. Given that the hydrogen-bonding patterns are totally different for the two conformations, this might be expected.

On the other hand, it's possible that a residue or cluster of residues have similar phi-values, or lose their protection at a similar GdHCl concentration, between the two conformations. This would not be completely probative, as the similarities could quite easily be restricted to the observables and represent two different underlying structures. However, if veridical this might suggest that the latest common intermediate lies somewhere other than in the random coil. This would be more similar to the funnel view, in which a conformational search over the outcomes available to an intermediate gives rise to the ultimate choice of native structures. I've cartooned the idea over to the left. In this case I* represents a partially-folded intermediate that selects an end state based on the conditions.

After all, lymphotactin in both its native forms exists in physiological extracellular conditions. Knowing whether the protein must pay the full energetic cost of completely unfolding, or if it can switch conformations by taking a less-costly move to a common intermediate may be of significance to understanding the biology. And while these two alternatives (like the underlying models) are not as different as they may seem of first blush, the answer may do much to distinguish whether the conformational search of the funnel model or the deterministic folding of the pathway model is the best representation of the folding process.

Another major implication here is for the protein structure prediction crew. After all, the CASP-type experiments are all geared to the idea that a given sequence should give rise to a single folded structure. Lymphotactin is a clear counterexample to this idea, and while it may be unique, we certainly don't know nearly enough about proteins to let the dogma go unquestioned at this point. This makes for a much larger computational problem. I'm not a huge expert on these experiments, but my impression is that the programmers don't concern themselves too much about the characteristics of the solution the proteins are in. The assumption that co-solutes don't much matter is vastly simplifying, but as Brian's work shows, may ultimately limit the predictive power of these algorithms in significant ways.

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Sorely tempted

For the first time I really have an urge to get an XBox. It's a very curious thought, since the 'feel' of the XBox library doesn't much coincide with my gaming interests. A console where the highest-profile exclusive offerings come in the form of FPS, racers, and Western-style RPGs generally doesn't appeal to me. But it seems now like the XBox is catching some of the quirky RPGs that more suit my style. I'm specifically talking about Eternal Sonata, a new cel-shaded RPG set in the mind of Frederic Chopin shortly before his death of tuberculosis. The concept alone -- and the idea of music informing the RPG mechanics -- is pretty intriguing, but I've also been very drawn by the game art I've seen so far and the descriptions of the combat system. Ultimately it's not quite enough to convince me to blow hundreds of dollars on a system that still doesn't have much that I want. That said, if the XBox continues to get more of this kind of game, a sort that used to be more or less the exclusive province of the PS2, the case for it will get stronger and stronger in my mind.
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USAir... begins with you!

And ends with me, as far as I'm concerned. My trip down to Charleston was a comic travesty of airline mismanagement. I don't mean to say that it was the worst flying experience ever, or even that it was my worst flying experience ever. It was just a classic example of the pathetic failures of modern airlines.

The trip began with a 5AM phone call telling me my flight out of Boston had been cancelled, but that I had been put on another flight an hour later. At the gate, it became clear that the 12:30 had also been cancelled, and that the (I imagine) sparse occupancy of three different planes had been crammed together to make one very full 11:30 flight to Philly. Of course, it was also worth noting that the plane looked like it was falling apart on the inside - a large number of the overhead panels (with the lights and air nozzles) were pushed out of line with each other, or tilted about 3 degrees relative to the luggage compartments.

The flight from there to Charleston was unremarkable (although my cousin Eleanor was not so lucky and her luggage disappeared when she took the same flight a day later). We all had a good time with the Warner clan, and most of the Lyons and Heinsohn branches were also represented (though cousin Frank couldn't make it). Eleanor disappeared for an hour and a half and freaked out her parents, and I ate way too much of Aunt Peggy's shrimp salad.

On my way back to Boston, my flight out of Charleston got cancelled, supposedly for a mechanical problem. The clerk said the usual spiel about how it was better that they stopped the flight than flew with a bad plane, but this misses the point. In a competent company, the plane doesn't have a mechanical problem in the first place. Planes get serviced, and unservicable planes get removed from the fleet. At the very least, the company should have spares available regionally to pick up the slack. My own suspicion, however, is that the plane was fine except for not being full enough to make the flight profitable. Thus, people were shunted onto alternate routes. I ended up flying to LaGuardia rather than Charlotte and got home 3 hours late. My bags arrived on time, however; they took the 1 PM LGA-BOS flight while I had to wait till the 2 o'clock. This fact was announced only after the later flight's bags had rolled around on the conveyor belt -- a paltry 20 or so bags for a nearly-full flight. I found my bag sitting in front of the luggage office and ran while I still could.

Was anything really awful about this? No. But it's not really awful experiences that convince me to stay away from something. Truly terrible experiences, like wonderful ones, tend to be statistical anomalies and shouldn't be focused on unless they recur. It's the grating, repetitive march of insensitive and unprofessional behavior that really puts me off air travel in general, and now US Airways in particular. I'll probably have to fly with them in the future, but dang if I don't hope to avoid it.
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