An enzyme with a monkey's tail

It is rare, but not unheard of, for a human baby to be born with a tail. Atavism of this kind is generally understood to be the result of mutations in regulatory genes that cause an ancestral pattern of development to re-emerge. A physiological step backwards through the path of descent is often easy to recognize, because many of the evolutionary relationships are known. It should also be possible to identify atavistic events in particular molecules. For instance, one can imagine that a mutation to CLC-0 might result in a reversion to the ancestral transporter function. In a recent article in PLoS Biology, researchers from Florida State University and Brandeis University identify just such a relationship in the bi-functional enzyme inosine monophosphate dehydrogenase (IMPDH). PLoS Biology is an open-access journal, so open it up and follow along.

IMPDH plays a critical role in the synthesis of guanine nucleotides, an essential component of DNA. Two reactions take place in the active site — first, the inosine ring is oxidized to xanthosine, forming a covalent linkage with the enzyme, and then this bond is broken by a hydrolysis. The enzyme active site changes shape to carry out the reaction, bringing a catalytic arginine (R418) into position to activate the water for nucleophilic attack. Any time you see a complicated mechanism like this, it's natural to wonder how such a system could have evolved. Min et al. performed simulations and experiments to find out.

Using a crystal structure of IMPDH as a starting point, Min et al. performed hybrid QM/MM simulations in which the atoms taking direct part in the reaction were treated with quantum mechanics, and the rest of the protein was simulated using molecular mechanics. As one would expect given the enormous reduction in catalytic rate that occurs when R418 is mutated, the reaction proceeded through the arginine when the simulation had a neutral R418 side chain. The water is stabilized by two additional side chains from T321 and Y419, and reacts almost instantaneously, without the formation of a stable hydroxide intermediate. Although this is unusual, this prediction of the simulation is consistent with isotope effect experiments.

When the arginine was replaced by a glutamine in the simulation, the mechanism changed, naturally. Under these conditions, it was Y419 that activated the water for the hydrolysis, although the energy barrier was much higher (leading to a slower reaction). Again, the characteristics of the reaction indicated by the simulation line up pretty well with the results of biochemical experiments. Of course, Y419 enters the active site the same way R418 does, so the question of how the hydrolase activity could have evolved remains open.

Something very interesting, however, happens when the simulation is performed with R418 in a charged state. A fully protonated arginine will have a very hard time activating water for a nucleophilic attack. The simulation indicated that under these conditions, T321 performed this role, after being activated by a nearby glutamate (E431). T321 is adjacent to cysteine 319, which is essential for the oxidation reaction, and is not located on the mobile flap. If T321 really can catalyze hydrolysis, this would mean that it is possible that IMPDH possessed an (inefficient) hydrolysis activity before it evolved the mobile flap.

Because T321 only plays a signficant role in catalysis when R418 is protonated, blocking this pathway should result in decreased IMPDH activity at low pH. This is precisely what Min et al. observe in enzymatic assays (Figure 5) on a mutant in which E431 is mutated to glutamine. There is other experimental support as well: IMPDH enzymes that have been mutated at R418 usually have large isotope effects, which makes sense in light of the fact that the alternative T321 pathway involves the simultaneous transfer of two protons (rather than just one).

Things get even more interesting when IMPDH is compared to one of its cousins, GMP reductase. Although GMPR catalyzes a very different reaction, the C319/T321/E431 triad is also present there. This, along with other data from sequence alignment, suggests that these three residues were also present in a similar configuration in the ancestor of these modern proteins. Over time, progressive optimization of the two proteins resulted in the T321 pathway being supplanted by the more effective R418 in IMPDH, while remaining essential in GMPR.

If T321 really is a remnant of an earlier water-activating pathway, why is it conserved now that IMPDH has a much more efficient catalytic residue available? T321 is probably preserved because it stabilizes the water while it is being activated by R418. However, the other essential residue of that activating pathway (E431) is usually an inactive glutamine in eukaryotic forms of IMPDH (and some prokaryotes, as well). In these species the T321 activation pathway has been completely supplanted by the arginine pathway. Yet in the other forms of IMPDH this alternative mechanism still lingers, perhaps because of the additional activity it affords at low pH, or because it confers resistance to a particular inhibitor of the enzyme. In that sense, IMPDH's "tail" might provide an adaptive advantage quite different from that which gave rise to hydrolytic activity in the first place.

Donghong Min, Helen R. Josephine, Hongzhi Li, Clemens Lakner, Iain S. MacPherson, Gavin J. P. Naylor, David Swofford, Lizbeth Hedstrom, Wei Yang, Daniel Herschlag (2008). An Enzymatic Atavist Revealed in Dual Pathways for Water Activation PLoS Biology, 6 (8) DOI: 10.1371/journal.pbio.0060206 OPEN ACCESS
Disclaimer: Although I have little contact with Dr. Hedstrom's group, I am also working at Brandeis.

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Guided by the (blue) light

The ability to sense and respond to magnetic fields is a fundamental aspect of behavior in many animals. While migratory birds famously use the earth's magnetic field to navigate during, magnetic field responses occur in all manner of animals, from eels to invertebrates. Even the lowly fruit fly, best known as a reminder that you really should have taken the garbage out two days ago, can react to magnetism. While various explanations have been put forward in different species, magnetosensitivity remains fairly mysterious. In this week's Nature, researchers from the University of Massachusetts Medical School show that the blue-light photoreceptor cryptochrome plays an essential role in allowing fruit flies to detect magnetic fields.

Cryptochrome (or Cry) inherited the ability to receive blue light along with its photolyase domain, which is homologous to a prokaryotic, light-dependent DNA repair protein. Cry proteins, which are present in all animals, do not perform any DNA repair work, but instead play a role in regulating the circadian rhythm. While it is not clear in all cases whether Cry's ability to absorb blue light is biologically significant in clock regulation, it is known that fruit flies (Drosophila melanogaster) use Cry to synchronize their circadian clocks. Previous experiments had suggested that the ability of fruit flies to detect magnetic fields was somehow related to photoreception, and that short wavelengths (like those sensed by Cry) had different effects from longer ones.

Gegear et al. devised a relatively simple experiment to test the importance of Cry in Drosophila magnetosensing. They placed a T-junction in a box, with a magnetic coil on one side and a non-magnetic coil on the other. They released flies into the junction, with (trained) or without (naive) performing an earlier run where the magnetic field was associated with a sucrose reward. They shined a light into the box and used filters to investigate the role of specific wavelengths.

They discovered that several strains of Drosophila could be trained to go to the magnetic field, although the degree of preference and the nature of the naive response differed substantially between strains. Gegear et al. chose the strain that showed the greatest response in full-spectrum light (and displayed a tendency to avoid the magnetic field in the naive state) to perform the filter experiment. Cutting off all wavelengths of light shorter than 500 nm abolished both the naive and trained responses to the magnetic field in these flies, as did filtering out all wavelengths shorter than 420 nm. If only wavelengths shorter than 400 nm were cut off, some of the trained and naive response returned. Simply dimming the light was not enough to replicate the effect of filtering. These experiments indicate that magnetic sensitivity in these flies requires light in the blue to ultraviolet range.

In order to prove that cryptochrome specifically is necessary for this magnetic sensitivity, Gegear et al. took advantage of our tremendous knowledge of fly genetic manipulation to create mutant flies that did not have a functional Cry gene. No matter what wavelengths of light were used in the T-junction experiment, these flies did not respond to the magnetic field. Crossing these Cry-null mutants with normal flies restored magnetosensitivity. The authors also performed experiments to show that the circadian rhythm was not itself essential to magnetic response in the flies.

Because this is a genetic experiment, it cannot address the question of whether Cry is both the blue-light photoreceptor and the magnetosensor. Going just on what we have in this paper, it is also possible that Cry acts upstream of another magnetosensor protein or is part of its downstream signaling pathway. However, in light of research that shows the flavin photoreception in other cryptochromes induces the formation of magnetically-sensitive radicals, some of which I discussed last year, it certainly seems possible that Drosophila cryptochrome does the whole job itself. As I mentioned in the case of the previous article, though, there is not yet any understanding of a mechanism by which information about magnetic field could be transduced from Cry radicals into the nervous system.

Dorosophila Cry differs from other plant and animal Cry proteins in significant ways, so it's unclear whether these results have any relevance for other organisms. However, the finding that Cry is essential to Drosophila magnetosensitivity suggests at least the possibility of parallel systems in migratory birds and other species that use magnetic fields.

Robert J. Gegear, Amy Casselman, Scott Waddell, Steven M. Reppert (2008). Cryptochrome mediates light-dependent magnetosensitivity in Drosophila Nature, 454 (7207), 1014-1018 DOI: 10.1038/nature07183

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How to help an enzyme crack cocaine

In addition to the adverse consequences of addiction and the inconvenience of serving several years of jail time for possessing it, cocaine can cause a fatal overdose. Although this condition can be treated, no therapy presently exists that attacks the overdose by removing cocaine from the bloodstream. One possible approach to eliminating cocaine from a patient would be to accelerate the process by which it is degraded. Unfortunately, the enzymes that perform this activity in the human body are not very efficient. In an upcoming article in the Journal of the American Chemical Society, however, a group from the University of Kentucky (assisted by researchers at the University of Michigan) have remodeled the active site of butyrylcholinesterase (BChE) to achieve a 2000-fold increase in rate. This raises the possibility of producing therapeutic enzymes as a treatment for cocaine overdose.

A cocaine overdose typically results in an elevated pulse rate, seizures, and hyperthermia, among other possibilities. The usual course of treatment involves addressing the symptoms — diazepam to reduce the heart rate, cooling protocols to address hyperthermia. These steps are proven to work, but they don't address the core problem: there's still a lot of cocaine floating around in the bloodstream. Treating with sedatives amounts to using one giant truck to stop another giant truck... both trucks will probably stop, but there might be a lot of collateral damage. Instead, it would be advantageous to either block the receptors that cocaine binds, or clear cocaine from the bloodstream somehow.

Plasma butylcholinesterase does most of the work in metabolizing cocaine, by cleaving it into two products that no longer exert the same pharmacological effects. If BChE was a highly efficient enzyme it's unlikely that people would experience cocaine overdoses at all, but it breaks down the main form of cocaine quite slowly, with a catalytic rate (k_cat) of 4.1 /min, resulting in a very long half-life for this substrate. The chemical mechanism of BChE (Figure 1) will be familiar to anyone who has taken biochemistry, being basically the same as a serine protease. Instead of a peptide bond, however, it is the ester linkage of cocaine that undergoes nucleophilic attack from an activated serine, while hydrogen bonds stabilize the evolving negative charge in an oxyanion hole.

Previous efforts to optimize the activity of BChE by mutation focused on eliminating steric clashes, but Zheng et al. noted that the hydrogen bond lengths in the oxyanion hole were not optimal for stabilizing the putative transition state. They therefore decided to focus their efforts on improving the energetics of this region. To do so, they used combined quantum mechanics/ molecular mechanics (QM/MM) simulations to determine the energy barriers in simulated reaction coordinates for a number of different mutants. This has the advantage of screening potential mutants for a specific effect, which may be quicker than wet lab work, but it requires the researcher to know the catalytic mechanism and to define a region of interest in advance.

By working through a series of mutations, Zheng et al. arrived at one multiple mutant of BChE that had favorable interaction energy for every residue in the oxyanion hole. When they generated this mutant in the lab, they found that it had a vastly increased catalytic rate towards cocaine, with k_cat now about 5700 /min. Based on these in vitro results they decided to test the mutant BChE in vivo using mice. They found that injecting mice with 30 µg of BChE protected them from seizure and death due to cocaine overdose. While the n for this experiment is small, and the BChE was injected prior to cocaine exposure rather than after, these results suggest that the mutant BChE has potential as a therapy for cocaine overdose in humans.

Obviously, further improvement would be needed before these protective effects could be realistically equaled in humans. To match the dose used in this experiment, a 180-pound man would need to be injected with 82 mg of the protein, which is a rather large amount. However, if used in conjunction with existing treatments, the required dose of BChE may be lower. If not, then translating these results into a useful therapy will require either further catalytic optimization or an enormous production effort. A significant amount of additional clinical research is required before this or any other mutant of BChE is introduced as a therapy for overdose or addiction. Nonetheless, these results illustrate the promise of enzyme optimization and design as a tool for medicine in the future.

Fang Zheng, Wenchao Yang, Mei-Chuan Ko, Junjun Liu, Hoon Cho, Daquan Gao, Min Tong, Hsin-Hsiung Tai, James H. Woods, Chang-Guo Zhan (2008). Most Efficient Cocaine Hydrolase Designed by Virtual Screening of Transition States Journal of the American Chemical Society DOI: 10.1021/ja803646t

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Two great mechanisms that go great together

The watery interior of a cell is separated from the watery exterior of a cell by a thin double layer of lipids called the plasma membrane. The oily interior of this membrane prevents water and charged molecules from escaping the cell, while allowing hydrophobic (oil-like) molecules through. This system has many significant advantages, but cells frequently need to move charged atoms (ions) across the membrane. This job is primarily performed by two kinds of protiens: channels that create specialized tunnels through the membrane, and transporters that mechanically transfer ions across the membrane. These are distinct activities, usually carried out by different families of proteins. Recent results in the ClC family of membrane proteins, however, have demonstrated that these activities are not as distant from each other as it might seem.

Ion channels basically work like tiny pipes that stick through a cell membrane. They have interior pores that are full of water, and usually possess some form of selection mechanism that lets only a particular kind of ion through. Although they can be gated — opened or closed by particular voltage states or molecular signals — they can only move ions with an electrochemical gradient. That is to say, they can only allow their particular ions to move across the membranes in a way favored by both concentration and voltage. By contrast, transporters physically translocate ions without using a watery pore. This allows them to move ions against a concentration gradient as long as they have an energy source, such as another ion's concentration gradient.

The figure cave drawing at right shows a simplified situation. A membrane divides two compartments, one of which has a high concentration of negative ions (red), while the other has a high concentration of positive ions (blue). The selective channel (C) can only allow negative ions to move from the high to low concentration in this situation, because the concentration and electrical gradients oppose a movement in the opposite direction. The transporter (T), on the other hand, can push negative ions out into the area where they have a high concentration. It does this by simultaneously transporting a positive ion from high to low concentration; it uses the favorable energetics of this transport event to power the unfavorable one. Of course, there are many different kinds of transporters -- the example here is a symporter, but there are also antiporters or exchangers.

Aside from dealing in ions, these two kinds of activity might seem to have little in common, and in fact most ion channels are not very closely related to ion transporters. However, the ClC family of chloride channels is unusual in that it also includes several members that are transporters. Because all these proteins are presumed to have similar structures in the membrane, there is considerable interest in understanding the key differences that separate ClC channels from ClC transporters. In the case of these proteins, it seems that the line between transporter and channel is easily blurred.

You got transporter in my channel!

The ClC-0 protein from the electric ray Torpedo marmorata has always been classified as a gated channel, but an odd one. Unlike many channels, it has two pores in its active configuration. These pores are closed off or gated by two processes. The fast gating occurs on the millisecond timescale and opens and closes the two pores independently of one another. The slow gating occurs on the timescale of seconds and opens and closes both pores.

The odd thing is that there is a thermodynamic imbalance in this system. All things being equal, we expect that if we track the number of pores that are open over time, we should see the pattern 1 → 0 → 2 → 1 (called J₊) with the same frequency that we see the pattern 1 → 2 → 0 → 1 (J_-). Instead, researchers have found that J₊ is observed more frequently than J_-. Originally it was thought that the electrochemical gradient of chloride controlled this asymmetry, but this model didn't quantitatively match the observations. Now Lísal and Maduke have shown that the asymmetry arises from the proton electrochemical gradient, and that this is a molecular vestige of ClC antiporter function (1).

The evidence for this comes from an experiment in which the chloride electrochemical gradient was held constant while the proton electrochemical gradient was changed. If the chloride gradient controls the asymmetry, we should see no differences in the J₊/J_- ratio during the experiment. Instead, it was observed that switching the direction of the proton gradient changed the behavior of the channel gating from almost exclusively J₊ to an even mixture of J₊ and J_-. Further experiments demonstrated that the J₊/J_- ratio was proportional to the proton electrochemical gradient, and that it leveled off at 1 when the direction of the gradient was changed to favor movement of protons from inside the cell to outside the cell. This suggests that the gating mechanism is unidirectional.

Because the proton gradient provides energy to produce gating asymmetry, CLC-0 must be a proton transporter as well as a chloride channel. The authors suggest that this reflects a vestigial antiporter activity (similar to several existing members of the family) that has been repurposed as a regulatory mechanism for channel gating. Although channels have very different thermodynamics from transporters, it appears that a single protein can have characteristics of both.

You got channel in my transporter!

But how does an antiporter transform into a channel? Because the structures of these different types of proteins tend to be quite dissimilar, it seems unlikely that the transition between these mechanisms could be accomplished with just a few point mutations. However, sequence homology in a family of proteins usually implies structural homology (with some exceptions), so the ClC channels probably evolved from antiporters in this fashion. This could happen by the destruction of gating mechanisms in the transporter.

In the Cl^-/H⁺ antiporter CLC-ec1, a central binding site for chloride is blocked by two gates. One side is blocked off by Glu148, and the other is putatively formed by the interaction of Tyr445 and Ser107. In principle, removing these gates could give rise to a watery passage through the membrane, and high transport rates similar to those of ion channels. Jayaram et al. therefore performed a series of mutations at these sites and measured the effect on transport rates (2).

From previous work it was known that altering these residues uncoupled chloride transport from proton transport, which is one step towards becoming a channel. Here, Jayaram et al. find that mutating Tyr445 to a smaller side chain does not have a significant effect on chloride transport rates. Mutating Glu148 to smaller residues actually decreases the chloride transport rates. Mutating both residues, however, leads to a 100-fold increase in chloride transport over the wild-type protein. The fastest mutant, moving chloride ions at a rate of more than 35,000 /s, is not quite as fast as a channel. Still, the acceleration is significant.

Jayaram et al. found that increasing the size of the substituted side chain at either site decreased the rate. This is what you would expect for a simple case of larger side chains leading to more constriction of a channel. In order to confirm that there was such a channel, they crystallized one of the mutants and analyzed it to determine whether there was a continuous pathway that water could permeate. Below, you can see a figure I shamelessly stole from their paper, with the WT protein on the left and the E148A/Y445A mutant on the right. The red dots represent a surface made with a 1.4 Å probe designed to mimic water. As you can see, the probe can go all the way through the A/A mutant, but is stopped by the intracellular gate in WT. The double mutant is a tight, but genuine channel.



Because these residues are conserved in both channel and antiporter members of the ClC family, mutations like these are not likely to be the means by which one kind of protein evolves from the other. Nonetheless, they establish that the crystal structure of CLC-ec1 is likely to be a good model for occluded states of gated ClC channels. Moreover, the ease with which a specialized ClC transporter is made into a channel suggests that a progenitor protein could have switched from antiporter to channel in just a few mutations.

The thermodynamics of ion translocation by channels and transporters are quite different, so it was a surprise to discover that the ClC family contained both kinds of activities. These recent papers show that the entanglement is even closer than previously thought. The ClC channel CLC-0 still retains vestigial proton transport activity, and the ClC antiporter CLC-ec1 is only a few mutations away from becoming a channel itself. These findings suggest that substantial changes in operating thermodynamics may result from small evolutionary steps, and point to a shared antiporter past for members of the ClC family.

1. Jiří Lísal, Merritt Maduke (2008). The ClC-0 chloride channel is a 'broken' Cl−/H+ antiporter Nature structural & molecular biology, 15 (8), 805-810 DOI: 10.1038/nsmb.1466

2. H. Jayaram, A. Accardi, F. Wu, C. Williams, C. Miller (2008). Ion permeation through a Cl--selective channel designed from a CLC Cl-/H+ exchanger Proceedings of the National Academy of Sciences, 105 (32), 11194-11199 DOI: 10.1073/pnas.0804503105
Disclaimer: I work on the same floor as all these guys.

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How media resemble real life in your head

How does the human brain react to the communication of emotion? Does the observation or imagination of emotions have anything in common with the personal experience of them? It is possible that the brain uses a setup in which seeing a person experience an emotion, imagining that emotion, and feeling that same emotion all use completely independent circuitry. Yet since all of these experiences make references to the same emotional state, it is also reasonable to think that some of the pathways are shared. In a recent article from PLoS ONE, a team of researchers uses functional Magnetic Resonance Imaging (fMRI) to determine similarities and differences in the patterns of brain activation following various means of communicating disgust. PLoS ONE is open access, so go ahead and open the article up in another window.

First, a word about fMRI, for those unfamiliar with it. As the name would suggest, fMRI is an elaboration of the standard MRI techniques used image the interior of your body without the use of potentially harmful radioactivity. Neuronal activity in the brain causes a local depletion of oxygen from the blood, followed by a localized increase in blood flow. Because the magnetic properties of iron in the blood change with its oxygenation state, it is possible to detect these hemodynamics using magnetic resonance imaging. Thus, fMRI is able to indirectly detect neural activity, although the fMRI signal lags behind activity by a few seconds. A given fMRI signal also encompasses a large number of individual neurons and therefore can only serve as a rough map to where things are happening in the brain. These temporal and spatial limitations limit the conclusions that can be drawn reliably from fMRI, but the observed correlations can provide valuable insights.

Jabbi et al. used fMRI to map the neural response of subjects to various encounters with disgust. Previous research had shown that a particular region of the brain (the IFO) showed increased activity when subjects either tasted something disgusting, or viewed a short clip of someone else tasting something disgusting. For this study, Jabbi et al. had participants read short scripts (samples can be found in the supplementary materials) intended to make the reader imagine being disgusted, pleased, or not feeling anything. They found that reading disgusting passages induced a neural response in this region of interest, just as it had for the cases of tasting or observing disgust.

While this may seem completely unsurprising, it bears some consideration. The experience of personal disgust differs significantly from the experience of observing disgust in others. Similarly, imagining or reading about disgust creates a very different subjective experience than, say, drinking quinine. Given that these are all quite different feelings, it is somewhat surprising that a single area is activated by all three.

Of course, there is a fine line to consider here -- the passages meant to make the subjects imagine disgust may have actually disgusted them. The paragraphs that the authors make available in the supplementary materials are written in second person and involve things like accidentally ingesting animal waste. Because the subjects are reading passages that ask them to imagine themselves being disgusted, and the passages are themselves disgusting, the act of imagination may be contaminated by an immediate personal experience of disgust. In a more elaborate experiment it might be of value to use passages written in the third person. Additionally, it might be useful to employ passages in which the characters, because of particular phobias or personal experiences, are disgusted by items or actions the reader is likely to find innocuous.

Whether the readers where themselves disgusted or not, the overall response in the brain differed for each of the stimuli, as shown by a map of correlated activity (Figure 2). While the area outside the IFO activated by observation was relatively small, both the disgusting taste and the disgusting scripts produced widespread activity relative to a neutral taste or script. In general there was not much overlap between the networks, except for a small region shared by the imagination and experience groups. The authors propose that the similarities of imagining, observing, and experiencing emotion are due to the common activation of the IFO, while the differences between these are due to the largely distinct networks of correlated activity. Different modes of exposure to disgust may therefore act in complementary, rather than independent, ways.

Additionally, this result appears to be consistent with the view that our recognition of observed disgust and our imagination of disgust rely on an internal simulation of our own feelings of disgust. However, these experiments cannot establish exactly what a particular region of the brain is doing, so this remains an open question.

While this research does not indicate whether these results can be generalized to other emotional states, this finding may interest developers of media that make use of multiple modes of communication, specifically video games. Games often rely on video cutscenes to convey story and emotion, but this approach may be wasting a significant amount of potential. The participatory nature of games makes it possible to approach emotional communication not only through the observational route, but also the experiential route.

Consider the case of Agro's fall in Shadow of the Colossus. Observing the cutscene, and hearing the voice of Wander, the player can understand that Wander feels grief at this event, in much the same way that anyone watching a movie could understand it. Additionally, the emptiness of the game's landscape and the forced collaboration between the player and the Agro AI has helped to create a relationship between the player and the horse. Thus, in observing Agro's fall, the player may feel his own sense of grief at the event, increasing the emotional resonance of the moment.

This suggests a possible, if lengthy, experiment. It would be interesting to compare the fMRI profile of a subjects observing Agro's fall under two conditions: one in which they have actually played the game up to that point, and another in which they have watched the game as a movie, with exploration and battles recorded previously from an expert player's run. Would the first group have activity in both the observational and experiential networks, or would each group activate a different network? What implications might these outcomes have for the development of emotionally fulfilling games?

Of course fMRI studies are not some holy grail that makes everything clear. The work of Jabbi et al. has given us a rough map to where things are happening, but understanding exactly what is happening and how it is happening will require additional experiments and possibly new investigative techniques. Nonetheless, this is an interesting piece of the puzzle, and perhaps some food for thought.

Mbemba Jabbi, Jojanneke Bastiaansen, Christian Keysers (2008). A Common Anterior Insula Representation of Disgust Observation, Experience and Imagination Shows Divergent Functional Connectivity Pathways PLoS ONE, 3 (8) DOI: 10.1371/journal.pone.0002939

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Do conformational changes precede or follow binding?

The binding of a ligand to a protein rarely occurs with the simplicity of a block sliding into an appropriately-shaped hole. Protein and ligand often engage in complementary conformational changes to adapt their shapes to each other. As a result, the structure of a protein bound to its target may differ substantially from the structure of the free protein. Unfortunately, it is virtually impossible to view the binding process in fine structural detail; as a result, most of our knowledge comes from the relatively stable bound and free states. Improving biophysical techniques, however, have brought a change in the way we view some binding events.

Most alterations of conformation during a binding event have historically been interpreted using the induced fit model. In this view, the protein stably maintains the free or "open" structure until it comes into contact with a ligand molecule. This encounter stimulates a conformational change so that the protein adopts the "closed" conformation that tightly holds onto the ligand. Thus, the ligand induces the conformational change necessary to form the bound, closed (BC) structure from the unbound, open (UO) structure, and the intermediate on this path is some kind of bound, open (BO) structure. This model is physically reasonable and has been very successful in interpreting many systems.

However, for the past few decades an increasing amount of evidence has suggested that this is not the whole story. NMR investigations indicated that instead of remaining in a single, well-defined backbone conformation most of the time, many proteins experienced significant changes in their structure while floating free in solution. These results suggested an alternative mechanism of population shift. In this view, the protein actually samples the "closed" conformation (or something very similar) while unbound, and it is this conformation that binds to the ligand. We still go from UO to BC, but now the intermediate is an unbound, closed (UC) structure.

This sounds very arcane, but it is not without functional relevance. Consider, for instance, a protein that is activated by a particular ligand. If we wish to make a drug that binds exclusively to the BC form, then we may experience unforeseen side-effects if our target protein occasionally samples a UC state. It would be useful to have a general idea of what kinds of circumstances are likely to favor a population shift model vs. an induced fit model. That is precisely what Kei-Ichi Okazaki and Shoji Takada aim to provide in an upcoming paper in Proceedings of the National Academy of Sciences (1).

Okazaki and Takada performed a coarse-grained molecular dynamics simulation of glutamine binding protein. In the bound and unbound states they employed a double-well Gō model, a simplified representation of molecular forces, to represent "opening" and "closing". To switch between these states (i.e. to represent binding) they used a Monte Carlo algorithm. This approach has the advantage of being quick and relatively inexpensive from a computational standpoint, but the results must be interpreted cautiously because the physics of the model are greatly simplified. They observe UO ↔ UC and UC ↔ BC events in this system, but they also observe UO ↔ BO and BO ↔ BC events. This suggests that the simulation will be able to make predictions about both population-shift and induced-fit mechanisms.

In order to try to make some predictions about the circumstances in which a particular mechanism is favored, Okazaki and Takada varied the strength and range of the binding interaction. By monitoring whether the simulated system entered the BC state from BO or UC, they could tell whether the system obeyed the induced-fit or population-shift mechanisms, respectively. They find that as either the strength or the range increase, the induced-fit mechanism is increasingly favored (Figure 4). These results make sense. If the protein regularly samples the closed state while unbound, then the amount of energy needed to reach that state is probably small, so it makes sense to see a population-shift mechanism associated with low-energy binding. Similarly, if a ligand is to associate productively with a non-optimal protein conformation, it makes sense that key interactions will be effective at long range.

From these results Okazaki and Takada suggest that the binding of small hydrophobic ligands is generally likely to proceed via population shift, while the binding of large, charged ligands (such as DNA) will likely proceed via induced fit. They acknowledge, however, that the simulation is limited, particularly in its view of conformational change. Unitary transitions in which the whole protein changes its structure simultaneously are probably not the norm, particularly in the case of very large conformational changes. These changes may instead be stepwise or hierarchical. For instance, a protein or complex recognizing multiple features of a DNA strand may proceed by an apparently induced-fit mechanism, even though each individual binding event more closely resembles population-shift behavior.

An additional limitation of this study is that it considers only one protein, but mechanisms of binding and conformational change may be idiosyncratic properties of particular folds. One could consider the behavior of lymphotactin, which displays clear hallmarks of the population-shift mechanism despite binding to macromolecules (heparin and a GPCR) much larger than itself, as a counterpoint to the predictions developed here. Similarly, the population shift of NtrC involves a charged phosphate group likely to have long-range interactions, although this is a post-translational modification and not a strict ligand-binding event. While the authors point to some examples that match their expectations, overall the data are not unanimously in support of their predictions. Still, the general rules laid out here provide a starting point for experimental work.

Despite the limitations of the simulation, it provides a relatively efficient tool for assessing these processes in other proteins. While no simulation can yet replace experimental data, coarse-grained models like this can serve as a means to formulate testable hypotheses about the energetics of protein-ligand systems.

1. Okazaki, K., Takada, S. (2008). Dynamic energy landscape view of coupled binding and protein conformational change: Induced-fit versus population-shift mechanisms. Proceedings of the National Academy of Sciences 105(32) 11182-11187. DOI: 10.1073/pnas.0802524105

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NSAIDs vs. Alzheimer's: Multiple modes of action?

Loads of interesting stuff is going on in Alzheimer's research right now. While the hot news is about a trial showing significant benefits from going after tau tangles, a recent paper in PLoS ONE continues to investigate the pathology of the amyloid-β peptide. As I've mentioned in previous posts, cleavage of the amyloid precursor protein to a 42-residue peptide (called Aβ1-42 in this paper) initiates the formation of peptide oligomers and eventually plaques. Recent research has indicated that these oligomers are sufficient to cause the development of Alzheimer's disease, but the mechanism by which they do so remains uncertain. Sara Sanz-Blasco and colleagues show that Aβ oligomers disrupt calcium homeostasis in neurons, damaging the mitochondria and promoting apoptosis, and that certain NSAIDs can suppress these adverse mitochondrial effects (1). PLoS ONE is open access, so go ahead, open the article up in another window, and follow along.

Although the appearance of plaques and neuronal death are classic hallmarks of Alzheimer's pathology, the relationship between these features is not well understood. For instance, it is possible the plaques themselves kill neurons or impair neural function. However, it seems equally likely that the appearance of plaques and the death of neurons are two distinct effects with a single cause. This view is supported by the oligomer toxicity study, but that study fails to resolve the question of exactly how Aβ oligomers kill neurons. Previous work has associated Aβ with derangement of cellular calcium (Ca²⁺) management — a 2005 paper by Demuro et al. (2) showed that soluble Aβ induced an increase in intracellular Ca²⁺ in a neuroblastoma cell line. Sanz-Blasco et al. therefore decided to directly test whether Aβ oligomers were increasing Ca²⁺ levels in neurons, and specifically in mitochondria. In order to do this last bit they used a low-affinity aequorin targeted specifically to mitochondria.

Allow me digress... to many of my readers that probably sounds like a terrible idea. If you're trying to detect a particular chemical in the cell, it seems like the best thing to do would be to get a high-affinity binding partner. And if figuring out whether there is any calcium in the mitochondria is what you want to do, then a high-affinity detector makes sense. However, when you're using a small amount of a sensor to detect changes in the concentration of a large amount of ligand, a low-affinity sensor is what you want.

To see why, take a look at the graph on the right. This is just a rough calculation based on a situation where the detector is at a concentration of 100 µM and the concentration of its ligand (that you're trying to detect) changes from 10 mM to 100 mM. Note that the concentration of the detector is at most 1% that of the ligand. If the dissociation constant K_D of this complex is 1 mM (blue) (a lower K_D means higher affinity), then the detector is almost saturated when you start, and the percentage occupied doesn't change very much over the course of the experiment. This means that it will be very difficult to tell the difference between, say, 50 mM ligand and 100 mM ligand, because that amounts to a signal difference of 1% of the maximum response. The situation gets a little better if the K_D is 10 mM (green). The lowest affinity detector here (K_D = 50 mM, red) actually does the best job of distinguishing between 50 mM and 100 mM ligand, because the difference in response amounts to 17% of the total dynamic range. Ideally, you want to tune the K_D of your detector in such a way that its response to changes in ligand concentration is large and linear over the range you are likely to be observing. For the last detector, this range lies between 10 and 40 mM of ligand, so that would likely be the ideal range to investigate with it.

The precise numbers are different in the present paper, but the principle is the same. The affinity you want in your detector will depend on what you are trying to detect and the circumstances under which you are trying to detect it. In this case, the researchers are trying to measure changes in calcium ions over a fairly wide range, which have a pretty high concentration in mitochondria, and they're doing it using a luminescent protein, which isn't very concentrated. As a result, a relatively low-affinity detection system is best.

So, what did they find? The results in Figure 1 show that Aβ oligomers and fragments cause an influx of calcium into the cytoplasm of cultured neurons, but preparations of Aβ fibrils did not cause this effect. Moreover, exposure of the cells to Aβ oligomers caused a clear influx of calcium into the mitochondria (Figure 3). This is a problem for a cell because Ca²⁺ overload in mitochondria can cause programmed cell death, or apoptosis. Using the classic TUNEL assay, the authors of this study showed that the Aβ oligomers caused apoptosis. In addition, they showed that treatment with the oligomers caused the release of mitochondrial cytochrome c (a step in the apoptotic pathway) and that the addition of cyclosporin A, which inhibits the release of proteins from the mitochondrion, blocked cell death (Figure 4). Together, these pieces of evidence support the idea that Aβ-induced Ca²⁺ influx into the mitochondria activates the apoptotic cascade, leading to neuronal death. These results are consistent with a very cool study published this week in Neuron (3) showing that amyloid plaques correlated with high neuronal Ca²⁺ levels in vivo (in live mice).

On its own this is pretty interesting, but Sanz-Blasco et al. push it a bit further. Because they had shown previously that some NSAIDs prevent mitochondrial Ca²⁺ uptake in a cancer cell line, they decided to find out if they would work in this instance, too. As you can see from Figure 6, the three NSAIDs tested kept the mitochondria calcium-free, even if the cells were treated with Aβ oligomers. NSAIDs also prevented cytochrome c release and cell death (Figure 8).

Some readers may recall that Kukar et al. showed that certain NSAIDs prevent oligomerization of Aβ1-42, hinting at a possible explanation of these results. However, the controls performed by Sanz-Blasco et al. show that under the conditions of these experiments the NSAIDs they used have no effect on cytosolic Ca²⁺ concentrations (Figure 7). If it is amyloid oligomers that let Ca²⁺ through plasma membranes, then this would appear to rule out structural disruption as a mechanism. Instead, Sanz-Blasco et al. propose that these NSAIDs specifically alter the polarity of the mitochondrial membrane in such a way as to prevent Ca²⁺ uptake.

If this is true, then NSAIDs may be able to perform a double-whammy on Alzheimer's disease. On the one hand, they appear to be capable of altering Aβ cleavage patterns to reduce the formation of toxic oligomeric precursors. In addition, they appear to have an ability to block mitochondrial breakdown and subsequent apoptosis directly. While this is encouraging, and speaks to the value of pursuing refinements of existing NSAIDs as possible Alzheimer's treatments, this experiment doesn't necessarily prove any therapeutic value. Even if the neurons are saved from death, the calcium flood may impair their function to such a degree that their continued survival doesn't matter. Only clinical trials and further research can firmly establish whether current or optimized NSAIDs can provide significant protection against Alzheimer's disease.

1. Sara Sanz-Blasco, Ruth A. Valero, Ignacio Rodríguez-Crespo, Carlos Villalobos, Lucía Núñez (2008). Mitochondrial Ca2+ Overload Underlies Aβ Oligomers Neurotoxicity Providing an Unexpected Mechanism of Neuroprotection by NSAIDs PLoS ONE, 3 (7), 0-0 DOI: 10.1371/journal.pone.0002718 OPEN ACCESS

2. A. Demuro, E. Mina, R. Kayed, S.C. Milton, I. Parker, C.G. Glabe (2005). Calcium Dysregulation and Membrane Disruption as a Ubiquitous Neurotoxic Mechanism of Soluble Amyloid Oligomers Journal of Biological Chemistry, 280 (17), 17294-17300 DOI: 10.1074/jbc.M500997200 OPEN ACCESS

3. K Kuchibotla, S Goldman, C Lattarulo, H Wu, B Hyman, B Backsai (2008). Aβ Plaques Lead to Aberrant Regulation of Calcium Homeostasis In Vivo Resulting in Structural and Functional Disruption of Neuronal Networks Neuron, 59 (2), 214-225 DOI: 10.1016/j.neuron.2008.06.008

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"Run, you pigeons! It's Robert Frost!"

The title of this post comes from a line uttered by Manny Calavera, the main character of Grim Fandango as you fail to solve a puzzle. Because Grim Fandango is an adventure game, failing to solve puzzles is something you'll do an awful lot, so it was nice of the developers to at least give you a good laugh as you flail your pathetic way through the misadventures of a skeleton man in a film noir world. Adventure games always attempted to complement good stories with hard puzzles, and a widespread failure to achieve the right kind of difficulty probably contributed significantly to their fall from grace. As I've been playing Grim Fandango for the Vintage Game Club, I've been reminded of what kinds of problems they had.

This post will echo some things I said in the VGC forum, and it will also spoil the first two puzzles of Grim Fandango, so keep that in mind before you read on.

Adventure games had two kinds of hard puzzles. Some of them required you to pay careful attention to the game world, remember bits and pieces of dialogue, think creatively about the items you had found, and work it all together into a solution that gave you a great "Aha!" moment. The rest were bullshit. The problems Manny must solve in the first chapter of Grim Fandango mostly involve the first kind, but you start off, unpromisingly, with the second kind, and it is these that turned people off adventure games, with good reason.

At the start of Grim Fandango, Manny is desperate to get somewhere, but in order to get there he must get his boss to sign a work order for a car modification. To this point, the problem proceeds relatively naturally. However, in order to get the work order signed, Manny must leave his office building, discover a rope that his boss has used to sneak out of his office, climb up that rope, and change his boss' automated message so that the secretary will sign his name on the work order. This solution falls into the bullshit category for several reasons, the most significant of which is that there is no reason for the player, or Manny, to suspect that the boss isn't in his office, that he is using an automated message system to pretend that he is in, or that the message can be changed to make his secretary sign the order. In fact, because Manny has received an emergency work order from his boss just moments before, both he and the player have a very strong reason to believe that the boss is in, a view which the secretary reinforces by stating outright that she is only at work because the boss is. Without knowing that the boss sneaked out of his office, or that he did so in a way that allows Manny to sneak in, the decision to leave the building (which is required in order to find the rope) is nonsensical. A key step to solving the puzzle requires an unnatural response to Manny's problem.

When people say adventure games are "hard", this is a significant reason why. It's not that the puzzles are too intellectually demanding, it's that they require you to act in ways that are utterly unnatural to the solving of the puzzle. If there's an emergency and your boss isn't taking calls, you knock on his door. You don't go outside the building to check if he might have climbed twenty stories down a rope made out of ugly neckties. And if your wallet is locked in your girlfriend's car, you ask her for the keys. You don't try to combine a nearby cat with a mango from your inventory in hopes that this will produce a tool you can somehow use to unlock the passenger door.

Note that my problem is not that the solution requires Manny to go outside per se. In fact, getting Manny to go outside during this puzzle is an important part of making sure the player has the information he needs to solve the remaining problems of the early portion of the game. The illegitimate difficulty of this puzzle arises from the fact that the game never gives the player or Manny any reason to believe that going outside constitutes a step towards solving his problem.

Contrast this with the second main puzzle of the same game, in which Manny decides to steal a co-worker's client by intercepting one of his messages. To do this, Manny must find the room of pneumatic messaging tubes, break the system using chemical foam, find a way to keep the door to the system from closing after repairs are finished, and figure out how to stop the messages so he can read them first. Stealing the client requires several more steps than getting the work order signed, but the dialogue in the game and the items Manny notices in the game world all provide definite (if subtle) clues as to how the problem can be solved. Each step towards the solution makes sense in the context of the game world. The puzzle is difficult, but legitimate.

This distinction matters because the kind of difficulty involved has an effect on how the player feels once the puzzle is solved. Solving a difficult, natural puzzle makes the player feel like he's accomplished something—even if the player is frustrated while trying to solve the puzzle, success brings positive emotions because the player feels like he was in control of finding the solution. By contrast, puzzles that require unnatural solutions which are too obscure or don't make sense in the game world steal this feeling from the player. They make the successful solution of a puzzle seem like a matter of luck, rather than skill. In my personal experience, solving a puzzle under these circumstances can increase frustration, rather than relieving it.

Another problem with unnatural puzzles is that they break immersion. The work order problem can't be solved by thinking like Manny Calavera, but it can be solved by thinking like a guy playing an adventure game, who knows that you should explore everywhere to find the solution of a puzzle. Unfortunately, once I'm thinking like myself, my mind ceases to be active in the world of the game. While the game can get me back, each exit from the world of the game prompts the question of whether I should continue. If the player's mind disengages at a point of frustration, then he is always more likely to quit, at which point your work has gone to waste.

I started this post talking about adventure games, but I think these two points speak more generally to the use of difficulty in games. A high degree of difficulty, even unfair difficulty, is acceptable in a game so long as the player's sense of accomplishment and engagement in the game world are preserved. In games with very low difficulty, the player's sense of achievement may not be a design value of the game; however, even games with low difficulty must take care not to disengage the player with unnatural puzzles or other kinds of gameplay problems.

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The coltan controversy

The gaming internets were buzzing the last few days about a widely-publicized report from Toward Freedom that civil war and ongoing militia activity in the Democratic Republic of the Congo (DRC) have in part been fueled by the demand for coltan, a source of the tantalum powder that is necessary for capacitors in consumer electronics. Of course, this isn't a new issue, just a new press release, in which the Playstation 2 in particular is singled out as a cause of conflict. This would be an excellent reason to dislike gaming consoles were it not inflammatory nonsense.

It would be foolish to deny the destructive effects of coltan demand, of course. Although the civil wars in the DRC had political origins, the conflict was sustained in part by demand for this mineral resource. Neighboring countries used militias in the DRC as proxies to ensure access to coltan, which they then sold to a number of foreign companies, which then resold it to various manufacturers, including many American makers of capacitors. Prices for tantalum spiked significantly in the years 1999-2000, providing an even stronger incentive for the DRC's neighbors to seize its rich reserves of coltan. Conflict and deficiencies in infrastructure have at times limited industrial mining of coltan, meaning that it is often mined using less safe artisanal techniques, and often by children. While many responsible companies have taken steps to ensure that they do not use any coltan illegally seized from the DRC, monitoring any supply chain of this kind is very difficult and there are strong incentives for bad actors to lie. Moreover, the greatest damage was done by the '99-'00 price spike; adopting supply controls now can't undo what happened. The Toward Freedom article attributes the spike (and ongoing demand) to the Playstation 2. This is at best a highly selective interpretation of history.

Consider that as of 2007, the total number of cellular phone subscriptions worldwide was estimated at 3.3 billion, while the total number of PS2s sold to date is something like 150 million. According to statistics found at the Cellular Telecommunications and Internet Association website, the number of cellular phone subscriptions in the United States in 2000 was more than 100 million. By 2003 the number of cell phone subscriptions in the United States alone exceeded the number of Playstation 2 units sold worldwide in the entire production run to date. Keep in mind that those cell phone users are changing their phones regularly: the number of used and discarded phones in the United States (which has a low cellular penetration rate) substantially exceeds its human population. As such, we should expect that the actual number of cellular phones used by human beings since their introduction is significantly higher than the number of subscriptions, perhaps by a factor of two or more. Why blame game consoles for the coltan demand, then, rather than the far more prevalent cellular technology?

Of course, the demand for coltan doesn't end with game consoles or cellular phones. Tantalum is used in virtually every modern piece of electronics equipment, including pagers, laptop computers, digital cameras, and inkjet printers, as well as artificial joints, film, and various tools. Why blame game consoles for the ongoing conflict when their sales and usage are completely dwarfed by these other applications? The turn of the century saw a massive expansion in the use of all manner of electronics, all over the world. All of these factors contributed to the tantalum price spike. Yes, the 2000 shortfall of PS2s played a role, but the 6-fold change in price that year could not have happened without an existing huge demand for phones and laptops, and serious structural problems in the tantalum market.

How, then, did this conflict come to be known as the "Playstation War"? Reporter John Lasker doesn't provide any answers. He certainly doesn't quote anyone on the subject and it's not clear to me that the words "Playstation War" were used in reference to this conflict at any point before he did so in this article. In a fit of fairness late in the piece, Lasker acknowledges that the spike in tantalum prices that provided incentive for neighboring countries to interfere in DRC affairs was the result of multiple concurrent technology booms, but any good journalist knows that readers remember the lede. I don't know why Lasker made the choice to put the console reference so high up in the story, but doing so was inflammatory, irresponsible, and counterproductive.

The world does not have vast and plentiful reserves of tantalum, and existing electronics that contain tantalum are, in general, not properly recycled. Continued strong demand for consumer electronics and the still-increasing penetration of cellular phones in the global market are combining to bring the price of tantalum back up. Thus, a rejuvenation of resource warfare in central Africa is becoming increasingly likely. The best defense against this scenario is for consumers to keep strong pressure on manufacturers and regulators to prevent "blood coltan" from making its way into consumer goods. The typical purchaser of a cell phone doesn't know anything about tantalum or its uses, and in fact the typical American probably couldn't tell you what continent the DRC is on. Awareness, then, is a precious commodity, especially for an advocate-journalist. However, awareness is diminished when you make game consoles into a scapegoat.

Even if you manage to heighten awareness of mineral wars in Africa with a piece like this, no good is done if the reader doesn't make the proper connection with his own life. Can you imagine a person reading the lede paragraph on his laptop or cell phone and cursing those darn gamers for carelessly supporting resource wars? I can imagine nothing else. Calling the conflict a "Playstation War" allows the non-gaming reader to shift the blame onto those darn gamers and ignore his own contributions to resource scarcity and conflict.

Are gamers to blame for the sustained conflict in the DRC? Sure they are — to precisely the same degree as anyone who owns a cell phone, laptop, digital camera, artificial joint, anti-lock braking system, etc. All of us should try to be conscious of the origins (and destinations, you non-cell-phone-recycling jerks) of the materials in our electronics, and pressing the companies that make them to ensure that our sale price doesn't subsidize war in Africa. Inordinately emphasizing the importance of game consoles in the financial origins of war in the DRC, however, lets non-gamers think they are off the hook, and amounts to little more than blaming the "other" for the sins of all. A putatively progressive organization like Toward Freedom should be capable of recognizing that, and ought to feel ashamed at having done so.

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Great Game Music

For those of you who have missed it, Greg Tannahill has a fun series of posts about great gaming music up at his blog, The Dust Forms Words. While I don't agree with all his selections (I prefer "Hikari" to "Sanctuary", for instance), and I think he ommitted some really good ones (more on that below), it's a great series of posts. Greg, who will soon be standing for election in Canberra, brings back happy memories of some really good games with great music, but he also dug up some very interesting renditions, such as the one below the fold.

Yes, those are musical tesla coils, and yes, that is the Mario Level 1-1 theme.

One of my favorites among the games he's mentioned is the opening theme to Katamari Damacy. You should follow that link and watch the clip, but be forewarned that hearing this music even one time will burn it permanently into that part of your brain that keeps songs in your head all day. I also think this is one of the best introductory sequences to a video game. Watching the intro doesn't prepare you in any way for the gameplay or the story, but the wacky visuals and catchy music put you in the right mood.

As I said, Greg missed a couple of great soundtracks. Rather than leave a snarky comment on his blog, I thought I'd include put some of the omissions in my own post.

The most obvious of these is Kō Ōtani's soundtrack to Shadow of the Colossus. You can find the whole soundtrack about eight times over on YouTube, but the following video will give you a good start.

One of the best parts about the sound design in Shadow of the Colossus is that it doesn't make every battle an exercise in overwhelming horns and heavy, pounding rhythms. Several combat sequences feature calmer music that builds tension with sinister or mournful undertones. Shadow of the Colossus would probably make the shortlist for best game of all time even if the soundtrack consisted entirely of kids beating sticks together, but featuring some of the best music in the history of gaming certainly doesn't hurt. "Revived Power" is one of my favorite heroic themes ever.

Second on my list of omissions is Christophe Heral's soundtrack for Beyond Good and Evil, which is simply amazing. One of the recognized high points is "Home Sweet Home", which plays over the credits and out on the water before you complete the Black Isle Mission. And my god, this piano arrangement of the main theme by Mythili Mahendran is fantastic:

The game that really cemented my love affair with computer RPGs was Betrayal at Krondor, and this was certainly helped by Jan Paul Moorhead's excellent midi soundtrack. This is harder to get these days, but I remember coming across some .ogg files from it not too long ago. Amazingly, YouTube actually had a clip of the credits (German version) to this great game, with the main theme playing over them. I actually prefer the funkier "Jimmy the Hand", which plays during this walkthrough, starting at about 0:18.

The PS2 had an amazingly vast library of RPGs to choose from, but perhaps the best, and certainly the most unique, was Shadow Hearts: Covenant. Playing fast and loose with history, it featured cameos from Roger Bacon, Anastasia, and Rasputin, blending its bizarre humor in with three convoluted and emotional love stories. And then there was the character who fights with a doll that can only gain new attacks if you give a particular tailor gay porn. The soundtrack was fairly diverse, ranging from creepy electronic themes like this one from the battle with Astaroth, to very placid piano and string pieces. Almost all of it was excellent, but I am inordinately fond of "Getsurenka", which played over the closing credits. Yes, it's almost hilariously overwrought, but after the "good" ending of Covenant a cathartic love song is precisely what you need:

There are certainly more that could go in here, but that's enough embedding for one post. If you're interested in more technical discussions of video game music, you should check out Cruise Elroy. Click his category link on the side to find the music posts.

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Microwave Pfu CelB 5 minutes for highest activity

Like many bachelors, I regularly eat meals heated up in a microwave oven. I'd like to think I eat a somewhat lower percentage of frozen dinners than others in my situation, but even when it comes to food I cooked myself I usually don't have the time or patience to cook a recipe for one every night. That means I'm often eating reheated leftovers from the trusty Radar Range. The microwave oven works using dielectric heating, a process in which the movements of dipolar bonds are coupled to an oscillating external field. Water, for instance, is a molecule with a large dipole moment, and when you cook something in a microwave a lot of the heating occurs by the accelerated movements of water molecules. In principle this kind of excitation should also occur for other kinds of polar molecules, and thus we come to an interesting study from the lab of Alex Dieters, in which microwave radiation was used to activate a cellulase from a hyperthermophile.

Protein backbones consist of series of peptide bonds which include a carbonyl group, a classic example of a polar bond. Naturally, one might expect that the motion of these groups would be excited by microwave radiation. However, it does not directly follow that additional motion of the peptide backbone will actually accelerate chemical reactions, because this motion may be chaotic or unproductive. Moreover, from the fact that microwaves cook things (like eggs), we know that microwave radiation does a good job of denaturing proteins, sometimes at lower temperatures than we expect. Both of these problems can conceivably be avoided by studying a hyperthermophilic protein.

Proteins from hyperthermophiles such as Pyrococcus furiosus tend to be stable and optimally active at very high temperatures, at or even exceeding the boiling point of water. At lower temperatures, they retain their stability, but tend to become inactive. In many cases this reduction in activity appears to result from squelching internal motions that may be necessary to bind or properly orient substrates. Young et al. decided to study the β-glucosidase CelB from P. furiosus as a way of understanding whether microwaves might enhance enzymatic catalysis. Because CelB has optimal activity at 110° C it should be possible to see a significant difference in activity if microwave activation works. The stability of this protein at high temperatures also suggests that you will not accidentally cook it.

Sharp readers will have noticed an obvious problem with this idea—because heat activates this protein, and microwaves heat aqueous solutions, we must incorporate some kind of control in order to determine the pure effect of the radiation as opposed to the temperature. Young et al. resolve this problem by monitoring the heating of the sample during microwave irradiation, and then using a normal thermal apparatus to match this temperature profile (Figure 2). When a reaction reached 40° C using either heating method, it was quenched by the addition of a basic solution and the concentration of products was measured. Simply heating the Pfu CelB reaction to 40° C produced negligible activity, but microwaving it increased the activity by 4 orders of magnitude (i.e. a factor of 10,000). Less dramatic, but still significant, effects were observed for two other hyperthermophilic enzymes, but an enzyme from a mesophilic organism (the almond) was not activated by microwave irradiation.

That the microwaves caused increased backbone motion was supported by the finding that irradiating Pfu CelB at 75° C caused it to denature; this temperature is well below the normal melting temperature of this enzyme (115° C). The authors attribute the differences in activation between CelB and the other hyperthermophiles to their lower optimal activity temperatures, but it is also possible that the particular motions enhanced by microwaves are simply not as productive in those molecules. Although all the dipoles should be affected in similar ways by microwaves, they are all oriented differently with respect to each other in the protein molecule. As a result, the induced motion may be chaotic, perhaps specifically so, and therefore the activation of a particular thermophile may depend on the nature of the motions needed for its catalytic cycle. Enzymes that require large ensemble motions of subdomains, such as adenylate kinase, might not be activated as much as a protein that simply needs to be melted a little. Examining the differences in structural dynamics of enzymes differentially activated by microwaves may be an interesting area of future study.

While microwave activation is unlikely to revolutionize some of the more common uses of hyperthermophilic proteins (i.e. PCR), it does have promise. In ligations, for instance, hyperthermophilic enzymes can not be used at present because many DNA inserts denature at the optimal active temperature. With microwave activation, it may be possible to employ extremophilic ligases in these reactions, gaining the benefits of their speed and durability without having to worry about accidentally melting your DNA. Depending on the enzymes available, this technique may also prove valuable in improving mobile medical laboratories and developing novel diagnostic tools for field work.

1. Young, D.D., Nichols, J., Kelly, R.M., Deiters, A. (2008). Microwave Activation of Enzymatic Catalysis. Journal of the American Chemical Society DOI: 10.1021/ja802404g

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It really isn't

"I don’t think riding in a fighter plane and getting shot down is a qualification to become president."—Gen. Wesley Clark (ret.)

I'm finding it difficult to see why this statement is considered to be at all controversial, despite the firestorm it has ignited in the conservative set. It is not an attack on John McCain's service nor is it a smear against him. I could almost find the outraged Republican blowback on this quote to be funny, if I didn't have a memory. Instead, I find it positively revolting.

It amazes me that anyone has to defend this quote at all, as our history has amply demonstrated that military service has no bearing on Presidential quality. Yes, the Army gave us Washington and Eisenhower. It also gave us such perennial entries on the shortlist for worst President in U.S. history as Franklin Pierce and Ulysses S. Grant. Even successful leadership at the rank of General does not indicate that one will perform admirably as a President. For Clark, a military man (and former Presidential candidate) himself, to point this out hardly qualifies as a smear. It's fair to say this comment is unnecessary, but absent any additional context, the histrionics of the McCain campaign are just unwarranted foolishness.

However, we are not without additional context, because in an amazing coincidence, one of the men delivering those histrionics was Bud Day. Yes, the very same Bud Day who was a member of the Swift Boat Veterans for Truth and appeared in one of their commercials. This man, who through ignorance or foolishness attacked Clark's perfectly sensible statement, had the gall to follow it up with a defense of the Swift Boat smears as "the truth".

That's why I can't just dismiss the McCain response as a good laugh. The outrage, feigned or not, of conservatives is positively revolting in light of the Swift Boat campaign. To see those who winked at the SBVT's outright slander of an American serviceman treat Clark's statement as some kind of blood libel is a repellent display of hypocrisy. For McCain, who claimed to repudiate the SBVT tactics, to employ SBVT members and take their money in this campaign only heightens my disgust. The maverick, the straight-talker, the man for whom I felt a good deal of respect all seem to have perished in the pursuit of the Presidency.

McCain's experiences in the Vietnam War, heroic as they were, have no bearing on his qualifications for the Presidency. His reaction to Clark's statement of this obvious fact, however, has me more firmly convinced than ever that he lacks the judgment, the restraint, and the integrity to lead this nation.

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