It's only temporary

Far Cry 2's Africa burns impermanently. The lifelike flames consume plants and buildings and men, leaving great black scars on the landscape of a nameless, war-torn nation. The rickety shacks will char, the rusted jeeps will explode, and gas pumps will erupt like volcanic vents. In the course of the game you will leave dozens of checkpoints and ersatz military bases as smoldering ruins in the wake of your bloody path. Come back after a few minutes, however, and these sites will be transformed. The grass regrows, the buildings repair, the vehicles return to working order, and the men stand as they once did, hostile, guns ready. It's as if you were never there — and all this is fitting, because Far Cry 2 is, at its core, about futility.

Far Cry 2 casts the player as a mercenary sent to some African powder-keg to murder a conflict-stimulating arms merchant named "The Jackal". This mission fails spectacularly within the first ten minutes of the game, and then the powder-keg ignites, leaving the main character a penniless, malaria-afflicted gunman in a country that fortunately has a great deal of demand for his talents. It's a decrepit nation, without any apparent functioning agricultural or industrial infrastructure, and nearly all the civilians have already fled. As for the forces tearing the country apart, they occupy territories with indistinct boundaries, and lack any kind of uniform, as their armies consist almost entirely of mercenaries. Black natives hold the leadership positions in these ragtag armies, but their aides-de-camp and chief lieutenants are all white soldiers of fortune — making war for money, because this land has nothing else left worth fighting over anymore.

And yet the fighting continues. Far Cry 2 makes a half-hearted effort at embodiment, but for all that your hands can do in the game, what they will do most of the time is hold a gun. Missions are available from the two warring factions, your friends, arms dealers, a civilian underground, and mysterious voices that talk to you through cell towers. No matter who you're fighting for, everyone will attack you — a serious concern since almost every major intersection has its own contingent of mercenary guards. Their habit of regeneration means you must kill them coming and going, because your missions are handed out in central locations, and you will traverse the fastest routes to these places repeatedly, in cars or on foot.

Off the roads, this world has its nooks and crannies, hiding scattered diamonds or traces of your elusive target. The Jackal, strangely, sat for a series of interviews with an ignored journalist. Speaking clearly, but with alacrity that reminds one of high school policy debates, the arms dealer indicts all sides of the conflict, from the warlords to the first-world nations that ineffectually police them. The Jackal is as raw and uninhibited a capitalist as Bioshock's Andrew Ryan, more dedicated to the pursuit of the dollar than any abstract moral ideal. He sells guns to both sides, then buys them back when the cease-fire hits and sells them again somewhere else. And why not? As he points out: "[Weapons] aren't biodegradable. Only the dead are biodegradable."

Yet your weapons do degrade, in frustratingly short order at times. They jam, misfire, or even just explode in your hand as you pull the trigger. In Far Cry 2's Africa, the guns are temporary, but the men are permanent. The only ones that stay dead are the ones whose names you know — Frank Bilders won't come back if you let the morphine take him. Eventually, though, another buddy will take his place in the foreigner's bar, asking you for favors while offering none.

Far Cry 2's petty annoyances become apparent almost immediately, and conspire to drive the player away. Walking or driving, the player must make a long, dull trek to receive his next repetitive mission, performing murderous chores at each checkpoint along the way. With time, however, one acclimates to these discomforts as one might become inured to the oppressive heat implied by the game's setting. The visceral combat, the Jackal's rantings, the selfishness of nearly every person who speaks, and the sheer futility of each bloody journey across the African wilderness impart an awful momentum to the game, an irresistible force driving it towards an inescapable conclusion.

Far Cry 2 departs from formula by accepting the dark finale its every moment implies. This game stars a murderer who comes to Africa and kills almost everyone he meets, and fittingly, he dies anonymously in the jungle. Are his few good deeds enough to redeem him? Does it matter that he manages to save a few civilians here and there? You can't know that. You are allowed no certainty but this: that the next man to drive down these roads will find the guard posts restaffed, the mercenaries rearmed, and the war continuing blindly, needlessly, mindlessly, until the guns at last biodegrade.

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Rare codons give domains time to fold

The ribosome produces proteins by matching tRNA that has been correctly loaded with an amino acid to a codon (triplet of DNA bases) in the mRNA that contains the gene sequence. The triplet code allows 64 combinations of nucleotide bases, but proteins are made from only 20 amino acids (plus a "stop" signal). This means that most amino acids are coded by multiple codons, and hence have multiple tRNAs. Not all codons are created equal, however; in bacteria some codons are found much less frequently than others that represent the same amino acid. The tRNA associated with these "rare codons" is also less abundant than other tRNA, and this means that when a ribosome hits a rare codon, it often has to pause while it waits to encounter a loaded tRNA. To structural biologists like myself, who do their work by overexpressing proteins in bacteria, rare codons can be a nuisance because they slow down protein production, or even prevent it entirely. In a recent paper in Nature Structural & Molecular Biology, however, researchers from Germany suggest that the slowdown due to rare codons may have a functional advantage in vivo.

As a first step, Zhang et al. used a bioinformatics approach to survey the sequences of bacterial genes so that they could identify patches that would be slow to translate (the Methods section appears to contain an error in the description of this technique). They found that for proteins longer than about 300 amino acid residues, nearly every transcript contained at least one cluster of slow-translating codons. When the authors used a cell-free E. coli expression system to make some of these proteins and allowed only one round of translation initiation per ribosome, they saw a pattern of translation intermediates that matched the sizes predicted by the location of slow-translating patches.

In order to find out whether these translation intermediates had any significance, the authors examined the multi-domain protein SufI. In their prediction of the translation speed, which is on top in this figure that I have shamelessly stolen, there are four slow spots. Aside from the first one, these appear to correspond to the boundaries of different structural domains in the protein (lower part of the figure). Experiments with proteases suggested that these domains actually folded during the pauses, as the ribosome-bound translation intermediates were resistant to proteolysis.

Interestingly, when two rare leucine codons were replaced by more common ones (the authors call this SufIΔ_25-28), the whole protein became vulnerable to degradation. Similarly, when extra tRNA for these rare codons was added to the cell-free expression system, the full-length protein became protease-sensitive. This suggests that the slow patches are actually necessary for proper folding of the protein. It's often the case that lowering the incubation temperature can improve the expression of certain proteins in E. coli. The authors of this study find that is also true for SufI, as the protease resistance of SufIΔ_25-28 can be restored by lowering the temperature, and thus the overall translation rate. When analogous experiments with SufIΔ_25-28 and tRNA supplementation were carried out in living E. coli, the translocation of SufI into the periplasmic space was reduced by a factor of 10 even though the overall protein concentration was not affected, indicating that the co-translational folding allowed by the rare codons is necessary for proper functioning of the protein in vivo.

Of course this is a single case study, and it would be premature to conclude that every patch of rare codons corresponds to an important co-translational folding event. Indeed, that doesn't even appear to be true of SufI, which folds properly when one of its other slow patches is removed. However, at certain key locations these stretches of rare codons may be an important part of the folding machinery in multidomain proteins. In addition, the more frequent appearance of rare codons in β-strands (as opposed to α-helices) may also be related to folding due to the slower kinetics of β-sheet formation. As the authors note, the intrinsic kinetics aren't everything — pauses in the translation process may also buy time for the complex to encounter essential chaperones or cofactors. Regardless of the mechanism, it appears that rare codons, in at least some instances, provide a way for the folding process to catch up with the translation process.

Zhang, G., Hubalewska, M., & Ignatova, Z. (2009). Transient ribosomal attenuation coordinates protein synthesis and co-translational folding Nature Structural & Molecular Biology, 16 (3), 274-280 DOI: 10.1038/nsmb.1554

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A deadly halo in Alzheimer's disease

The observation of plaques composed primarily of amyloid-β (Aβ) peptides in the brains of Alzheimer's patients long ago gave rise to a hypothesis that Aβ was the agent that caused the disease. The plaques themselves, composed of long, insoluble fibrils of Aβ, were believed to cause the synapse loss and nerve death characteristic of the disease, and some data supports this model. However, several experiments have suggested an alternative possibility: that the symptoms of Alzheimer's may be attributed to soluble Aβ oligomers. In this view the fibrillar deposits may be an incidental feature of Alzheimer's disease, or even a defense mechanism whereby the body tries to get rid of the oligomers by forcing them into insoluble aggregates. In the March 10 edition of PNAS, a team led by researchers at Massachusetts General Hospital claim to have reconciled these two models. Using fluorescence microscopy, they find that amyloid plaques are surrounded by a "halo" of Aβ oligomers that kill the surrounding synapses.

The authors of this studied used fluorescence labeling to identify plaques, oligomers, and synapses in thinly-sliced tissue sections and living brains. They performed their experiments in mice that had been genetically manipulated so as to develop amyloid plaques. When they examined the brains of live mice, Koffie et al. noticed that the fibrillar plaques were surrounded by a cloud of the oligomers, as you can see for yourself in the figure below. On the left you can see the plaque core labeled by a fluorescent dye, and the middle image shows fluorescence associated with an antibody that specifically binds to amyloid oligomers. When these images are merged, the diffuse "halo" of oligomers becomes obvious. The authors see a similar result when they perform a similar experiment using thin slices of brains.


The authors also used a fluorescent-conjugated antibody to identify elements of the post-synaptic density (PSD), so that they can identify healthy synapses in the brain. Experiments in tissue sections demonstrated that the number of healthy synapses was reduced not only right next to the plaque, but also in a region extending up to 50 µm away (a length comparable to the diameter of a human hair). Aβ oligomers are also enriched in this region, and the relative concentration of the oligomer roughly correlates with the loss of synapses. By comparing the pattern of Aβ fluorescence to that of the PSD, the authors determined that oligomers were associated with many synapses, and that interactions between PSD and Aβ oligomers resulted in decreased synapse size. The relationship between Aβ binding and reduced synapse size was also shown to hold in control mice expressing normal levels of native amyloid precursor protein.

The observation that the presence of Aβ oligomers correlates with synapse loss, and the apparent degradation of synapses by Aβ, indicates that the soluble oligomers are a significant cause of Alzheimer's symptoms, although this study does not rule out the possibility that the plaque itself is also toxic. Even if the plaques have no immediate toxic effect, the authors propose that they serve as reservoirs, releasing synaptotoxic Aβ oligomers into the surrounding neural tissue, increasing the size of the lesions beyond the extent of the plaque itself. In this way Koffie et al. believe they have reconciled the previous models — oligomers are directly toxic, plaques release toxic oligomers, so both can serve as causative agents in Alzheimer's disease.

If this model is accurate, it implies that Alzheimer's disease may be quite resilient to attack. Antibodies or drugs that break up the Aβ oligomers will be effective in mitigating the synaptic damage, but as long as the plaques persist they will continue to replenish the pool of oligomers. Treatments that successfully break up the plaques will probably result in worsening symptoms due to the release of toxic oligomers as the fibrils disintegrate. These possibilities reinforce the idea that the most treatment for Alzheimer's will involve reducing the concentration of amyloidogenic Aβ peptides to prevent them from forming plaques in the first place.

(1) Koffie, R., Meyer-Luehmann, M., Hashimoto, T., Adams, K., Mielke, M., Garcia-Alloza, M., Micheva, K., Smith, S., Kim, M., Lee, V., Hyman, B., & Spires-Jones, T. (2009). Oligomeric amyloid associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques Proceedings of the National Academy of Sciences, 106 (10), 4012-4017 DOI: 10.1073/pnas.0811698106

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Urea binds to the peptide group

I've mentioned urea and guanidinium (Gdm) before on this blog, usually with reference to questions about their mechanism of action. These small molecules cause proteins to denature, or lose their higher levels of structure and become unfolded chains. The complete unfolding of a protein typically requires a fairly high concentration of denaturant, almost always more than 1M, and the explanation for this is that the denaturant molecules preferentially associate with the polypeptide chain with low affinity. In a recent issue of PNAS, a paper from Walter Englander argues that urea, but not guanidinium, associates with the backbone of the protein via hydrogen-bonding interactions.

Lim et al. reached this conclusion using hydrogen-exchange experiments. Amide nitrogens in proteins freely exchange their covalently-bound hydrogens (protons) with the surrounding water. The rate of this process can be measured (among other ways), by placing a protonated amide group into a deuterated solvent and tracking the decline in proton signal by NMR; this is called an HX experiment. In the case of a folded protein chain the observed rate will depend on the intrinsic chemistry of the particular amide and the stability of the protein structure, because this structure excludes water from the backbone and makes hydrogen bonds that lock the protons in place. Rather than deal with all of that, the authors used a small peptide mimic that (probably) has no complex structure. This had the additional advantage that the simple spectrum could be tracked by 1-D NMR, substantially increasing the time-resolution of the measurements. The authors measured the rates as they varied the pH — because we're talking about D₂O, it's called the pD instead — and added various cosolutes that are known to denature or stabilize protein folds.

As expected, the dialanine itself had a V-shaped rate profile in these HX experiments, with a minimum at a pD of 4. The hydrogen exchange reaction can be catalyzed by acid or base, so the rate increases as you go up or down in pD from this minimum. When urea was added to the solution, the authors found that acid-catalyzed HX accelerated while base-catalyzed HX decelerated. The most reasonable explanation for the latter result is that a hydrogen bond between the carbonyl of urea and the amide proton protects it from water attack. The authors do some mathematical modeling to establish that the effect on rate reflects a bonding association between the peptide and urea, not just random collisions or thermodynamically neutral associations.

The acid-catalyzed result is interesting, because in theory one would expect that urea would accelerate acid-catalyzed HX more than it actually does, because under acidic conditions it can accept a hydrogen from the amide nitrogen. While there are some confounding factors, the most likely explanation for this result is that the NH₂ groups of urea form hydrogen bonds to the carbonyl of the peptide. Because acid catalysis of HX hinges on the favorability of protonating this carbonyl, a hydrogen bond would be expected to reduce the HX rate. The authors argue that the ability of urea to serve as an acid catalyst is therefore mitigated by its propensity to bind to the carbonyl.

The formation of hydrogen bonds between urea and the peptide group meshes well with evidence that it denatures proteins through interactions with the backbone, some of which I have mentioned before. From HX experiments under native conditions we know that even a folded protein chain regularly undergoes excursions from its water-excluded, hydrogen-bonded state. Urea may bind to the backbone during these fluctuations, preventing or slowing a return to the folded structure.

Lim et al. also tested a number of other cosolutes, and found that none of them had a similar effect on the HX rate. In the case of the stabilizing molecules (glycerol, sorbitol) this is entirely expected, as their action cannot be explained in terms of a preferential association with the backbone anyway. The surprise concerns guanidinium, which is a more powerful denaturant than urea. The authors noted that Gdm has a small effect on the rate, but not in a pD-dependent way, and one that was little different from an equivalent concentration of NaCl (ordinary table salt). Gdm has no groups that can hydrogen bond to the amide, so the absence of an effect on base-catalyzed HX is expected. However, it should be possible for guanidinium to hydrogen-bond to the carbonyl, so it should seemingly have an effect on acid catalysis. This is not in fact the case.

The authors note that existing evidence does not support the idea that Gdm forms hydrogen bonds with water (although urea is known to do so). Lim et al. suggest instead that the planar Gdm molecule forms favorable stacking interactions with other planar groups. These include the peptide bond and several side chains. They argue that the stacking of Gdm with these groups pries the protein apart without requiring hydrogen bonds.

As a means to investigate diseases that result from protein misfolding, many groups are now trying to structurally characterize the unfolded state of protein molecules. Many of these experiments model the in vivo denatured state by using chemical denaturants such as urea or Gdm. The possibility that direct interactions between the denaturant and the protein will give rise to experimental artifacts should be taken seriously. Urea's promiscuous formation of hydrogen bonds with the backbone, itself, and water, may give rise to loose networks of hydrogen-bonded molecules that act to condense the chain. By contrast, Gdm's stacking effect will likely act to artificially extend the chain by steric obstruction. Because of the difference in these mechanisms, it may be of value to cross-validate findings from structural studies on unfolded states by repeating experiments with alternative denaturants.

Lim, W., Rosgen, J., & Englander, S. (2009). Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group Proceedings of the National Academy of Sciences, 106 (8), 2595-2600 DOI: 10.1073/pnas.0812588106

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Activated caspases stick together

In a post last week I mentioned a technique for obtaining the high-resolution structure of a protein inside a living cell, but I also pointed out that this technique was difficult and expensive, and might not be applicable to large proteins. Techniques improve and become more powerful, of course, but you might not want to wait for NMR to catch up to your question. Fortunately, high-resolution in vivo structures may not be necessary if you already have relevant dilute-solution structures of your protein and merely want to distinguish between different known conformational states. In a recent paper in PNAS, researchers from San Francisco used conformation-specific antibodies to locate activated caspase-1 in cultured cells.

Caspase-1 is a cysteine protease that plays a role in the immune response, as well as being released during apoptosis. From crystal structures we know that this protein can adopt two different structures, of which only one represents a catalytically competent state of the enzyme (the on-form). Caspase-1 also possesses an allosteric site where an inhibitor can bind, locking the enzyme in an inactive conformation (the off-form). When it's not bound to anything (the apo-form) caspase-1 is presumed to have a conformation similar to the off-form. Like many proteases, caspase-1 has a large, inactivating tail when it is made (the pro-form) that must be cleaved off before activation is possible. The structure of the caspase-1 proenzyme is not known. Current models of inflammatory response propose that after processing, the on-form binds to scaffolding proteins in an "inflammasome". In order to confirm this proposition, the authors decided to generate antibodies that would bind specifically to the on-form or the off-form of caspase-1.

The key to this experiment was combining irreversible inhibitors that could essentially lock the caspase into one conformation with the phage-display technique for optimizing antibody recognition. The authors had the advantage that both the active site and the allosteric site have cysteines in them. In an oxidizing environment, small molecules can covalently bind to the protein via disulfide bonds, thus locking the enzyme into the on-form or off-form. The authors immobilized these "locked" forms of caspase-1 and used them to screen antibody fragments (Fabs) using phage display. In addition to the typical selection approach, the authors performed anti-selection at one point using the "wrong" conformation to increase the specificity. After several rounds of selection, and some controls to ensure that the antibodies were binding to caspase and not the inhibitors, Gao et al. had several candidates for further optimization and screening. After they completed that process, they had two antibodies, Fab_on and Fab_off, specific for the two conformations. Each antibody bound to its intended target with a K_D of less than 5 nM. The authors also made full antibodies (IgG_on and IgG_off) from these Fabs for expression in mammalian cells.

The authors took these new antibodies for a spin with the apo-form of caspase-1. One might naively expect that only Fab_off would bind to this protein, but in fact Fab_on bound as well, albeit with substantially reduced affinity relative to the on-form. One possible interpretation of this finding is that the apo-form is equivalent to the off-form, but that Fab_on can convert it to the on-form via an induced-fit mechanism. If this is the case, then we would expect Fab_off to have the same affinity for the apo-form as it has for the off-form. However, the authors find that Fab_off has reduced affinity for the apo-form relative to the off-form. This indicates that the apo-form is an ensemble of conformational states, most of which more closely resemble the off-form than the on-form. Consistent with this view, the authors found that they can activate or inhibit apo-form activity by adding Fab_on or Fab_off, respectively.

By contrast, IgG_on did not bind detectably to a model of the pro-form, suggesting that this form's conformational ensemble contains no members that are close in structure to the active form. The weak affinity of IgG_off for the pro-form suggests that there are substantial differences between this conformation and the off-form as well.

At this point we know that IgG_on will bind tightly to the on-form of caspase-1, weakly to the apo-form, but not to the pro-caspase. This means it will likely be an effective probe of active caspase-1 in cells. The authors performed this experiment in THP-1 cells that they differentiated into macrophages. While IgG_off produced diffuse fluorescence in these cells, IgG_on stained small, concentrated bodies in a fraction of the cells. This suggests that active caspase-1 is localized to supramolecular structures in these cells, which the authors argue are identical to a structure previously identified as the "pyroptosome".

Although this particular experiment took advantage of binding-site cysteines that are particular to caspase-1, it should be possible to extend this approach to other proteins. Even non-covalent inhibitors or activators should be useful in this approach as long as the concentration is held high enough to saturate the target site during the selection step. Of course, the conformational change must alter the structure enough that the antibodies have something to grasp — it may not be possible to get specific antibodies if the shift is too subtle. If this requirement is met, however, it should be possible to determine the distribution of specific conformational states in cells, or even (as the authors suggest) to use antibodies as activators or inhibitors in vivo.

J. Gao, S. S. Sidhu, J. A. Wells (2009). Two-state selection of conformation-specific antibodies Proceedings of the National Academy of Sciences, 106 (9), 3071-3076 DOI: 10.1073/pnas.0812952106

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Get by with a little help from your friends

Fog blankets the sleepy little town of Inaba, hiding a secret and a killer. On rainy midnights, the televisions in the village show strange images of citizens who have recently disappeared. Days later, when the fog lifts, these people turn up dead. Only a small group of teenagers know the truth: inside the televisions lies a perilous world where the hidden aspects of a person's soul become murderously real. Someone is kidnapping the town's citizens and forcing them into that world, and only the main character of Persona 4 can save the victims and nab the killer. By himself, this would be impossible, but he has an amazing skill that guarantees his victory: he's a good friend.

The world inside the televisions of Inaba is crawling with monsters, and in order to confront the powers that are behind the disappearances the characters must defeat these creatures in turn-based battles. Alone, they'd never stand a chance, but the teen heroes of the game are assisted by "Personas", spiritual manifestations of their personality that have enormous power. The main character, named by the player, can acquire hundreds of these in battle or by "fusing" other Personas, thus gaining access to new abilities and stronger attacks. His friends, however, have just one Persona each, and that can only be obtained by traversing a dungeon and confronting their own souls in the form of a "shadow" born from the suppressed or disliked aspects of their personality.

The characters' shadow selves lack subtlety and nuance — one reflecting a young man's uncertain sexuality runs around in a towel and lisps outrageously — but they're not supposed to have those things. They represent the clumsy stereotypes common to the worldview of teenagers, and the oversimplifications of mass culture. In a game that depicts television's numbing effect as a world-obscuring fog, they're completely appropriate, though they can (and arguably should) make a player feel uncomfortable. Typical forms of teen angst, from jealousy to power struggles, inform most of the shadows' behavior, though the early dungeons are dominated by expressions of uncertainty about sexuality. Persona 4 also reflects on the inadequacies of its own medium in these dungeons. One of the game's antagonists is a disturbed, game-playing teen; his shadow takes the form of an infant cocooned in armor representing the hero of an 8-bit era RPG.

Persona 4 requires a significant time investment due to the level grinding in these dungeons. The relative ease of escaping and re-entering them means this can be discretized into play sessions lasting an hour or less, but the repetitious trekking through empty, uninteresting hallways to fight the same enemies over and over again will wear on many players. Losing your focus can lead to a frustrating game over, however. Like most Shin Megami Tensei games, battles really depend on correctly identifying elemental strengths and weaknesses. High-level monsters can be toppled easily if you know their vulnerability, but routine battles can quickly turn into disasters if the enemy exploits yours. Selecting the appropriate Personas and allies for a particular area or battle is a critical strategic task — the right friends make the difference between a long, difficult boss fight ending in victory and a short, frustrating boss fight ending in defeat.

Persona 4 also invites the character to develop relationships and explore problems outside the TV world through the social link system. In deference to traditional RPG mechanics, each key relationship has a "level"; by spending time with your friends and talking through their problems with them your relationship levels up, bringing you closer to resolving their problems. In terms of the game mechanics, your reward for success in the relationships is that you are able to create more powerful Personas. In reality, the social link conversations become their own reward. They're believably written, moving, and diverse, though the majority are built around repairing relationships or coming to terms with events or situations that cannot be changed. The main character makes a pretty good emotional facilitator, however, and in their culmination most of the social links give the impression that he's done something good for these characters just by listening to their woes and being supportive.

The mini-plots of these relationship arcs work better than the central story, which plods along predictably for the first 2/3 of the game or so, coming across more as an excuse for changing scenery and enemies than an actual mystery. Late in the game the pace picks up, with layers of double-crossing and deception that actually manage to elicit the feel of a caper. The eventually-revealed culprit (who the player must pick from a list in order to avoid the game's bad ending) manages to surprise, but this is only because the clues to the murderer's identity amount to only a few sentences of dialogue out of a game that can last upwards of 100 hours. As a result, forcing the player to choose the solution feels a bit unfair. Yet this reveal has its own reward; the story the culprit tells about his exploits gives the impression that he's someone who never faced down the dark parts of his personality. One gets the impression that all of this could have been avoided if he'd had a friend like the main character to help him.

Don't let Persona 4's demons and monsters fool you — this is a game in which the mechanics and story are built around the positive power of friendship. The main character supports his friends through hard times and tough decisions. In return, they fight beside him and give him the strength to fight on his own. All the creepy imagery and uncomfortable situations disguise a core message that comes straight out of an after-school special, and you'll only realize it when the game's warm (and yes, somewhat hokey) finale puts it right in front of your face. As the train rolls out of the station, you may find that the fatigue of Persona 4's long hours fades away, and you'll find yourself wanting to play it again, just to spend a little more time with your friends.

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High resolution protein structure from a living cell

Virtually everything we know about protein conformation comes from experiments performed in environments that do not resemble the biological context of proteins in action. Our data generally come from solutions that lack the significant array of salts, sugars, and metabolites that fill the cytosol of living cells, and often these data are acquired at a pH far removed from cytosolic. In addition, the dilute solution conditions used in almost all structural biology experiments do not capture the crowding and excluded volume effects that are likely to play a role in determining protein conformation in densely-packed cellular environments. As techniques in biology and NMR have advanced, however, the determination of protein structures in living organisms has become possible, despite the significant challenges. This week in Nature, a research team from several institutions in Japan and Germany reports that they have solved a high-resolution NMR structure of a protein in the cytosol of living E. coli bacteria (1).

The authors chose a relatively small and simple protein to work out their technique, in this case a 66-residue metal-binding protein from a thermophilic organism. They expressed the protein in E. coli using standard methods, exchanging the bacteria into isotopically-enriched media once they reached the appropriate density for induction. At the end of the induction period, the bacteria were gently centrifuged, resuspended into a thick slurry, and put in an NMR tube. Samples produced in this way were stable for about 6 hours, which is generally not enough time to perform the kinds of 3-dimensional experiments necessary for NMR structure determination.

To get around this problem, the authors used non-linear sampling and maximum entropy processing. This approach allowed them to reduce the number of data points they took, without losing much of the frequency discrimination that is vital to successful NMR. In this way they were able to compress the essential assignment and structural experiments to about 3 hours, although they found it necessary to repeat experiments and add them together in order to get enough signal to proceed. In order to ensure that the data were not contaminated by sample degradation, they ran short two-dimensional experiments to check sample quality in between the 3-D spectra. With this approach they managed to take 9 assignment spectra, several relaxation spectra, and several NOESY spectra for structural data. Apparently, each spectrum required its own, new sample due to the short lifetime of the bacteria under these conditions.

The authors performed control experiments in order to address some of the problems that affected previous research on proteins in living E. coli. They found that removing the cells from the NMR tube eliminated most of the protein signal, and that lysates of the bacteria contained protein signal. These experiments showed that the data collected in their experiments genuinely came from protein inside the bacteria rather than protein that had leaked out.

After all this work, the authors were eventually able to solve a structure of TTHA1718 in live E. coli, which you can see to the right (explore this structure at the PDB). This result would not have been possible, however, had the authors not employed specific methyl labeling in order to get additional long-range restraints, a technique typically used for very large proteins. As you can see in the supplementary information, attempts to solve the structure without the methyl NOEs gave rise to a fairly disordered ensemble. Even this ensemble is nowhere near as tight as the in vitro structure that the authors also solved. Because the in vivo structure used many fewer NOEs than the one from dilute solution it is difficult to tell whether differences between these ensembles reflect real conformational changes or simple uncertainty. The loop near the metal-binding cysteines (shown as fat sticks in this image) is a case in point — it looks quite different from the solution structure, especially in the positioning of the critical side chains, but there are almost no NOE restraints for this loop in the in vivo structure (Figure 4e). Chemical shifts support the idea of a conformational change, and inside the cells many of the signals in that region are too broad to detect. This, in conjunction with some metal-enrichment experiments the authors performed, suggests that the protein is regularly binding to metals in vivo, but the structure of this bound state is essentially a mystery. There are also a few clear structural differences in well-defined regions of the protein, but their significance is also unclear at this time.

This experiment serves as proof of principle, but NMR spectroscopists weary of years of promising experiments that turn out to only work on ubiquitin might rightly question whether this approach has any further applicability. In order to address this, the authors expressed the protein to a lower level in order to demonstrate that the procedure could still work for less-concentrated proteins. In addition, they show spectra from calmodulin in the supplementary data, suggesting that this approach will at least be applicable to proteins up to the 20 kilodaltons. However, the relaxation data the authors show in the supplementary data indicate that tumbling in the bacteria is significantly slower than in dilute solution, and the T₂ of the protein is 5-6 times shorter in vivo. If this result is general, then structural work on larger proteins may not be possible.

Why did this experiment work when other experiments on globular proteins in E. coli have led to leaking protein or an absence of signal (2)? Part of this may be that not all E. coli are created equal: the authors of this study used the JM109(DE3) rather than the popular BL21(DE3) strain. Genetic differences between the cells used may be responsible for the altered outcome. This will be a difficult thing to nail down, however, as overexpression typically involves the introduction of foreign DNA, an antibiotic, and an exotic activator of some kind, not to mention that isotopic labeling requires nutrient-poor minimal media. The difference between CI-2 that leaks out of cells and TTHA1718 that stays in may be as simple as the amount of magnesium sulfate in the M9. Until the factors causing the excretion of overexpressed proteins are more fully understood, careful controls will be an absolute necessity of in vivo experiments.

Because of the difficulty and expense this is clearly not an approach to be taken up lightly. For the time being, at least, you want to save this sort of experiment for systems where there is a real inconsistency between structural data from dilute solutions and results in vivo. As we improve the NMR approaches and increase our ability to manipulate E. coli behavior, however, this technique will grow more powerful and broadly applicable. Moreover, the Japanese part of the team reports in the same issue of Nature that they have managed to acquire spectra from proteins transferred into cultured human cells (3). This suggests the possibility of purifying labeled proteins at high yield, transferring them into living human cells, and then monitoring their structural and dynamic properties in their biological context.

1) Daisuke Sakakibara, Atsuko Sasaki, Teppei Ikeya, Junpei Hamatsu, Tomomi Hanashima, Masaki Mishima, Masatoshi Yoshimasu, Nobuhiro Hayashi, Tsutomu Mikawa, Markus Wälchli, Brian O. Smith, Masahiro Shirakawa, Peter Güntert, Yutaka Ito (2009). Protein structure determination in living cells by in-cell NMR spectroscopy Nature, 458 (7234), 102-105 DOI: 10.1038/nature07814

2) Li, C., Charlton, L.M., Lakkavaram, A., Seagle, C., Wang, G., Young, G.B., Macdonald, J.M., Pielak, G.J. (2008). Differential Dynamical Effects of Macromolecular Crowding on an Intrinsically Disordered Protein and a Globular Protein: Implications for In-Cell NMR Spectroscopy. Journal of the American Chemical Society DOI: 10.1021/ja801020z

3) Kohsuke Inomata, Ayako Ohno, Hidehito Tochio, Shin Isogai, Takeshi Tenno, Ikuhiko Nakase, Toshihide Takeuchi, Shiroh Futaki, Yutaka Ito, Hidekazu Hiroaki, Masahiro Shirakawa (2009). High-resolution multi-dimensional NMR spectroscopy of proteins in human cells Nature, 458 (7234), 106-109 DOI: 10.1038/nature07839

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