cAMP gives CAP a twist

The catabolite activator protein (CAP), which plays a significant role in telling bacterial metabolism to digest sugars other than glucose, is a classic example of allosteric activation. Binding of the small signaling molecule cyclic AMP (cAMP) switches CAP into an active state that recruits RNA polymerase to certain metabolic genes. The biochemistry of cAMP activation is well understood, but the structural basis is not as clear, because a structure of the inactive protein was not available. This week in PNAS, researchers from Rutgers University and the University of Wisconsin-Madison report a structure of the free state of CAP that illuminates this allosteric effect.

Many excellent structural studies have examined CAP in its activated and DNA-bound form. CAP is a dimer, and each monomer has two domains: a DNA-binding domain (DBD) that recognizes its specific sequence, and a cAMP-binding domain (CBD). The monomers bind to each other through a coiled-coil interaction between two long helices. When CAP is activated it binds to DNA, with two helices (the recognition helices) sliding into the major groove and specifically identifying the sequences to which it should recruit the transcription apparatus. Without cAMP bound, CAP can still interact with DNA, but this interaction is of low affinity and not specific for any particular sequence. There are a number of ways this could conceivably happen, but it's difficult to be certain about any model in the absence of a structure of the free (apo-) protein.

In order to determine the structure, Popovych et al. used NMR. The 50 kDa size of the dimer means that it requires some extra effort for NMR work, but it is still well within the capabilities of the technique. The fact that the protein is a symmetric homodimer makes assigning the spectra somewhat easier, as the researcher only needs to deal with 209 residues rather than 418. The authors determined the structure using short-range distance restraints from nOe experiments, long-range restraints from paramagnetic relaxation enhancement, and angular restraints from residual dipolar couplings (RDCs). These angular restraints allowed the authors to unambiguously determine the relative orientation of the DBD and CBD in each monomer.

Getting that orientation right is key to the story here, as you can see from the image to the left. Here I'm showing you the DBD and coiled-coil helix (lower left) of a single monomer in the two different states. The activated CAP is in green (PDB code: 1G6N, and the apo- structure is in red (PDB code: 2WC2). You're looking down the coiled-coil, and the recognition helix is in a brighter color right at the front. If you superpose these structures on the bottom end of the coiled-coil, you can see that the recognition helix is rotated by 60° when cAMP binds. This twist of the DNA binding domain gives the recognition helices the right orientation and spacing to slide into the major groove of DNA and identify genes to activate. In the apo- state, these helices cannot both fit into the major groove simultaneously, explaining the low affinity and lack of specificity in that state.

Although the DBDs undergo a radical change in position following cAMP binding, they don't actually have any direct interactions with the signaling molecule, which binds down in the CBD near the coiled-coil helix. This helix, which links the CBD to the DBD, turns out to be key to communicating the allosteric signal. In the apo- state, the top part of this helix (near the DBD) is actually somewhat disordered and loop-like, not helical. Binding of cAMP to the CBD forms several contacts and causes several structural shifts that result in the formation of regular helical structure at the top of the coiled-coil. This in turn swings the DBDs around so that the recognition helices are in position to interact with target sequences (the authors provide a short movie of this in the Supplementary Information). A similar molecule, cGMP, that does not activate CAP, fails to make the key contacts with T127 and S128 that mediate this structural change.

The fact that the upper part of the coiled-coil is unstructured suggests that CAP may sample a range of conformations in the apo- state. This possibility is supported by one of the mutational studies in the paper. As you can see from Figure 5, a G141S mutation and binding of various effectors to the mutant causes the NMR resonances of DBD residues to shift on a line between the WT apo- and WT cAMP-bound states. This, in conjunction with the broadening of those intermediate peaks, suggests that the DBDs are exchanging between the two states on a timescale of microseconds. It seems quite likely that one or both DBDs in the inactive dimer occasionally samples the active conformation. In this model, the function of cAMP would be to stabilize, rather than enable the active conformation. The negative cooperativity of cAMP binding may help keep CAP switched "off" in the face of this conformational heterogeneity.

This study only dealt with a single protein, but the results are likely to be applicable to a number of systems. The allosteric mechanism described here seems to fit observations in at least some other members of the protein family to which CAP belongs. As such, this structural work and the dynamics investigations that will probably ensue are likely to provide important insights into a number of regulatory pathways in bacteria.

Popovych, N., Tzeng, S., Tonelli, M., Ebright, R., & Kalodimos, C. (2009). Structural basis for cAMP-mediated allosteric control of the catabolite activator protein Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0900595106

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Blog of the Round Table: Garden of Eden

In the April Blogs of the Round Table, Corvus asks us to design a game about a social issue we personally find troubling. There have been several stellar entries already, which you should check out from the main hall or using the pull-down menu at the end. Be aware that the subject matter of some of these games is fairly disturbing and may not be appropriate for all readers. My own design is rated E, because it is a light little planting sim I'll call Garden of Eden, which is a metaphor about sustainability and resource consumption.

The Design

In Garden of Eden, a player attempts to create the most aesthetically pleasing garden possible with a limited set of resources. The garden consists of several equally-sized patches of soil that the player chooses to seed with certain plants. Each plant requires a certain richness of dirt. The player can increase the richness of each patch's dirt by adding topsoil or fertilizer. By adding these elements the player can enrich the soil; every season of growth depletes the soil. The garden receives a score every season. So long as this score increases which each successive garden, or decreases by a small amount, the game continues. If the score falls by too much for 2-3 consecutive seasons the player is fired and the game ends. An online leaderboard built into the game interface shows high scores.

At the beginning of the game, the player has 8 patches under his control, and 16 different kinds of plant that he can grow (4 for each different season). The player has a warehouse containing a large but finite number of sacks of fertilizer and topsoil. A set number of these sacks (more topsoil than fertilizer) are deposited in hoppers on screen each season, and the player can move them from the hopper to a patch in order to enrich that patch of soil. Topsoil can only enrich the soil to a certain level, but fertilizer can enrich it infinitely. The only limit on soil richness, therefore, is the number of fertilizer bags in the hopper. The richness of each patch can be represented to the player either as a number or by the color of the soil.

Each plant requires a certain minimum soil richness to grow, and at this level will produce small flowers and a baseline score. Plants can be placed in soil that is too poor, but they will die and give no score. Making the soil richer than the minimum will produce a plant with more and larger flowers, and a much higher score. The return on investment is non-linear: enriching by X amount will increase the score by X^1.5 or more, up to the maximum available for a particular kind of plant. The minimum and maximum scores for each plant are known to the player, as is the amount of soil richness needed to achieve these scores.

Once the soil has been adjusted and the plants chosen for each patch, the plants grow to full bloom over the course of about 30 seconds. At this point the player can pause to admire his garden, or move to the next season. Higher-scoring gardens are always more interesting to look at — the plants themselves are larger, more detailed, and more beautiful. In addition, high-scoring gardens draw animals such as singing birds, deer, butterflies and bees, and human visitors who ooh and aah over the garden. Once a season is completed, the now-dead plants are removed into a rubbish bin. The player can choose to move them from there to a composting heap; after one additional season each plant left there will turn into one bag of topsoil. The intrinsic richness of the soil in each patch will decrease, and the cycle begins again.

With a high enough seasonal score or net score (added up across all seasons), new plants become available: these plants always have higher soil richness requirements, and also give higher scores with the same degree of non-linearity as soil enrichment. The player is shown when he will receive a new plant. The scores required are low initially so that new plants come rapidly, but as the game progresses it requires a higher and higher score to get a new plant. The player's highest-scoring season is also recorded on an online leaderboard, but it is not stored permanently. The score is only on the board as long as the player's game continues. The score is also removed temporarily after a few minutes (say, 10) of inactivity. The player sees the top ten, as well as his own position on the leaderboard and the 10 players who flank his score.

At a certain point, new patches become available, always delivered in groups of 16 — there are 5 extra groups of patches. Each time you receive one you get a one-time delivery of additional fertilizer and topsoil to the warehouse. In addition, because you now have more patches of soil overall, your hopper contains proportionally more fertilizer and topsoil sacks. This means that the maximum enrichment possible for a given patch of soil increases. It also means that more fertilizer and topsoil are removed from the warehouse each season. Something must be planted on every patch you have, and that plants on these new patches only count half as much towards your score as plants on your original patch. Additionally, the intrinsic soil richness of these patches depletes more quickly than the player's original 8 patches. The player cannot choose to refuse new patches.

Obviously the fertilizer will eventually run out, and as the intrinsic strength of the soil gets depleted the return from composting will not match the investment of topsoil needed to grow anything. If the player manages things properly he can quite easily keep the scoring decline slow enough that the game continues indefinitely. Once a section of soil is completely depleted and the player has no more topsoil, the game runs on its own. The soil in the six different groups of patches becomes cracked, dry, and barren as the seasons continue. The original set of 8 patches expands to take the shape of North America; the other groups of 16 patches, as they dry out, fill in a Mercator projection map of the earth. Once this happens the game ends; the player loses. If, however, the player manages to run the game for a very large number of turns, then he is told that he has won, and his best garden score from that game is permanently entered in the leaderboard. At no point prior to winning the game is the player told that this outcome is possible.

Aims of the Design

The game is meant to describe the problem (not a solution) of sustainability. In the metaphor fertilizer represents non-renewable resources and topsoil represents mostly-renewable resources. The composting process represents recycling efforts — the player must make a conscious mental effort to compost, and the recovery of resources is not totally efficient, in that many resources are lost and composting only returns the less powerful topsoil, not new fertilizer. The mechanics associated with the additional patches of soil represent a global drain of resources from poorer to richer nations, here represented by the currently-worst offender (the USA).

The mechanics of the game are meant to encourage the player to value a high-scoring garden with large, rich plants that suck down resources. This value is communicated to the player by the direct presentation (more beautiful garden, more visitors, more interesting sounds) and also socially, via the ever-present leaderboard. The requirement that the player actively interact with the game in order to keep himself on the leaderboard is meant to increase the volatility of the board, but also to keep each individual game moving forward. Additionally, ever-increasing growth or carefully controlled losses are valued by the game (by allowing progression).

Contemporary values romanticize the idea of a continuous march of progress, in which our success as a society is measured by how much bigger or better our things are compared to the things previous generations had. This is not just a view that informs our attitude towards technology, although it is most evident there; it also informed our latest real-estate disaster and the associated plague of suburban sprawl. The game is meant to communicate the idea that these values are self-defeating in a world of finite resources. Even if a small area can be sustained at a high level, this can only be achieved by taking resources from other areas at the cost of making their standard of life lower. Ideally I would tune the game so that it would be possible for a dedicated and careful player to run a modest garden indefinitely. To achieve this kind of success, or to "win" the game, the player must reject the "bigger, better" values implicit in the design. Because the game means to infect the player with these values, the upshot is to make the player question himself.

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Who's in the party?

Along with the Vintage Game Club, I have recently been playing Chrono Trigger, a game widely hailed as one of the best console RPGs ever made. While the game has many attributes worth praising (not least the continuity of world and battle screens), I was particularly interested by its combination attacks. Almost any two characters that are in the combat party can combine their unique individual battle techniques into more powerful "Dual Techs". For instance, Lucca can charge Crono's sword with fire while he performs his "Cyclone" attack to create the "Fire Whirl" tech. Frog and Ayla can combine their weak individual healing skills into the "Slurp Kiss" that slightly heals the whole party. I think it odd that this approach has seen so little use subsequently, since it seems like modulating abilities based on party composition has great potential for infusing characterization into the combat mechanics.

Of course, most role-playing games, especially in the JRPG genre, give the player characters with complementary abilities or specialized roles, so that certain rosters are somewhat more effective than others. Having the characters explicitly combine their attacks is much less common, and is typically reserved for high-powered finishing moves, as in Kingdom Hearts II or Tales of Symphonia. Chrono Trigger differs from these games in that its combination techniques span a spectrum of opportunity costs (also, the animations are short). Some of them do require a significant investment of MP and can only be used sparingly, but others have a low enough cost that they can be used almost every turn. As a result, the shared abilities are almost as important as the individual abilities to the tactical evaluation of a particular party composition.

This had an interesting effect later in the game, after I managed to get Magus to join my party. Magus is a strong and versatile character with hard-hitting magic attacks of several different elements. Considering just his individual abilities he's a good character to have in the party. Yet, I almost never used him because he didn't have any dual techs at all. He couldn't increase his effectiveness by teaming up with anyone, and rather than give up the tactical flexibility, I left him out. It felt mechanically like he just didn't fit in with the party, an appropriate note because he is a major antagonist for much of the game. The mechanics of the game isolate him in battle just as the story suggests he would be isolated in the group.

This seems to me like the sort of thing that should be done more often. The enormous cast of many JRPGs means that most of the playable characters and their relationships never really get fleshed out, despite the hours of cutscenes. Why not use the combat system to do some of this work? Envision a set of party dynamics, and use the availability of combination attacks to illustrate those dynamics. If you imagine that Marle is jealous of Ayla and Lucca, creating friction in the group, express this by limiting their combos or making them less powerful. Then, when the player needs Lucca in the party for her fire attacks, the tactical limitations would encourage the player to respond by removing Marle from the combat group. The player's choice of party members remains an act of play, but through the manipulation of the mechanics it becomes an act of narrative characterization as well.

We can push this further by making the available combos respond dynamically to narrative. Events in the story that bring two characters closer could make new combination attacks available to them. When the relationship between two characters changes, the set of shared techniques could be adjusted to reflect the difference. This need not be tied to the planned story: a relationship system like that of Tales of Symphonia could be used to guide the evolution of combination techniques.

I've seen a couple of excellent essays (from Jay Barnson and Nayan Ramachandran) recently trying to dissect what's wrong with the JRPG and suggest ways that the genre could be revitalized. My own advice is to let the gameplay tell the story, too. Persona 4 and The World Ends With You both did this, in different ways, but you can still apply the lesson even if you aren't so unconventional. Use the gameplay of party dynamics to reinforce the story of personal relationships. Make "these characters belong together" part of the player's thinking in every phase of the game, and the stories of those characters may gain more resonance.

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Drugs disrupt DHFR dynamics

One of the most-studied cases of the relationship between dynamics and catalysis is the bacterial dihydrofolate reductase (DHFR). DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate while oxidizing the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). As part of this catalytic process, a region of the protein called the "Met 20 loop" switches from a "closed" state that shields the active site from solvent to an "occluded" state that separates the substrate from the cofactor. NMR studies of DHFR structural dynamics have correlated the protein motions with the chemical changes. In a recent study appearing in Structure, researchers from the University of North Carolina show that the binding of inhibitors such as methotrexate (MTX) and trimethoprim (TMP) appears to uniquely disrupt the dynamic networks of DHFR.

Previously, seminal work from the lab of Peter Wright surveyed the dynamics of DHFR in every step of its reaction pathway. Boehr et al. determined that structural fluctuations in each complex represented motions towards the next step in the reaction. The conformational exchange rates they obtained from their relaxation-dispersion experiments closely resembled the rate constants that had been independently determined for the chemical steps. In almost every complex the conformational exchange was widespread, affecting residues in both the substrate and cofactor binding sites, as well as important distal locations such as the Met 20 loop.

Because the existing work from the Wright lab hewed as close to the natural substrates and products as possible, Mauldin et al. chose to examine the dynamic effects of inhibitor binding to DHFR. Like Wright's group, they used relaxation-dispersion experiments to identify conformational changes taking place on the μs-ms timescale. In the NADPH:DHFR complex the motions are widespread, encompassing the substrate binding site, the Met 20 loop, and distal locations. Binding of either inhibitor eliminates about half of this dynamic network and dramatically reduces the fluctuation rates of those residues for which conformational exchange continues to occur.

Based on their fits of the exchange rates, Mauldin et al. conclude that the substrate binding pocket moves in a way that mimics the enzyme's normal motions in the transition from its closed state to its occluded state. The long-range conformational changes that actually complete this transition, however, have been completely quenched. With the inhibitors bound, DHFR is like a car that's turning over but won't start. Part of the enzyme is still moving in exactly the right way to proceed along the reaction coordinate, but for some reason this motion doesn't catch on throughout the protein.

In order to gain a more complete understanding of the dynamic effects, Mauldin et al. performed experiments to identify the motion of the protein on the ps-ns timescale. Analyzing the dynamics of methyl and amide resonances using the Lipari-Szabo model-free formalism, the authors realized that inhibitor binding did cause long-range changes in dynamics, just in a faster regime. Where the natural substrate complexes have motions that occur hundreds or thousands of times per second, the inhibitor-bound forms have (smaller) motions that occur millions of times per second. Because these altered motions encompass the Met 20 loop and surrounding residues, the authors argue that they reflect abortive attempts by the protein to transition into the occluded state.

Although these inhibitors do not appear to change the protein's overall conformation, they produce long-range dynamic effects on short timescales and quench distal motions on intermediate timescales. The binding pocket appears to still be experiencing fluctuations related to the transition between the closed and occluded conformational states, but the mechanism that couples the binding site dynamics to the motion of the loop that defines these two states appears to be broken.

The million-dollar question is this: do drugs alter DHFR dynamics because they inhibit the chemistry, or do these drugs inhibit the chemistry because they alter DHFR dynamics? Quenching dynamics costs energy in the form of conformational entropy, and it may be possible to tune a drug for improved efficiency by blocking the binding site without altering the dynamics. This is only true, however, if the dynamics don't matter to successful inhibition. On the other hand, if blocking the conformational switching of the Met 20 loop inhibits the enzyme, then drugs can be designed for that angle of attack as well. In the case of a protein like DHFR, where the bacterial enzyme has similar activity but a very different structure from its human equivalent, drugs that target regions other than the active site may significantly reduce side-effects. As a result, protein targets that were previously off-limits due to shared chemistry may become tractable due to divergent dynamics and structure.

Mauldin, R., Carroll, M., & Lee, A. (2009). Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State Structure, 17 (3), 386-394 DOI: 10.1016/j.str.2009.01.005

Boehr, D., McElheny, D., Dyson, H., & Wright, P. (2006). The Dynamic Energy Landscape of Dihydrofolate Reductase Catalysis Science, 313 (5793), 1638-1642 DOI: 10.1126/science.1130258

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Critical Thinking Compilation: BioShock

BioShock is the rare game that really does change the way we think about video games, if for no other reason than that it has turned up as an example in almost every discussion of game style, mechanics, story, or design that has been written since its initial release in 2007. BioShock has received excessive adulation, a much-discussed backlash, and even a backlash to its backlash. Discussions of the game spawned the most popular jargon in games writing. So much has been written about BioShock that I could only put my hands on a fraction of the material without driving myself nutty as a splicer. Thus, this constitutes only the first draft of a survey of critical thinking on a game that will likely be regarded as a classic.

"Is a man not entitled to the sweat of his brow?"

One of BioShock's most compelling features is that it details an interesting philosophical system and then uses it to frame an ethical question. The underwater utopia of Rapture was founded by an industrialist named Andrew Ryan on a system of principles much akin to Randian Objectivism, so much so that John Lanchester argues in the London Review of Books that BioShock is the only popular work in recent years to give Rand a drubbing. Lorenzo Wang fleshes out this case in his rich and interesting essay "BioShock Explained". In his view, the game attacks two key flaws of Rand's philosophy: society can never sustain its ideal state (somebody must, after all, scrub the toilets), and free will, even in the land of plasmids, is limited. Jay Barnson felt that the game critiqued the intrinsic short-sightedness of the market, which is regarded as all-wise by the lovers of laissez-faire economics.

This interpretation of the game's attitude towards Objectivism was not universal, however. Shamus Young interviewed an Objectivist on the subject, who argued that BioShock really aims its criticisms at the idea of philosophical certainty. In his very interesting Marxist critique, Richard Terrell lays out a case that the game conditioned the player to accept the principles of Rapture's economy. In his view, the choice to attack the Big Daddies (and possibly the little sisters) makes the player part of the oppressive capitalist regime. Justin Keverne argued that the mechanics of the game suggest that one's goal is to acquire power in order to gain the ability to acquire further power.

At this point, designer Clint Hocking felt that BioShock went off the rails in a certain sense. In his essential essay "Ludonarrative Dissonance in BioShock", Hocking argues that the game presents the player with two conflicting contracts. The gameplay establishes that the player must serve his own interest in order to advance, while the story forces the player to serve others in order to advance. Our attempts to deal with or ignore this tension are them mocked by the game's central twist.

"How can you do this thing? To a child?"

The philosophy of rational self-interest provides context for the story, and also for a moral choice that the player makes. When Rapture's lumbering Big Daddies are defeated, the player may choose the fate of "little sisters" they protect. If he rescues a little sister, he receives a small amount of ADAM that he can use to purchase plasmids, tonics, and other upgrades, and the girl survives. If he chooses to "harvest" the girl, he gets a great deal more ADAM, but she does not survive. For Leigh Alexander, saving the girls is like saving Rapture's last bit of innocence. In her view, the whole saga with ADAM was like a child's wish, and saving the girls is a way of forgiving Rapture for making that wish, despite the destruction it caused. Yet many did not find this choice to be compelling. As Wes Erdelack notes, in BioShock as in many other video games, the moral choices are too simplistic and do not feature a sufficient challenge to "goodness".

One frequent complaint about the rescue / harvest choice is that if the player rescues enough little sisters, then the twisted Dr. Tenenbaum will leave a Teddy bear at a nearby vending machine, stuffed with plasmids, ammo, and extra ADAM she has presumably harvested on her own. In a thorough critique of this moral choice, D. Riley argues that the benefits of rescuing are sufficiently great that the whole system is neutered. Duncan Fyfe concurs, and wonders how the developers could have gotten this so wrong.

This opinion is not universal. Leigh Alexander believes that approaching the rescue / harvest choice with a cost-benefit analysis is too limiting. In her view the question to ask is not what you want to get but who you want to be. As she says, "The merit of choice in games may not be what we get from it, but when done this richly, how it feels." Bonnie Ruberg feels that the gameplay is really there to expose the selfishness of gameplay tactics in general. I also felt that the near-equivalence (in economic terms) of the two choices was making a point about the hidden values of games.

Some also argue that the harvest choice is not compelling because the player is spared having to watch the act or even see the body that remains. D. Riley's previously noted essay includes a comment on this point, and I make a similar case in my own essay, "Ecce, soror". Nels Anderson acknowledges that this may be so, but points out that on-screen child murder by the protagonist of a game simply could never get by ratings boards or the easily-outraged public. Nonetheless, he argues that the nursery scene provides some of the necessary emotional impact.

And perhaps the little sisters aren't even the real focus of the moral choice. Justin Keverne argues that the player's real moral choice is whether to attack the enslaved Big Daddies for his own personal gain. Unlike the other denizens of Rapture, the extremely dangerous Big Daddies won't harm you unless you attack them first. For Glenn Turner, the choice to put down a Big Daddy was harrowing because of the little sisters' reaction. Gene Koo felt that becoming one of these lumbering behemoths brought the game's emotional and philosophical threads close to each other. In his view, however, this didn't quite succeed, because the player has no choice about whether he becomes a big daddy.

Whether the rescue / harvest choice was compelling or not, BioShock at least invited a fresh consideration of the meaning of moral agency in games, according to Leigh Alexander. She also pondered whether our behavior in the game would change if our peers were aware of it (perhaps through achievements or trophies).

"A man chooses; a slave obeys."

Choice is the subject of BioShock's most compelling moment, the confrontation with Andrew Ryan. This moment twists the preceding exposition of the ideas of rational self-interest into a commentary on the nature of gaming itself. Wes Erdelack views BioShock as parable about gaming, highlighting the fact that the feeling of agency is always an illusion. The Graduate School Gamer notes in an essay comparing BioShock to Braid that "The player can only converse with the text within the confines of the game's design and always remains at the will of the designer." The illusion of choice is not a subject unique to gaming, according to Roger Travis. He notes that the non-choice of killing Ryan resembles Achilles' non-choice to join battle in the Iliad.

The first-person perspective heightened the impact of the climax. Matthew Gallant regards the first-person viewpoint as essential, especially in moments like the confrontation with Ryan. The personal choices at the center of the game couldn't be the same if the player's eye into the game world didn't seem to make him a part of it. Sinan Kubba shares Gallant's skepticism about a BioShock movie, feeling that the immediacy and immersion of the game's perspective could never truly be replicated in film. The praise was not universal, however: Brad Gallaway felt that the silent, first-person protagonist interfered with the narrative at several important points. He especially felt that the internal logic of the game collapsed when the player injected himself with a plasmid for the first time.

The more widely-expressed complaint about the plot, and specifically the encounter with Ryan, is that there is far too much game after it. The denouement of the game is widely recognized as its weakest segment, a fact that Josh Birk explains by pointing out that BioShock, like many other games, has more backstory than story. The confrontation with Ryan is the culmination of the fascinating backstory, leaving the rest of the game to become little more than the tale of a man with a gun out for revenge. Moreover, the game doesn't exactly free the player up to make his own choices after its climax. The player continues to obey a character, only now it is Tenenbaum rather than Fontaine. BioShock refuses to engage this dilemma, which is a significant source of frustration for Duncan Fyfe. Chris Dahlen also found this troubling, because the game's most compelling character (Ryan) has so much agency and the player has so little. As he puts it, "...within the game, you never become a man. The only choice you have is to stop playing."

Aside from Ryan, surprisingly few writers have gone in depth on the characters of the game. A worthy exception to this rule is Leigh Alexander's examination of the bizarre Sander Cohen. She relates his personality to people she knows from her theater background, seeing in him a metaphor for the whole backstory of the game. In her view, Cohen is "a brilliant character not only for his spot-on characterization, but for the way his endless wrestling with 'the muse' is a perfect metaphor for the consumptive nature of Rapture in general."

"I know why it has to be children, but why just girls?"

Without focusing a spotlight on particular characters, several interesting pieces have examined the role of women and femininity in BioShock. Although creepy little girls are a staple of the horror genre, as Leigh Alexander has noted, BioShock uniquely gives the player power over their fate. Bonnie Ruberg found the female enemies in the game particularly disturbing, and wondered whether their horrific power drew from the simple fact of their gender. She also found the power relationship between the player and the little sisters to be troubling, and agreed with a Penny Arcade comic suggesting that it had overtones of pedophilia.

Nels Anderson indicates that the design of the girls is intended to evoke sympathetic feelings, but what attitude does this imply on the part of the player and developers? In a comprehensive critique, Richard Terrell argues that BioShock pervasively trades in patriarchal values because it "depicts women as weak, emotional, submissive, and nurturing and men as strong, and protective...". The little sisters are portrayed as helpless human commodities, and for much of the story Diane McClintock equates her self-worth with physical attractiveness. Moreover, Dr. Tenenbaum's redemption comes through an acquiescence to patriarchal ideas of motherhood. Terrell's analysis encompasses the mechanics of interacting with female characters as well.

"I chose the impossible. I chose... Rapture"

It's easy to understand how Cohen, Tenenbaum, and so many others could have chosen Rapture. For Ed Borden, the environment was key to BioShock's immersion of the player. The crumbling city arrests the player's attention and inspires his curiosity. Glenn Turner felt the same way, arguing that the art design was perhaps the game's best feature. For Richard Naik also, the selling point of the game was Rapture's auditory and visual design, overwhelming all of the game's shortcomings. The art design unified the disparate levels, making the world of Rapture feel like a coherent whole and maximizing the emotional impact on the player, as Tom Cross explains in "Surviving Rapture".

Part of the power of the environment was the way in which it was used to tell a story. Steven O'Dell compared the player's journey through Rapture to a guided tour of a dying city, one in which the enormously detailed spaces tell a story through the way they are designed. Wes Erdelack points out that the much-loved environmental storytelling of Fallout 3 has some roots in the construction of Rapture's spaces. While the audio logs constitute the most powerful storytelling in BioShock, the game's spaces allow the player to play detective and reconstruct his own history for the game world. Careful construction of spaces and traversals played a significant role in player experiences as well, as Simon Cooke explains. Every time the player encounters a massive set piece, the developers use clever design to make sure he is able to see every bit of it.

"...bugger gets into his 'ead that he's gonna go down guns blazing."

Also making sure that the player can see every inch of the city is the fact that he simply can't die in it. In some respects, the Vita-Chambers that resurrect the player after every fatal encounter resemble a streamlined checkpoint system (much like Elika in the later Prince of Persia), and Scott Juster places them among a number of ways that BioShock used narrative elements to disguise common gameplay tropes. Nonetheless, the Vita-Chambers were widely criticized. This goes beyond the hardcore player's lament, articulated for us by Josh Bycer, that resurrection makes the game 'too easy'. Josh Birk complains that they interfere with the scare factor in the game and the logic of weapon collection. Justin Keverne points out that they encourage the player to take the path of least resistance, though for him the draw of using the plasmid powers was enough to keep him playing fair. Not so for Richard Terrell, who felt that the chambers too strongly encouraged the wrench / revive / repeat approach to combat, and demoted the central activity of the game, which he felt to be shooting. Moreover, he argues that the effective immortality of the player weakens the psychological impact of the game in his psychoanalytic evaluation.

Another way to conceive of the Vita-Chambers might be as a resurrection spell, an apt comparison since BioShock had so many RPG elements. In fact, Richard Terrell felt the game's mechanics tended more towards the role-playing side, in particular because the almost nonexistent cover system forced the player to behave like a bullet sponge. He compares tactics in BioShock to the attack / attack / heal approach common in RPGs. Justin Keverne comments on the oddity of this, as the Vita-Chambers actually make healing and health packs totally superfluous in all but the final battle. Writing for Eludamos, Matthew Weise connects BioShock with RPG roots originating in Ultima Underworld.

BioShock resembled RPGs in even less flattering ways as well, specifically because of its fetch quests. These quests added numerous objectives that muffled the story, in Duncan Fyfe's view. Rather than engaging in a breakneck pursuit of Ryan (or Fontaine), the player spends his time mucking about Rapture looking for 7 vials of bee spit. This quest temporarily scared Tom Armitage right out of the game, even though it ultimately led to some interesting exploration. He warns, "I was thrown by the instructions the game gave me..." saying that even a good game can be derailed by players' bad memories of similar quests.

And despite the "too easy" vita chambers and exciting plasmid powers, the conventions that BioShock embraces limit its audience. Although Lanchester praised BioShock's consideration of Randian philosophy, he criticized the game because even its modest difficulty would keep it from being experienced by a broader culture that might genuinely appreciate it.

Author's Note

If you go to any of these blogs and search them for the word "BioShock" you will come up with dozens of posts. The game has become a kind of yardstick by which we measure others, and a rich source of examples to illustrate points. I attempted to limit what I included here by stipulating that the post must be at least 50% about BioShock, and that it should be in fully developed paragraphs rather than bullet points. I'm not sure I actually ended up holding to that, but that was at least the approach I tried to apply. I am positive I left some excellent posts out, mostly by failing to find them in the vast wilds of the internet. If I omitted your dissertation on the semiotics of dentistry in the context of Rapture, it is probably because I didn't run into it while I was doing my survey. Would you kindly let me know about it in the comments, via twitter, or with an e-mail?

Last updated: 4/6/09

Future updates of this compilation have moved to Critical Distance. Look for this and other great critical compilations (by myself and others) there.

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You can't put a price on your soul

BioShock's most famous moral choice concerns the fate of young girls who are wandering through the undersea city of Rapture. The player gets to decide whether these girls live or die, and in a game that features a civilization built on the principle of laissez-faire one expects economics to play some kind of role in this decision. Many writers, however, have complained that it does not, and that the choice has too little of an effect on one's ultimate experience of the game. Leigh Alexander and Bonnie Ruberg, among others, have suggested that something more sophisticated is going on here, and in other aspects of the player's behavior in Rapture. The difficulty of resolving the rescue / harvest choice in BioShock by a cost-benefit analysis illuminates the little-discussed value of selfishness embodied by most gameplay.

Ludonarrative dissonance and the player's choice

Clint Hocking's famous critique of BioShock accused it of developing a case of "ludonarrative dissonance", or tension between the message of the mechanics and the message of the story. As he put it, the mechanics offer a "ludic contract" that tells the player to seek out power (in the form of weapons, ammunition, plasmids, and ADAM) in order to advance in the game. This contract encourages the player to adopt a philosophy of rational self-interest much like that espoused by Andrew Ryan, the founder of the game's undersea dystopia. The story, by contrast, offers a "narrative contract" that tells the player to help others (initially Atlas, later Tenenbaum) in order to advance. In Hocking's view, the player can choose to accept or reject the ludic contract by harvesting the little sisters (maximizing his take of ADAM) or rescuing them, respectively. However, because the overall narrative of the game is fixed, the player cannot really choose to accept the philosophy of rational self-interest, and the climactic confrontation with Ryan seems to ridicule the player for putting up with that limitation.

Subsequent discussions of the game took a curious turn, however, in that aspects of the rescue / harvest choice itself were accused of being the centerpiece of dissonance. Wes Erdelack's excellent discussion of ludonarrative dissonance is a case in point, although I believe this take on the matter originates with Jon Blow. The essence of the complaint in this case is that the story sets up the player's choice to save or kill the girls as crucial, the consequences of the choice amount to almost nothing in terms of the gameplay. Although the player obtains significantly less ADAM when he chooses not to harvest the little sisters, repeatedly rescuing the girls causes Tenenbaum to give him gifts containing additional ADAM, ammunition, tonics, and a unique plasmid. As a result, in terms of strict gameplay consequences, the choice the player makes with respect to the fate of the little sisters doesn't matter. In essence, the game doesn't allow the player to make a choice about the philosophy of rational self-interest because both options are compatible with that philosophy.

These discussions all assume that dissonance ought be avoided, but as Michael Abbott has noted, it has its uses. The odd discordant note may offend the ear, but it draws attention and forces the observer to consider the artwork more carefully. The seeming mechanical equivalence of choice may be the conceptual error of a foolish team, or an intentional effort on the part of the developers to make the player consider the conflict presented more carefully. If we interpret this dissonance in BioShock as signal rather than noise, BioShock indicts the value systems of games and gamers.

You are only a link in the great chain

The philosophy of rational self-interest lies at the heart of both kinds of dissonance in the game. Andrew Ryan intended Rapture to be a utopia with no governing principle other than man's own self-interest, and regards those who seek the protection of a government to be little more than parasites sucking society dry. He builds Rapture for himself. His primary antagonist, Frank Fontaine, embraces this philosophy in a more nuanced way. He too only acts in his rational self-interest, but rather than Ryan's reactionary hatred of charity, Fontaine sees the feeding and housing of the poor as a transaction in which he purchases loyalty. They are very different men — Ryan wishes to build a society, Fontaine aims only to plunder one — but their heuristics for moral decision-making are identical. Whether either of them fairly depicts Randian Objectivism is an interesting but ultimately pointless debate; their self-interested philosophy most resembles the implicit morality of video games.

Obviously I don't mean to attribute a moral system to games like Guitar Hero, but most games that feature a player-controlled avatar operate on a moral system much like Fontaine's, in which all the world is the player's for the taking. Certainly most games try to cover this with a veneer of a story that somehow involves saving the world, but this motivation rarely takes center stage in the player's mind. In almost every game in existence, the player is encouraged to kill everything he encounters and plunder every chest he sees in order to win the game. Role-playing games often acknowledge this, usually through some abstracted morality system as in the Fable games. Yet in these cases only the extent of the behavior is criticized, not the fundamental attitude. So long as a chest or trove lacks any obvious owner, the player will not be criticized for taking it. The primary value of these games is always selfish advancement motivated by hedonism.

"Never play a man for the short con when you can play 'em for the long one..."

In keeping with this value set, we expect our options in games to be clearly differentiated in terms of their utility with respect to our self-interest. Because this is not operationally true of the rescue / harvest choice, many players were frustrated or confused. This statement from Duncan Fyfe, is typical of the protests:

It's hard to understand why this is the case because it's such a fundamentally simple and classic philosophical debate. You can make the rational, self-interested choice to gain as much resources as you can from this one interaction. Or you can sacrifice/minimise your short-term reward in favour of long-term benefit. This is how it's presented to you and it's basic political philosophy. It's realism versus idealism. You'll get less Adam for rescuing the little sisters but there's the promise of a greater reward down the line.

The problem with objections like this is that delaying gratification doesn't put philosophical ideas into opposition at all. The debate over long-term benefit vs. short-term benefit is just an argument over what constitutes self-interest, or what is the more "rational" route towards actuating it. Reducing the rescue / harvest choice to long-term vs. short-term gains merely recapitulates the contrast between Ryan's short-sighted anti-altruism and Fontaine's "long con", but neither of those options entails a rejection of rational self-interest.

It's not the choice you make, it's why you make it

If you want the player to really consider the implications of the philosophy of rational self-interest in the rescue / harvest choice, there are only two real ways to do it. The first is to make the rescue choice truly oppositional to the player's expected self-interest, i.e. give him no ADAM or very little for taking this route. The obvious disadvantage of this approach is that the game now becomes highly skewed. Although the vita-chambers relieve difficulty in the typical sense of dying and thus being unable to continue, the game will certainly become a much more frustrating and challenging experience. A gamer who played through such an experience might come away with the impression that the game was designed to convey the idea that Ryan and Fontaine were right, that he should have chosen killing.

The other alternative is to arrange gameplay in such a way that "rational self-interest" no longer constitutes a reasonable standard for decision, and this is what the choice looks like. The point is not to give the player a way to reject the philosophy in the decision, but rather to draw his attention to the fact that this was such a large part of his decision-making all along. Rational self-interest cannot tell us what to do with the little sisters because both options fit this mindset. Thus, the arbiter of judgment is your conscience, as it is in real life. The fact that costs and benefits of the options you can take with the little sisters don't conform to our expectations about "moral choice" in a game invites us to examine the values that inform those expectations, and the ways in which games express or exploit them.

Games have implicit values — do as you're told, take what you will — that we might disagree with were we forced to acknowledge them explicitly. Because these values are both hidden and integral, it is difficult to force the player to confront them without a shock or a feeling of confusion. The dissonance in the rescue / harvest choice may be intended to inspire the latter. If so, then both the confrontation with Ryan and the choice for the little sisters are commentaries on the nature of video games. Despite the ever-increasing promises of freedom, games have always played you the same way Fontaine played Jack. And regardless of how you feel about Ryan and Fontaine, you have always played games as if you were them. BioShock elaborately displays the callousness and evil of these men, and through them, you. How do you feel about that?

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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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