A point mutation remodels a binding interface

Decades of studies involving extensive mutagenesis of proteins and protein domains have impressed on us the idea that the folded tertiary structures of proteins are fairly resilient. While a particular mutation may abolish function by directly ablating a key chemical group, it is rare for a single mutation, or even a group of several mutations, to significantly change the overall conformation of a folded polypeptide chain. When a major change does result, it often takes the form of complete denaturation. Because of this, it may seem that protein folds occupy stable islands in sequence space, surrounded by a sea of sequences that form random coils or molten globules. However, there is some evidence that this view is mistaken, that substantially divergent structures may have very similar sequences. A paper recently published in PNAS adds to this view by describing a major change in the structure of a PAS domain resulting from 1-3 mutations.

PAS is a large family of protein-protein interaction domains contained in many signaling proteins. Although many of them have cofactors that modulate their binding, some PAS domains are constitutively active, which is the case for the domain under study here, the PAS-B domain from one of the founding members of the family, the aryl hydrocarbon receptor nuclear transporter (ARNT). The PAS-B domain was believed to dimerize with PAS domains from other proteins through one side of its β-sheet, and as a consequence Evans et al. decided to make several mutations on the outer surface of the sheet and monitor their effects on dimer formation.

One of these mutations, Y456T, had a strange effect on the domain's NMR spectrum: about 30 new peaks showed up in the ¹H-¹⁵N HSQC. Because this spectrum should show a single peak for each chemically unique proton-nitrogen pair, this result suggests that the pure proteins in the magnet exist in two distinct conformations. By lowering the temperature and repurifying the protein, Evans et al. were able to mostly separate these two conformations from each other. However, over time these conformationally purified samples became heterogeneous again, which means that the conformations can freely interconvert. This process was very slow, however — too slow to be detected using NMR relaxation techniques. From monitoring the HSQCs the authors concluded that the time constant for interconversion in the mutant is on the order of 16 hours.

Intrigued by what they were seeing, Evans et al. made additional mutations to ARNT PAS-B and found that the proportion of protein in each structure can be adjusted within a wide range by mutation. Using a triple-mutant system they were able to drive about 99% of the proteins into the new conformation. Using this mutant the authors were able to assign the resonances in the HSQC spectrum and solve the structure using NOEs. They learned that the chemical shift changes in the mutant are quite widespread, as you can see in the figure I have shamelessly stolen (left) for your benefit. In this figure the chemical shift changes are mapped onto their structure of the new conformation using color, ranging from green (very little or no change) to red (significant changes) — the sites of the three mutations are shown. As you can see, the chemical shift effect is widespread, covering almost the whole β-sheet of the protein and reaching to the helix on the opposite side.

Closer analysis of the structure shows why this is so. The strand of the β-sheet on which Y456 is situated has shifted its register by 3 spots. This has two effects. The first is that all of the hydrogen bonds involving that strand must be broken, at a substantial energetic cost. This is probably the reason the interconversion process is so slow. Moreover, because the register shifts by an odd number of residues, the strand must flip over, exposing to solvent the residues that were buried in the original structure, while burying the residues that were previously solvent-exposed. Although the backbone and side chain orientations in the strand overlay reasonably well between the two structures, the chemical groups available for interactions are completely different. Unsurprisingly, this alternate structure has very low affinity for its natural targets — titration experiments showed that the alternate conformation bound to its partner from hypoxia inducible factor at least 100x worse than the native structure.

Of course, in living cells with a wide variety of surfaces to bind to, the mutant PAS-B might find an alternative partner for which it has high affinity. Studies that attempt to understand protein-protein interfaces from an engineering or evolutionary perspective typically adopt the assumption that point mutations have little effect beyond adding a particular functional group here or there. This study indicates that this attitude underestimates the ability of point mutations to radically remodel interface surfaces. While a Y→T mutation may not seem particularly conservative, a Y→S mutation has similar effects and requires only a single nucleotide base change. It is not inconceivable that this alternate conformation could be reached in vivo, potentially giving rise to completely novel protein-protein interactions.

One might well wonder how common rearrangements of this kind are likely to be. As the authors point out, structural plasticity in the β-sheet is likely to be a common feature of PAS domains, making it difficult to assess whether this kind of mutational effect is widespread in other folds. However, the authors cite several examples of β-strand register shifts in other proteins. In addition, our decades of alanine-scanning mutagenesis have little to tell us about how common these kinds of rearrangements are, for two main reasons. First, the structural effects strongly depend on what a given residue is mutated to (see Table 1); had Evans et al. been content to leave things at alanine mutations they would never have detected this effect. Second, widely-applicable techniques for sensitively detecting small populations of alternate conformations have not been available until recently.

While the conformational transition induced by the Y456T mutation preserves the protein's overall fold and stability, it rearranges the hydrogen bonding contacts of the main β-sheet and shortens a loop. Obviously this is not as dramatic a change as found in lymphotactin. However, this alternate structure has significant consequences for PAS-B function. Unexpectedly, this single point mutation can radically remodel the PAS-B binding surface. Moreover, this result adds to the evidence that new structures (even in the context of known folds) may be accessed with only a few changes in amino acid sequence, without any need to detour through molten-globule intermediates.

M. R. Evans, P. B. Card, K. H. Gardner (2009). ARNT PAS-B has a fragile native state structure with an alternative β-sheet register nearby in sequence space Proceedings of the National Academy of Sciences, 106 (8), 2617-2622 DOI: 10.1073/pnas.0808270106

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Allostery in the CBP KIX domain

Classically, allosteric and cooperative effects have been identified with large complexes of multiple protein subunits, in which the binding of a ligand to one subunit enhances ligand binding in a different subunit. While some features of the models developed to deal with these systems do not translate well to cases of allostery within a single protein or domain, many of their core ideas continue to illuminate these single-subunit systems. In an upcoming paper in the Journal of the American Chemical Society, a team of European researchers examine an allosteric effect based on population shifts in a transcriptional activator, comparing it to a famous model for explaining allostery in hemoglobin (1).

The CREB binding protein (CBP) is a large molecular scaffold that brings pieces of the transcriptional machinery together in order to turn on a gene. Like many scaffold proteins it contains several protein-protein interaction domains linked together by large unfolded regions. One of these domains is KIX, a small bundle of helices that binds other proteins at two distinct sites. In one case, a protein called MLL binds to one site while a protein called c-Myb binds at the other. What is so interesting about this is that KIX is much more likely to bind c-Myb when it is already bound to MLL. Brüschweiler et al. used NMR techniques to try and understand how this happens.

In order to pull this off they performed relaxation-dispersion experiments on the amide nitrogen, α-carbon, and some methyl carbon atoms of the KIX domain bound to a peptide derived from MLL. Many of the amino acids in the protein showed a significant contribution to R₂ from exchange, suggesting a global conformational switch between two states. In order to cover their bases, the authors performed experiments to prove that this behavior was not related to the unfolding of the protein. Satisfied that the protein was stable, they used standard methods to calculate the rate of the conformational change, the population of the two states, and the chemical shift difference between them. They found that the minor state of the KIX-MLL complex is 7% of the total population of protein molecules. They also noticed that the chemical shift difference between the two states correlates very well with the chemical shift difference between the KIX-MLL complex and the KIX-MLL-c-Myb complex. Assuming that the conformation of KIX is the primary determinant of chemical shift in the bound state, this suggests that the dynamics are sensing a switch between a state that doesn't bind c-Myb and a state that does.

In order to determine whether MLL binding gave rise to this conformational switching behavior, the authors measured relaxation dispersion in KIX at several different concentrations of MLL. Excluding residues highly sensitive (by chemical shift) to MLL binding, they found that the exchange contribution to relaxation increases as MLL is added. Although Brüschweiler et al. were unable to fit this small number of residues quantitatively, these results strongly suggest that the addition of MLL increases the population of the c-Myb binding state. Moreover, under conditions where KIX forms a saturated complex with MLL and a peptide from another protein (pKID), the chemical exchange contribution to relaxation vanishes, suggesting that the protein has been pushed completely to the binding-competent state.

In order to identify the pathway by which the MLL binding site communicates to the c-Myb binding site, the authors examined the residues in KIX that had the largest chemical shift change associated with the chemical exchange behavior. As it happens, residues satisfying these criteria cluster in a region stretching from the MLL site to the c-Myb site, as you can see to the right (explore this structure at the PDB). Here, KIX is blue, the MLL peptide is red, and the c-Myb peptide is green. The side chains of the residues Brüschweiler et al. identify are shown as sticks inside the pink atomic surface. As you can see, these residues constitute a contiguous body stretching from one site to the other. Presumably, this set of residues provides a pathway for communication between the two sites. A trio of isoleucines at the core of this region (I 611, 660, and 657) are present in KIX domains from many different species (supporting information), suggesting that this communication pathway is evolutionarily conserved. Mutational studies centered on this trio of residues may teach us more about the mechanism of information flow in this domain.

Although this allosteric pathway is known to work in reverse (binding of c-Myb enhances the binding of MLL), the authors were unable to detect any exchange contribution to R₂ when only c-Myb or pKID was bound. While this may suggest that communication in the opposite direction uses a completely different mechanism, such that KIX has two unidirectional allosteric pathways, that's not a necessary conclusion from this result. Alteration of R₂ due to conformational exchange is dependent on the populations of the two states, the difference in chemical shift between them, and the rate of the switch. Actually detecting a dispersion curve requires that all these parameters lie within a 'sweet spot' that allows observation. This doesn't always happen, even when a dynamic process is occurring with a μs-ms rate. Because the chemical shift changes that result from MLL binding appear to be quite large (2) the exchange process may be slow on the NMR timescale.

One minor concern I have with the paper is that the experiments were carried out at a pH of 5.8, which is lower than the pH of cytosol (7.2). The only groups likely to change their charge over that range are histidines, but one of the key residues for this paper is H651 in the KIX domain. The experiments that established the allosteric effect of MLL on c-Myb binding (2) were performed at pH 7.0 so it is formally possible that the dynamics and allostery are a coincidence (although the chemical shift data argue against this). It would probably be worthwhile to perform HMQC experiments to clarify the protonation state of the histidine, or to repeat the binding experiments at a lower pH. The latter might be preferable; I assume that mildly acidic conditions are used for the NMR experiments because KIX has undesirable spectral characteristics nearer neutral pH. Additionally, it might be interesting to perform experiments that explore the effects MLL has on the kinetics of binding, seeing as this appears to be a dynamic process.

Brüschweiler et al. identify their results with the Monod-Wyman-Changeux model of allostery. Although this model was formally developed for systems with multiple subunits, what the authors really wish to emphasize is the idea from the MWC model that proteins in solution exist in an equilibrium of high-affinity and low-affinity forms. The evidence from the relaxation-dispersion experiments indicates that a very small proportion of free KIX exists in a (unfavorable) conformation that's ready to bind c-Myb. The binding of MLL enhances KIX affinity for c-Myb by stabilizing this structure — the allosteric effect arises because MLL binding defrays the energetic cost of adopting this fold. This manifests as a shift in the population of KIX proteins towards the binding-competent state. This kind of binding cooperativity may play a significant role in CBP's transcriptional activation function.

(1) Sven Brüschweiler, Paul Schanda, Karin Kloiber, Bernhard Brutscher, Georg Kontaxis, Robert Konrat, Martin Tollinger (2009). Direct Observation of the Dynamic Process Underlying Allosteric Signal Transmission Journal of the American Chemical Society DOI: 10.1021/ja809947w

(2) N. K. Goto, T. Zor, M. Martinez-Yamout, H. J. Dyson, P. E. Wright (2002). Cooperativity in Transcription Factor Binding to the Coactivator CREB-binding Protein (CBP). Journal of Biological Chemistry, 277 (45), 43168-43174 DOI: 10.1074/jbc.M207660200

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Blog of the Round Table: Hero's Blade

For the February Blogs of the Round Table, Corvus asks us to take a design provided by another contributor for the January Round Table and build on it. The idea is to develop a similar theme or mood in this second game, without necessarily remaining true (or relevant) to the literary work that inspired the initial pre-make post. In that vein, I wanted to riff off Corvus' "A Lego Orange" with an eye primarily towards its endgame. At that point he's trying to get across a sense of disenchantment with a once-loved lifestyle, which is what I'm going for in this idea for an unconventional fantasy RPG called Hero's Blade.

Hero's Blade is set in a fairly large valley with a road running through it. There's a village at the center and several outlying farms, a forest area, and a few fields and streams. The player controls an older hero in the "grizzled ranger" mode, armed with an excellent sword and bow, good armor, and so on. The game begins as the Hero wakes up in the village inn, learning that he got a knock on the head during a landslide that has blocked the road at one end of the valley. Don't worry, he doesn't have amnesia. The road at the other end has recently been taken over by bandits, so he is trapped in the valley until the road is cleared or he removes the bandits. The game world should at this point be presented in desaturated colors, as if the player is looking at the world through a filter that gives everything a gray tinge. Other than this, the graphics should be standard realistic 3D stuff.

The inhabitants of the valley are all farmers and, with one exception, are neither willing nor able to fight the thieves on their own. The exception is a single character (who I'll call the Kid) who is eager to go out and get rid of the bandits. He (or she) should have similar armaments to the Hero, but of much lower quality. The Kid is a very unskilled and totally untrained fighter, but possesses the same set of combat skills as the Hero. Combat in the game is turn-based and perfunctory — as little removed from a classic turn-based model as possible. There are no magical items or magic users, no elemental strength/weakness system, no need for any kind of strategy. The Hero's abilities are so far above those of the bandits that one of them can only stand against him for a turn or two. He earns no experience from battle at all. By contrast, the Kid starts off as a pretty bad fighter and earns experience points and skill boosts as is typical for an RPG. The Kid is AI-controlled, aggressive, and foolhardy; the Hero's only means of controlling him is a command that can knock the Kid down and take him out of the battle for a few turns. If either the Kid or the Hero dies in combat the game ends.

Each turn of combat and each bandit killed desaturates the color palette by a certain fixed percentage. If the saturation drops below a certain level, the game whites out, then fades to a steel-gray color and ends. This cutoff applies without respect to bonuses based on relationships or locations (see below). Thus, a player can get a game over by walking into the cemetery.

While the valley has several places where the bandits patrol, the rest of it is fairly open and features no real threats. The Hero can converse with the inhabitants of the village and the farm, and can establish relationships with each (about 50 total). Early stages of building a relationship involve just talking with the inhabitants, using a conversation system that allows the player to convey an attitude towards the NPC. For the most part these NPCs will do their own daily tasks without seeking out the Hero. The exception is the Kid, who can never be found if you're looking for him, but appears immediately if you call him (using a button solely dedicated to this task in the open world). The Kid always shows up out of nowhere to join in or watch if the Hero enters a fight or does a job that might be related to fighting or adventuring. As the Hero's relationship with an NPC improves, the inhabitant will offer quests to the player, some of them minor and some of them more involved, but never requiring combat. Several characters will also offer jobs.

These side-quests and jobs should take be the focus of the development. Each job should have some depth of gameplay, so that it carefully rewards strategy and player skill. Also, there should be some degree of challenge to each of them. Similarly, each quest should have extended moments of interaction between the character and the world. For instance, if an NPC has a missing cow, the player's job will be to find the cow and then guide it back carefully by coaxing it along and constantly nudging it in one direction or another. The goal is to require continual input from the player during each of these quests while varying significantly from quest to quest so that the player is always engaged during them (as opposed to the combat).

The performance of each job or sidequest will be discretized in some way so that as the Hero does more of each the colors of the world increase in saturation. Similarly, each time the Hero "levels up" his relationship with one of the NPCs, he the world saturates. In addition, NPCs with whom the Hero has a very good relationship will boost the color saturation in nearby areas. Spending time near his friends will literally make the Hero's day brighter. Only the Kid does not have this effect.

Taking a relationship with an NPC to a certain level will cause the Hero to open up to the NPC, with the Hero revealing some of his past. These flashbacks should take the form of slideshows of images illustrated in striking style and vibrant color, voiced over by the Hero narrating the event from his life. The life events he narrates will take a variety of tones, appropriate to the history and nature of the NPC. However, each of them will have a sad epilogue. His old friends have all died in battle, the women he danced with in taverns across the land have settled down and forgotten him, he outlived his favorite horse, etc. etc. The image shown in each epilogue should use the washed-out palette from the start of the game.

Taking a relationship to a high level will also help keep the Hero from dying by whiteout. In cases where the game would normally end due to the color desaturation, a good friend can find the Hero and boost the saturation level back to safety.

All of this is meant to make combat an unattractive thing to do, but after every couple of days that the Hero does not find and fight a bandit in the valley, the raiders will become emboldened and attack the town. If the Hero does not decimate them, one of the villagers will be killed. On the day after the attack, any bandits or villagers killed will be buried in the town cemetery in a scripted event; depending on who is getting buried the Hero's attendance or non-attendance will affect his relationship with all the NPCs. In the vicinity of the cemetery itself (as well as the bandit hideout) the color desaturates by a certain fixed amount.

In addition to these spots, the color will desaturate at any spot outside of the village where the Hero killed a bandit previously. These will be easily identified because the bodies will persist, slowly decaying with time. In town, bandits killed indoors will leave permanent bloodstains. No matter how faint the rest of the world gets these will always be clearly visible; they will fade to black rather than white.

The population of bandits is finite, however, and once they get reduced to about a fifth of their initial number they will hole up in their little fort and start sending out individuals to burn farms or kill villagers while the Hero is too far away to help. At this point the Hero will be urged to attack their stronghold. If he refuses to go, the Kid will go by himself and die there; the game ends. If the Hero accepts, the villagers will contrive a way to make the bandits open their fort and the Hero and Kid will take them on. The "boss" bandit leader is only marginally more skilled than the bandits themselves.

After this fight, victory celebration, yada yada yada, the Hero is awakened one morning by the Kid, who says that he is going to leave the valley and adventure across the world. The player can choose either "I'll go with you" or "You should take my sword." For the first option, the Hero's better friends drop by and say farewell. The Hero will start to walk out of the valley with the Kid at his heels, and the player's ability to redirect his motions will gradually diminish. As they pass by the bandit hideout, the player loses control entirely. A few straggling bandits attack shortly thereafter, with both the Hero and the Kid being completely scripted. Each turn desaturates the view significantly, and when the bandits are defeated in the 5th turn or so the screen whites out. Game Over.

If the player chooses the second option, however, the camera leaves the Hero behind and starts to follow the Kid, who is not yet in the control of the player. The colors return to the same washed-out look they had at the beginning of the game. As the Kid gets closer to the bandit hideout, however, the palette becomes more vibrant. As he continues on past, the saturation increases. When the bandits attack, the player is finally given control of him; at this point the Kid is better than them by a fair margin. Each turn of combat increases color saturation, as does each kill. After he triumphs, the Kid looks away from the valley into the very bright and vibrant lands beyond. Heroic music; game over.

The thing I'm really going for in this idea is the weariness of the Hero. He's good at fighting, much better by far than anyone in the valley. Once upon a time he found combat thrilling, but now it's lost whatever luster it had. He's tired of killing, but he's trapped in a particular life by the sword. The desaturation of colors with each moment of combat and each kill represents his growing weariness and depression. The combat is intentionally made un-fun and un-rewarding to encourage the player to feel the same way about it that the character does. The life the Hero finds in the valley refreshes and revitalizes him, hence the increased color, and so the conversations, jobs, and sidequests should be made rewarding and engaging to encourage a similar feeling in the player. The Hero finds one last use for his fighting skills and then passes his sword along to the Kid, who resembles the Hero's younger self in his outlook. Or, the Hero allows his spirit to fade and consigns himself to an empty life he has grown to hate. The choice at the end isn't really about player input, but about asking the player to reflect on his experience in the game and correctly identify what the character values.

Obviously, I'm also tweaking a couple of things about RPGs here. A typical RPG would be about the fresh-faced Kid who has made a decision to go out into the world and have adventures, and this bit back home in the valley with the weak enemies would just be the first chapter. The pointless monotony of combat grind is also something I'm trying to get at with the desaturation and the first ending to the game. Of course, this could end up just being a collection of minigames with a story attached, but that might be refreshing in its own right.

Please visit this month's other entries:

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Critical Thinking Compilation: Prince of Persia

The decision to completely reinvent the Prince of Persia universe, following the (mostly) widely-praised Sands of Time trilogy may have come as a surprise to many fans. Removing the time-manipulation mechanic, discarding the character of the Prince, and adopting a dreamlike, cel-shaded look all seemed like significant and perhaps inexplicable departures. Duncan Fyfe has suggested, in "Time after Time", that the franchise needs to keep rebooting itself because the simplistic core of its story and gameplay is at odds with the need to extend its stories into trilogies and series. Perhaps it was this, perhaps Ubisoft simply felt that it was time to go in a new direction. Given the great affection of many gamers and critics for Sands of Time, it was perhaps inevitable that Prince of Persia would be scrutinized extensively. Perhaps it was held to too high a standard, as Scott Juster thinks, but fairly or not several of the decisions in the new game's design have proven widely divisive.

A gentle journey upstream

Among hardcore gamers there is little doubt that Prince of Persia is easy to play, but a debate rages over whether it is too easy, whatever that might mean. The game is devoid of punishing "Game Over" screens or unforgiving platforming puzzles in the mold of Sands of Time's Tower of Dawn. The Prince's companion Elika has streamlined the experience of "death" in the game, as Richard Naik and others have pointed out. In addition, Joe Tortuga has identified an equivalence of action for the face buttons in different modes that is likely to make the control experience more sensible for the newcomer. Moreover, as a consequence of the open-world design, the difficulty curve is rather flat.

The upshot of all of this is a game in a hardcore mold that reaches out to more casual players. Michael Abbott, in "Prince of Noobs", relates that the striking visuals of the game attracted the attention of new players in his family, while the toned-down difficulty and simplified mechanics got out of the way of their enjoyment. Scott Juster believes that this approach fits with a new era of gaming, in which major titles primarily aspire to be accessible, rather than challenging. On the other hand, Jorge Albor and GSG have argued that Prince of Persia may be a bridge intended to stimulate the casual player's further interest in games. In this respect, Ubisoft may be recapitulating Nintendo's upstreaming strategy.

Gameplay as characterization

The idea that the challenge was stripped from a game in order to make room for casual players is not one that will make the hardcore crowd happy. Other writers have suggested, however, that the simplification of gameplay has aims beyond merely appealing to the pick-up-and-play crowd. Rather, the platforming and interactions with the world are designed to make the player feel a particular way. The compelling interactions with the game world grab the player's attention, according to Spitfire, while Angelo asserts that the platforming segments and interactions with Elika develop into a kind of motion poetry. Greg Tannahill argues that the fun of the game comes from experiencing the platforming, rather than overcoming it. For Thomas Cross, the exuberant movements of the Prince draw the player into his world. I go even farther to argue that the emotions the player feels while platforming are a way of characterizing the Prince himself. If these things are true, then analyzing the game as a puzzle-platformer is inappropriate because that's simply not the kind of game it is. Iroquois Pliskin understands it as more of a rhythm game than a character action title. For him, the physicality of the platforming gave rise to the romance of the story.

Haptic interactions between Elika and the Prince are an important component of the storytelling in the game. The way they touch each other while moving through the world makes the player feel like he's in control of an actual person interacting with another actual person. As Jorge Albor says, touch deepens their relationship in a way comparable to Ico. Because Elika streamlines some aspects of the game and slows down others, David Zhong views interactions with her as a mixed blessing in terms of the gameplay. In "Wait for Me!" he ponders whether this ambivalence makes her more real. For Allen Cook, her relentless helpfulness actually made Elika seem at once passive-aggressive and boring. Although Joe Tortuga liked them, he felt that the haptic interactions interrupted the flow of play to the game's detriment.

Because the feelings elicited by gameplay to a large extent depended on the flowing nature of the platforming, just about anything that brought that to a halt was viewed as a negative. Even the optional conversations, which were praised in some quarters, were criticized along these lines by Nels Anderson. Although he liked that these segments were optional, he detested the way they broke up the game and the fact that they were visually boring. I felt the combat fell flat for similar reasons. For me, the slow, halting nature of the combat was completely at odds with the rest of the gameplay, and also didn't really fit the character of the Prince himself.

An interactive storybook?

Despite all of this, the difficulty level remains the elephant in the room, repeatedly brought to mind by the more ludicrous "Achievements" the game offers. David Zhong, for instance, characterizes the game as practically playing itself. In a pair of essays, Sinan Kubba argues that the lack of challenge in the game goes beyond the simplicity of execution and actually descends to the level of removing meaningful player input. In his view, Prince of Persia becomes something of an extended quicktime event, more akin to an interactive story than an actual game.

Is there something to this? The game seems to fiddle with player agency in interesting ways, especially at the end. The Prince's decision to cut down the trees was at odds with the desires of many players. As discussed at Tangletown Games, this fact emphasizes the division between the Prince, as a character, and the player as an agent, and forces the player to examine his own values. David Zhong felt that the optional dialogue gave rise to a disconnect between the player and the Prince. His informal survey suggests that many people saw the Prince, rather than themselves, as the agent in the closing scenes. In general we think of games as being about player agency (even if it is only the weak agency of "continue or end"). Could the reaction to the game's difficulty result from a feeling that the game in its story and mechanics removes agency from the player, despite its open story structure?

The Prince or the Princess?

The choice to invest so much in the Prince seems odd in retrospect because few critics were impressed with him. Always a good point man on character matters, Michael Abbott brings two essays to this line of inquiry. In "Prince of Promises" he identifies an uneasy tension between the light-hearted wisecracking of the Prince and the depressing destruction of the world; he feels the game might be telling the wrong story. The Prince's character artlessly reaches for Han Solo territory, as Michael details in "Prince of Nada", which makes him much less interesting than Elika.

So, why not make the game Princess of Persia? In his essay, "Caring about the Prince" Tom Cross acknowledged that Elika is the real emotional core of the story, and this is a thread that you can find in many of these essays. In his consideration of Elika, Ben Fritz pointed out that she's so powerful one wonders why she needs the Prince at all. Yet the game's story works against Elika, and its narrative conforms to patriarchal values, as Scott Juster explains in his excellent "Prince of Patriarchy". In the game, nature and women are subjugated to the desires of men, and the King (at the beginning) and the Prince (at the end) undermine whatever agency Elika has.

Does Prince of Persia earn its ending?

The ending of the game puts all the decision-making power in the hands of the Prince, taking it away from Elika, and even from the player. Even people who agree about the difficulty seem to be sharply divided by the game's conclusion. Steve Amodio thought that Elika was clearly worth saving and the cold, dead world of the Ahura was not. As mtvernon points out, the feeling of freedom in the gameplay depends entirely on Elika; her absence in the closing segment of the game makes the player feel uncertain and wary during the platforming. Spitfire felt that the ending worked in part because the Prince was a dynamic character, and in part because the gameplay incubated affection for Elika in the heart of the player. When the Prince slapped the bier, Spitfire got excited because he was united with the Prince in not wanting the experience to end.

Voices from the other side of the spectrum were just as loud. Sean Beanland felt that the game actually didn't do enough to convince the player that the Prince would choose to save Elika. Eric Swain thought that the structure of the Prince of Persia's story worked against the development of the relationship because it prevented it from following any real trajectory (he also proposes a particular arc in an interesting piece). Although the vignettes with the various corrupted successfully create a vision of what Ahriman is and what can be accomplished through him, Swain wonders why the Prince doesn't learn anything from their hollow victories. In fact the Prince reveals on at least one occasion that he understands Ahriman's duplicity. In my own view, the game never does enough to build up the relationship between the Prince and Elika, or convince the player he's foolish enough to make this choice. Moreover, by destroying the player's time investment the ending makes him feel like a sucker.

The player's time investment comes up in Game-Boy's wide-ranging discussion of the game. He feels that Prince of Persia goes out of its way with mechanics like Elika's life-saving to respect the player's investment of time. As a result, he's confused by the choice to devalue that investment at the end. Joe Tortuga identified a different kind of tension at this point, in that the game has a very free and open structure up until the final battle, but at that point doesn't allow you any alternatives.

Or does it? The early credit scroll led to some extended discussion of whether turning the game off early was a valid approach. Joe didn't think stopping early was a legitimate response, and as a consequence he felt the ending ruined the game. In contrast, Michael Abbott chose to walk away from the game rather than submit to its epilogue. As Greg Tannahill notes in his equivocal piece "Closing the Book", turning off a game early can improve the experience, but the question remains whether you are gutting the intentions of the creator. At the same time, as I mentioned earlier, games are about creating a feeling of player agency. In a game like Prince of Persia, is turning it off the only relevant contribution a player can make?



Happy reading, everyone. Special thanks to Michael Abbott for setting up some cross-blogging about the game's finale. That link collection was a good starting point for the rest of this. As always, point me to anything you think I overlooked using the comments, twitter, or e-mail.

Last updated: 4/6/09

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A game divided against itself

Sparrow has a clockwork husband named Hugo in Bowerstone. He looks like a man and sounds like a man, but I can press a button and see his gears and escapements, the sliders that reveal his attitudes and the routines that show me how to change them. He didn't care for Sparrow at first, but my glance at his ticking innards told me the gifts to give, the expressions to perform, that would cause Hugo to display the red heart that meant he was ripe for marrying. Transforming him from a disinterested aristocrat into an ardent suitor was a fun, if unchallenging game, which was fine until Fable II asked me to care about him. Hugo is an uncharacter, an automaton, utterly unable to inspire emotional connection, a frustrating symbol of this game's imperfections and uneasy tensions.

Fable II clearly wants to be a simulated world, an organic experience that affords the player a real opportunity to live an alternate life. You can go anywhere! You can do anything! But why would you? The vast majority of Fable II's NPCs have no personality or meaningful dialogue. They can fall in love with your nameless hero (known only by his or her title) but there's nothing there for you to fall in love with. Even if you managed that trick, the game offers no way to communicate your love back to them, despite the array of "expressions" your character can use. What are we to make of a game that allows you to screw your husband in a room full of strangers but not to hold his hand? What kind of affection are we to feel for a world that lets you slap a child but has no option to hug him?

This strange attitude towards the open world extends into the quests. The game quite effectively builds the conceit that its world is centered around the main character — he decides the fate of several communities throughout the game, as well as the fate of the world itself. Yet the game also repeatedly insists on his impotence. The tragedies and petty cruelties of the main quest cannot be altered or avoided. The sidequests also abound in futility: you can rescue villagers from slavers a thousand times, but all your killing does nothing to diminish the number of kidnappings. And each time the main character traverses the Rookridge Road, bandits will be standing at their spawn points. He may slaughter hundreds of men on this narrow stretch of highway, enough to pave the road with their skulls, but the same bandits will always be waiting in exactly the same places, as if their numbers were replenished hourly from the wombs of impossibly fecund bandit queens back at the bandit hive. The slaughter is endless, mindless, and pointless.

The fact of the matter is that in Fable II player agency is just a red herring. For all the options the world presents, the game has at its core a linear quest that the player can do very little to affect, populated by NPCs with whom he cannot meaningfully communicate. You can become anyone you want, unless you want to become that guy who helped Lucien achieve his dream. You can ignore the roads, but only within the bounds of smallish areas. Fable II promises choice but only delivers choices that don't matter. The myth of the player's agency is window dressing for a by-the-numbers story that would be widely castigated for its limpness had it emerged from the studios at Square-Enix or Namco. These two sides of Fable II exist in an uneasy tension, always working against each other. The free-form elements ask you to look around and explore, while the main plot orders you (sometimes quite forcefully) to keep your eyes straight ahead.

Fable II's internal divisions extend further than this. Its simplistic combat and guiding golden trail, for instance, seemingly open the door for casual player. However, the complexity of the leveling system (in which you collect four separate kinds of experience points) and the wall of menus (you must traverse three of them in order to eat a carrot) slam that door shut instantly. It's as if the developers at Lionhead simply couldn't decide whether they wanted to make an open, accessible RPG-lite or a dense, impenetrable classic RPG, so they made both and stuck them together with Krazy Glue.

Moreover, there is a serious disharmony of tone. The bulk of the NPC dialogue, the absurdities of the expression system, the foul-tempered gargoyles inhabiting every corner of Albion, and many of the Fable II's side quests build a humorous tone for the world. Fable II regularly pokes fun at classic fantasy tropes, role-playing games generally, and even itself. The main quest, however, is serious business, a lengthy and depressing contemplation of death, obsession and choice. Although artlessly contrived, its moral dilemmas effectively convey the idea that a hero must choose between his own happiness and that of others. When Fable II allows you to smooth over your misdeeds by farting in public a few times, however, it becomes clear that the main quest's theme simply doesn't belong the same game with the rest of this silly shit.

That's a pity, because when Fable II plays for laughs it really works. The characters shine, the interactions make sense, and the writing sparkles in every moment of levity, and there are many. If Lionhead had gone all out and devoted themselves exclusively to making this a farce it would have smoothed over the uneasy divides between openness and linearity, accessibility and complexity. Fable II would have been better if it had merely asked you to enjoy playing with its clockwork men, rather than asking you to love them.

Fable II's dedication to its solemn main quest and its arbitrary cruelties makes that impossible, however, just as its insistence on RPG interface tropes blocks it from becoming a casual-friendly game, and the irrelevance of its free-form interactions prevents it from feeling like the open-world RPG it pretends to be. The player can't make the hero do anything meaningful to relate to the characters that are worth knowing, and the characters that can engage in dynamic relationships don't come with any systems capable of supporting a genuine emotional connection. When the game reaches for that level of investment it only ends up stumbling over its absurd expression system and nonexistent haptic interactions. In trying to be too many things — funny and weighty, accessible and traditional, open and epic — Fable II only achieves division and incoherence, despite its virtues. There's a moral in that story.

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Dark Age

Since Corvus decided to open the floodgates to second submissions for this month's Blogs of the Round Table, I thought I'd add another design idea to the pile. As a reminder, the topic of the month was to "pre-imagine" a game for a work of literature, i.e. a game that might have inspired a classic novel, play, etc. For the second submission, Corvus requested that we pre-imagine a vastly different kind of game, for a vastly different kind of work. As my previous proposal was for an expressive browser-based game to inspire the E. E. Cummings poem "l(a", I will now propose Dark Age, a turn-based strategy game meant to inspire one of my favorite works of science fiction, Isaac Asimov's Foundation series.

One could probably generate this game as an extensive mod of a Civilization, because really the idea is Civ in reverse. The game has a similar set up: a tiled world map dotted with many cities. Most terrain tiles that are suitable for agriculture have already been irrigated; most tiles that possess mineral resources already have mines. Before the first turn of the game this whole map is clearly revealed, and the civilization is relatively advanced. In the first turn, the "Civilization" collapses, leaving each metropolis as an independent city-state. The player's city-state is situated at a relatively lush site at the edge of this civilization that is poor in the mineral resources needed to develop a military. The player's task is to preserve his civilization, its knowledge, and its technology.

In order to successfully achieve this goal, the player must maintain his population, gather resources, preserve recorded knowledge, and defend himself from popular overthrow or military conquest. The knowledge level of the civilization is represented by a simple number. Particular kinds of knowledge and technologies are lost or gained in a set, specific sequence based on the knowledge number. Each turn of the game will also introduce a "fog of war" effect on the world map, slowly shrinking knowledge of the world until it encompasses only those areas directly under the control of the player.

Maintaining the population requires agricultural planning; time in the game is resolved seasonally, and the player must assign sufficient population to the fields in each season to feed everyone. The population can also be assigned to public works, military duty, or staffing buildings. In addition to manpower, the agricultural system responds to the climate. This operates in nested cycles -- a seasonal cycle within a larger cycle of wet and dry years within an even larger cycle of warm and cold eras. The wet/dry and warm/cold cycles will start off following a set pattern that is the same in every game. The player's agricultural success will rely on his ability to stockpile sufficient food during times of plenty (warm/wet) to last him during hard times (cold/dry). The player can also gather timber and stone to construct buildings that allow stockpiling (granary), decrease annual population loss (hospital, nunnery), and increase agricultural production (stable, blacksmith, cathedral). The city starts with a church, and by telling his local prelate to focus on themes of chastity or fruitfulness the player can roughly adjust the birth rate, but there is a lag of several years between sermon and population. Several years of famine or high disease rates (due to overpopulation) will lead to your overthrow, ending the game.

Preserving recorded knowledge requires "free population" (e.g. people who are not assigned to the military, agriculture, or construction) and support buildings. The player begins with a library, which requires a modest staff year-round but slowly loses knowledge. Optional buildings include a monastery, which requires a very low staff but loses knowledge more rapidly, a school which requires a modest staff and a library, but grows knowledge at a rate equal to the library's loss rate. Other buildings which increase or preserve knowledge may include a grand library, alchemical laboratory, and university. Some of these buildings cannot be constructed without a minimum population in the player's empire. If the knowledge number drops too far, then the game ends.

Certain technologies will be more easily lost or regained based on resources, most of which will have to be acquired through trade. The lack of metal will be the greatest immediate problem. Surrounding cities will lose technologies very quickly; the player can trade his civilization's knowledge for their resources. Despite their minimal technology the nearby states will have strong militaries. Trading with only one of them will cause other states to attack, but the states will not ally with each other so trading with several states will cause them to stand off or fight each other instead of you. Assuming you can gather enough mineral resources you can of course conquer these states, but moving population into your military means that you can't use it to preserve knowledge or grow food. However, each city-state has a "native knowledge" number; if you are trading with that state and their native knowledge falls too far below the player's knowledge, then they will overthrow their leader and join your empire.

Tactics required for trade negotiation and diplomacy will differ in a predictable way based on the local tyrant's personality; dialogue cues will reveal what this personality is with a clear 1 to 1 correspondence that is the same in every game. For each personality of tyrant there will be a predictable way to progress from enemy to trading partner to master. Although the precise location of nearby city-states will differ from game to game based on the map, the personality of the rulers of the first-encountered city-states will be the same in every game. As the player's empire grows larger the hostility of his neighbors will increase. Obviously, angering your neighbors to the point that they conquer you will cause the game to end.

The game will keep track of how well the player is doing and what methods he is using to expand his empire. In the early part of the game, the player will receive copious hints and tips about what is coming. The game will, for instance, give him hints about the larger climate cycles and the correct steps to take for a given personality of a rival ruler. At a certain point, based on time or an assessment of progress, the game throws a crisis at the player, which varies based on the player's style. For instance, if the player has primarily used a diplomatic/trade approach to winning the game, several nearby cities will unite into a military empire and declare war on the player, rapidly moving to conquer his territories. If the player has been using military force to expand, a debilitating plague will sweep through his cities over a period of years, decimating the population. There should be a wide suite of possibilities, including civil war, protracted famine, the burning of key intellectual facilities, simultaneous exhaustion of multiple resources, etc.

At this point all of the cycles and predictability that governed the first half of the game will be expunged and replaced. The climate will shift to a new cycle, responses to social engineering will change, libraries will become less effective, external city-states will become more resistant to the player, the cues for particular personality types will change, and the pattern of progression to conquer a city-state under a given personality will also shift. These will all still follow predictable patterns and cycles, but the rules will differ significantly from the earlier play segment. All the new behaviors will be chosen randomly, from highly degenerate sets when possible. The game will no longer give hints or tips of any kind, except a single warning that the behavior of "some" systems "may" have changed.

Winning the game involves keeping the civilization's knowledge at or above the game-start level for a set period of time after the crisis, conquering the entire map, or pushing the knowledge level to the maximum.

The Foundation series begins with a society meant to preserve knowledge in the face of a decline and eventual collapse of a galaxy-wide empire. Threatened by their neighbors, who are already sinking into barbarism, the Foundation survives by playing them off each other using a faux-religious trade in knowledge, then conquers them through their dependence on that knowledge. The reason for their success is careful planning by a man named Hari Seldon who is able to scientifically predict the future in general terms. In later novels, the Seldon plan is thrown off by an unpredictable crisis created by a psychic mutant, and then rescued by a secret "Second Foundation" of psychics. Then a great deal of work goes into steering the Foundation back onto the course laid out by Seldon initially.

I wanted to capture the secure feeling of knowing the future via easily predictable patterns reinforced by copious hints in the early stages, as well as the defensive footing of the early Foundation. The shock and confusion of the seemingly unpredictable crisis upends this feeling of security and inevitability. A new trepidation about the future is then reinforced by the altered patterns of the second half of the game and the absence of tips from the program itself. The goal is also a sort of poke in the eye at FAQs, the point being to make the generation of new behaviors so complex that it is more work to read an FAQ than it would be to figure it out yourself. The player's success at figuring out the systems on his own builds a new sense of confidence and control, one which is more honest than in the first half of the game where he is relentlessly guided. This matches the feeling of the Foundation in later books when they have bounced back from the Mule and their obsession with the Second Foundation.

Finally, I also wanted to put forward the idea of knowledge as a precious resource that can be traded and used to exert influence. This is something that has been used to some extent in the existing Civilization games, but not in quite the same way as it has in history. The Civ games believe in a straight-up transference of knowledge. What often happens instead is a kind of scientific and engineering colonialism, in which developed nations give the products of their ideas to the Third World without giving them the expertise itself. That's the fundamental underpinning of the Foundation's dominance in Asimov's books, and something for the player to think about in our world also.

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Man-made biological clocks

Numerous and diverse biological processes depend on the functioning of an internal clock. Biological timers determine your heart rate, the frequency of cell division, and the way you feel at 3 AM, among other things. Similarly, mechanical and electronic clocks serve essential functions in many kinds of man-made devices. As we begin to develop synthetic organisms for medical and industrial purposes, it will be useful for us to be able to construct timers within these micro-organisms to control their activity. In two recent papers, scientists have created molecular systems in mammalian and bacterial cells with tunable oscillation periods.

Although the methods used to construct these oscillators and the kinds of cells they were made in differ significantly, the two systems had one key similarity. Both oscillators used both a positive and a negative feedback loop. In principle, it should be possible to construct an oscillating system using only a negative feedback loop. For instance, you could have a system in which a transcriptional activator enhances the expression of a functional protein as well as that of a transcriptional repressor. As the concentration of the repressor increases, that of the activator falls, causing levels of the functional protein and the repressor to fall, allowing the concentration of the activator to rise again. By tuning the lag in this system one could in theory produce an oscillator with a range of possible frequencies. Yet many systems seem to have evolved with a positive feedback loop as well (in which the activator enhances its own expression).

This curious feature was the subject of a series of simulations reported by Tsai et al. (1) last July in Science. Their studies indicated that a system using only a negative feedback loop would produce a periodic oscillation just as expected. However, they also found that systems relying only on negative feedback were limited in that it was difficult to adjust the frequency of the oscillation without also altering its amplitude. Introducing a positive feedback loop stabilized the system so that the oscillator could be tuned to a wider range of frequencies without altering peak amplitude.

The benefits of this approach were demonstrated in data reported by Stricker et al. last November in Nature (2). They constructed a circuit that expressed green fluorescent protein in an oscillatory manner in response to stimulation by arabinose and isopropyl β-D-thiogalactopyranoside (IPTG). They created a circuit (see their figure, right) in which every component ran off a hybrid promoter that could be activated by AraC (which binds arabinose) and inhibited by LacI (which binds IPTG). Arabinose binds to and activates AraC, while IPTG binds to and inactivates the LacI repressor, with the result that all three genes are transcribed, and the cells fluoresce due to the presence of GFP. As the concentration of LacI increases, the activating power of the IPTG decreases, leading to an eventual repression of transcription and the end of fluorescence. As the LacI proteins get degraded the IPTG concentration again becomes sufficient to activate transcription, leading to a new fluorescent phase.

Stricker et al. found that they could alter the frequency and amplitude of this oscillation by altering the growth conditions of the bacteria (temperature and nutrient availability) as well as by adjusting the concentrations of the activating reagents arabinose and IPTG. By attempting to match computer models of their oscillator to the data they collected, they found that the time needed for translation, folding, and multimerization played a critical role in establishing the existence and period of the oscillation. Stricker et al. constructed an additional circuit using only negative feedback from LacI, proving that this was possible, but they found that in this case the period was not very sensitive to IPTG concentration and the oscillations were not as regular.

A similar system was constructed in Chinese hamster ovary cells by Tigges et al., who described their results recently in Nature (3). The circuit they constructed used tetracycline (TC) and pristinamycin I (PI) as activating molecules. The tetracycline-dependent transactivator (tTA) served as the positive feedback lood, activating transcription of itself, GFP, and the pristinamycin-dependent transactivator (PIT). In this system, increased levels of PIT cause the production of antisense RNA to tTA, causing its mRNA to be destroyed prior to protein production. This, in turn, diminishes production of all proteins until the reduced levels of PIT allow tTA to again activate transcription. They found that they could control the period of oscillation by altering the gene dosage (i.e. the quantity of DNA used to transfect the cells).

The oscillating systems constructed in these papers serve more as test cases and examinations of principles than as functional pieces of synthetic systems. You will not be using an E. coli alarm clock any time soon. However, it has always been true that you learn more from trying to build something than from trying to tear it apart. These attempts to construct artificial periodic oscillators have provided interesting insights into those that have evolved naturally. The knowledge gained from these experiments will help us to understand oscillatory systems like the circadian rhythm and cardiac pacemaker, in addition to illuminating design principles for synthetic biology.

(1) - T. Y.-C. Tsai, Y. S. Choi, W. Ma, J. R. Pomerening, C. Tang, J. E. Ferrell (2008). Robust, Tunable Biological Oscillations from Interlinked Positive and Negative Feedback Loops Science, 321 (5885), 126-129 DOI: 10.1126/science.1156951

(2) - Jesse Stricker, Scott Cookson, Matthew R. Bennett, William H. Mather, Lev S. Tsimring, Jeff Hasty (2008). A fast, robust and tunable synthetic gene oscillator Nature, 456 (7221), 516-519 DOI: 10.1038/nature07389

(3) - Marcel Tigges, Tatiana T. Marquez-Lago, Jörg Stelling, Martin Fussenegger (2009). A tunable synthetic mammalian oscillator Nature, 457 (7227), 309-312 DOI: 10.1038/nature07616

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More lab courtesy haiku

It's time once again to remind your labmates how to behave, seventeen syllables at a time.

Dust and detritus of past ages brushed away — leave balances clean!

Microbe-encrusted, reeking of death: your uncleaned centrifuge bottles.

If it cannot touch your skin, it must not touch my keyboard. Remove gloves!

Banshee scream boiling precious samples — don't mess with the sonicator!

Chemicals, hazards, trailing you in the hallways... Lab coats stay in labs.

Although you're wearing headphones, we can all still hear you singing along.

Unlabeled buffers may sometimes be used to make your morning coffee.

Washing glassware may not be your job, but please rinse that salty crust off!

The bear seeks a lost cub — I find my pipetman on your filthy bench.

Vapor will corrode them — store pipetmen upright, with tips ejected.

It takes five minutes to make destain — replenish the carboy when low.

Hiding that ruined column will not magically make it fix itself.

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Ecce, soror

During the course of play, the first-person shooter Bioshock repeatedly presents the player/character with a moral choice. Amidst the ruin of its underwater city, Rapture, a number of small girls wander, girls who possess a resource (ADAM) the player/character desperately needs. Upon defeating the girls' monstrous guardians, the player can choose to kill ("harvest") the child, gaining a large quantity ADAM, or he can choose to save the girl, receiving half as much ADAM and a vague promise of future help from another character. If the player saves a "little sister", he is treated to a short scene in which she thanks him, then runs off. If he chooses to destroy her he sees... nothing.

That's not strictly true, of course. The game shows the player/character reaching towards the girl (the on-screen hand seems to be subtly larger than in the "save" option), and transitions via a swirling effect to an image of the parasite the player/character has just ripped from her body. The girl's body is nowhere to be found. Although the player is meant to believe that the character has just eviscerated a small child in order to gain a strategic advantage, no evidence of the awful crime remains to confront him. Due to the limitations of the engine, the other dead bodies in Bioshock are often temporary, but they at least exist for a moment. The game's most horrible killings are also those that leave no physical trace at all beyond the advantage they confer on the player.

I find this disturbing because it speaks to an ethos that is at peace with the idea of child murder as an intellectual act, but not a physical one. Regardless of what some reactionaries may say about games, I sincerely doubt that any of Bioshock's players could actually go through with the "harvest" if doing so involved a graphic depiction of evisceration. If Bioshock only forced the player to face a dead, bloody body, many would only be able to "harvest" once. Even considered on a purely intellectual level many gamers found Bioshock's options to be too much, and were unable to choose "harvesting". Yet I feel that games which require the player to choose an ethic ought to confront him with the moral horror of his choice. Killing another person, adult or child, is an awful act, and if developers are to be true to the idea of giving the player a morality simulation then they should not hold back on a graphic depiction of the consequences.

I don't mean to imply that developer cowardice is the only interpretation of the design. One can also take the distancing of the player from the act to suggest that the player/character is in denial about his moral agency. Rapture's creator, Andrew Ryan, continued to insist on his objectivist beliefs even as he solidified his totalitarian rule over the city; he was blind to his betrayal of his own principles. The player/character's inability to visualize his own brutality and its consequences may mirror Ryan's refusal to countenance his own moral failure. Because the player/character is a mirror of Ryan in many ways this interpretation has some resonance.

In addition, the choice not to confront the player with the moral horror of the choice to kill may be making a point about our own moral judgments. Nothing beyond a slightly disturbing ending punishes the player for choosing to kill the little sisters. When one does not have to consider the negative consequences, the decision to murder a young girl becomes almost trivial. I would deny, however, that it is necessary or admirable to emphasize this view. The triviality of murder under such circumstances is already a critical element of this and every other shooter. Acknowledging that a lack of moral perspective or an elimination of consequence enables monstrous actions is hardly interesting at this point.

Developers, of course, must contend with factors beside the limits of their creativity and artistic desire. The ESRB would certainly recoil, and anti-game reactionaries howl in protest, were any game to realistically depict the evisceration and murder of a young girl, or even showed the dead body resulting from such an act. In my opinion, however, allowing players to perform the act and not confronting them with the consequences is exactly the sort of behavior the watchdogs should be trying to prevent. The bloodlessness of murdering children in Bioshock does not make that depiction less disturbing, but more so. In its larger story, Bioshock expresses the thesis that abstract ideals falter in the face of real consequences. To shy away from those consequences is to stab at the heart of the game.

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How we taste umami

Although we still do not know the full breadth of our flavor-sensing capabilities, human beings are known to possess receptors for at least five basic tastes. Probably you have known about the sweet, sour, salty, and bitter flavors since you were in grade school, but the fifth, umami, was less widely accepted in the West until recently. Umami is a savory flavor element that is found in many foods, including tomatoes, parmesan cheese, truffles, and many kinds of meat and seafood. The umami taste primarily detects the amino acid glutamate (hence the popularity of the food additive monosodium glutamate, or MSG), but the effect is also intensified by the presence of the nucleotide inosine monophosphate (IMP). In a recent (open access) paper in PNAS, researchers from two corporations examined the umami taste receptor to understand how this happens.

The umami flavor is detected by a pair of G-protein coupled receptors (GPCRs) that have an external venus flytrap (VFT) domain in addition to their classic 7-helix trans-membrane domain (TMD). This complex is closely related to the sensor for the sweet flavor: in fact one of the receptors (called T1R3) is the same in both sensors. It is the second receptor (T1R1 for umami, T1R2 for sweet) that determines what taste is recognized. What we don't know for sure is whether it is the TMD or the VFT of these receptors that identifies the flavor component.

In order to answer this question, the researchers performed an experiment known as a "domain swap". Using recombinant DNA technology they assembled two chimeric proteins, one with the VFT of umami and the TMD of sweet, and one with the VFT of sweet and the TMD of umami. They then inserted these proteins into cultured cells that would fluoresce when the receptors were activated. The authors suspected that the VFT is primarily responsible for binding the ligand. As you can see from figures 1 & 2 (this is an open access paper, so go ahead and take a look), the experiment bears this out. The chimera with the VFT of sweet caused a fluorescent response in the presence of compounds such as sucrose and aspartame, while the umami-VFT chimera reacted to glutamate and aspartate. You can also see in figure 2C that the presence of IMP dramatically enhanced the activity of glutamate in this chimera. This indicates that the VFT is also responsible for IMP synergy in the umami receptor.

The hurdle in going further than this is that no structure of the umami VFT is available, which makes it difficult to figure out exactly how everything fits together. However, T1R1 has a close evolutionary relationship to the metabotropic glutamate receptors (mGluR), and a crystal structure of that VFT is available. Using conserved and homologous residues as a guide, the authors made a model of the T1R1 fold from the mGluR data. Based on this model they predicted certain amino acids that would be essential for glutamate binding in T1R1 and then mutated them in order to measure the effect. Residues that were predicted by the model to interact with the zwitterionic amino acid backbone proved to be essential for ligand recognition. Interestingly, the amino acids that contact the side-chain carboxylic acid of glutamate in mGluR are not conserved in T1R1, and mutations at the matching sites do not alter glutamate binding. However, these mutations eliminate the effect of IMP.

In order to understand this behavior, the authors modeled the binding cleft in the closed state, with IMP and glutamate in place. Glutamate binds at the bottom of the cleft, with its side chain pointed outwards. This conformation puts several positively-charged residues from the two lobes of the VFT close together higher up in the cleft. The authors propose, in keeping with previous models of VFT behavior, that the binding of the glutamate lowers the energy barrier between the open and closed states of the domain, but that glutamate alone is not sufficient to hold the domain closed. Their model places IMP higher up in the cleft, where its negatively-charged phosphate interacts with the positive residues. Thus, IMP stabilizes the closed conformation of the VFT domain.

Some more work here would be welcome, particularly in the form of experimental crystal structures of the T1R1 VFT that can confirm the homology model. The VFT is rather large, but using a perdeuterated sample in a high-field magnet it might be possible to confirm the population-shift mechanism using NMR experiments. Lower-resolution techniques such as FRET may also be able to catch this stabilization behavior. If the model proves to be accurate, it would serve as an interesting example of positive allostery from a population shift.

Although these experiments only concerned the umami taste receptor, this allosteric mechanism may be a more general feature of certain GPCRs. The authors indicate that they have unpublished data showing similar behavior in the sweet receptor, and it may be possible to design an allosteric stabilizer for any GPCR with a VFT domain. Because the related mGluR receptors are involved in many neurological and psychological diseases, successful design of such activators may have some therapeutic value.

F. Zhang, B. Klebansky, R. M. Fine, H. Xu, A. Pronin, H. Liu, C. Tachdjian, X. Li (2008). Molecular mechanism for the umami taste synergism Proceedings of the National Academy of Sciences, 105 (52), 20930-20934 DOI: 10.1073/pnas.0810174106 OPEN ACCESS

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Downward Spiral

Corvus' January round table asks us to envision a game that preceded a favorite piece of literature. What game might have inspired The Iliad or Foundation? Well, I'm not going to deal in anything quite so epic. Instead I'm just going to imagine a game that might have inspired a poem I like by E.E. Cummings. This may not be what Corvus was going for, but it was an idea that came to mind and would not leave, so here you go.

I envision this as a browser-based flash game or a small downloadable. An impressionistic art style is what I would really prefer, given some of the early focus, but the later stages could also work with the heavily-lined visuals more common in these games.

The start-up screen for the game is an image of a tree standing alone in a lush field in summertime. Once the player has chosen to start the game by activating the window, the field of view zooms in on the top of the tree, finally focusing on a single yellow leaf, which immediately falls from its branch. From this point, all movements of the mouse result in gusts of wind. The player is free to move the mouse in any direction, but the maximum angle of the resulting "gust" will be 30° from horizontal. The ostensible aim of the game is to prevent the leaf from falling, but this is a losing battle for two reasons. Firstly, the leaf will not be able to exit the field of view, and the field of view will only travel downward in such a way that the leaf never enters the bottom fifth of the field. Secondly, the wind will not produce pure directional motion in the leaf, but rather chaotic tumbling.

The leaf begins descending through a green tree. The surrounding branches and leaves will move in response to the player's wind gusts. Starting about a fifth of the way down the green surrounding leaves will start transitioning to reds, purples, and dark browns. In order to assist the player's focus none of these leaves will be yellow. Once the color palette has transitioned completely the number of leaves on branches will begin to decrease (but none of them will fall off in the player's field of view). The player's aesthetic experience in this segment should be focused on the interplay of colors between leaves and sky, as well as light and shadow showing through them.

By about third of the way from the bottom the branches should be completely denuded (but still responsive to the wind gusts). The appearance of branches themselves should start decreasing in frequency until the field of view contains only the trunk and the leaf. The player's aesthetic experience in this region should be focused on the motions of the leaf itself. In keeping with this idea the chaotic tumbling of the leaf should be enhanced in some way.

As the leaf nears the ground the player sees that it is a white field of snow. Although this may have landscape features such as drifts, no living things should be in evidence: it is a fresh, deep snowfall. When the leaf lands on top of the snow the game ends. At this point the player's ability to perform any input ceases and more snow starts to fall, eventually burying the leaf.

During its descent the color of the leaf should fade from a bright yellow to a dull light brown. Its material behavior should also change. At the outset the leaf should be flat but flexible, changing its shape as it tumbles. During descent, however, it should gradually curl slightly at the edges and become a more rigid body. The color of the sky should fade from a light Carolina blue to a flat gray. Also during the descent the window should slowly zoom in on the leaf. At the outset the leaf should take up about 5% of the screen, growing to about 20% of the field of view at the end.

The E.E. Cummings poem I hope would be inspired by this game is "l(a":

l(a

le
af
fa
ll

s)
one
l

iness

I wanted the game to represent the explicit event while expressing the emotion. Obviously the verticality is important, but it's a natural part of the event (which is part of the brilliance of the poem). The simplicity of the game and spareness of the final stages are meant to connect with the linguistic efficiency of the poem. I suppose in one sense this isn't much of a game, because it's basically a way of playing with leaves, but I wanted it to display an unconventional attitude. The design relies heavily on gradients (of color, of shape, of element density), which doesn't really have much to do with the poem, but which I think would be critical to helping the emotion sneak up on the player.

I probably had more fun thinking that up than you had reading it, but fortunately everybody else has better entries. Go check them out!

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Long-range effects in the ribosome

Antibiotics such as chloramphenicol suppress infections by inhibiting bacteria from making proteins. They achieve this by binding to and blocking the peptidyl transferase center (PTC) of the ribosome, a large complex of RNA and protein that performs nearly polypeptide synthesis in living cells. Although PTC-binding antibiotics comprise several different families of compounds, mutations in the ribosome that confer resistance to one family often produce cross-resistance to other families. This is difficult to understand because the PTC itself is highly conserved and not very tolerant of mutations. In an upcoming paper (open access, read along) in the Proceedings of the National Academy of Sciences, a team of researchers from the Weizman Institute of Science analyze several crystal structures of the ribosome to understand how this cross-resistance arises.

Davidovich et al. mapped nucleotide mutations known to confer resistance to PTC antibiotics onto x-ray crystal structures of the large ribosomal subunit from D. radiodurans in complex with antibiotics. One interesting facet of the resistance mutations became immediately apparent: they were almost all clustered on one side of the antibiotic binding site.

You can see this pretty clearly in Figure 2 panels B&D. Although the antibiotics (large pink surface) are surrounded by nucleotides, most of those that are on the left side (thin tan sticks) do not confer resistance if mutated. Resistance-conferring mutations instead cluster around the "rear wall" of the PTC (to the right). The authors explain that in this region ribosomal functions primarily rely on the sugar-phosphate backbone of the rRNA. Because the backbone elements are the same for all ribonucleotide bases, mutations in this region are more likely to be tolerated without significant loss of function.

Another striking feature of resistance mutations is visible in Figure 2 and quantified in Figure 3A, namely that many of these mutated bases do not contact the antibiotics directly. In particular, mutation of G2032 appears to play a role in conferring resistance to several different antibiotics. Overall, however, it appears that numerous long-range interactions can interfere with antibiotic binding.

The lynchpin of these interactions seems to be U2504, a base that directly contacts the bound antibiotic in most cases. Mutations to U2504 itself do not appear to be well-tolerated, but many of the long-range mutations occur in the layer of bases surrounding it. The authors describe in detail several mechanisms by which the observed mutations might increase the flexibility of U2504, allowing it to adopt positions that could allow continued protein synthesis while reducing the binding of antibiotics. The commonality of interactions with U2504, and the importance of the structural context of the surrounding nucleotides, explains why many mutations can give rise to cross-resistance.

The practical upshot of these findings is that they may serve as a guide for the design of future antibiotics. Since the majority of the drug-resistance mutations lie on the rear wall of the PTC, the effectiveness of these antibiotics may be enhanced by improving their binding to other parts of the site. With further modeling it may also be possible to design antibiotics that can compensate for flexibility at U2504. These findings also remind us that dynamics and long-range interactions can be important to the function of any biomolecule with a folded three-dimensional structure, not just proteins.

C. Davidovich, A. Bashan, A. Yonath (2008). Structural basis for cross-resistance to ribosomal PTC antibiotics Proceedings of the National Academy of Sciences, 105 (52), 20665-20670 DOI: 10.1073/pnas.0810826105 OPEN ACCESS

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