Don't look for "the" structure

It surprises me how often I hear students, postdocs, and even professors talk about determining the structure of a protein. A singular structure has the advantage of being relatively easy to interpret, but the cost of this is often the loss of functional data. It's easy to understand how this terminology emerges from the discipline of crystallography, which after all only works when the protein molecules adopt only a small number of conformations. Yet even when it comes to NMR, a technique that should be very sensitive to the fact of structural multiplicity, the language of researchers and the structural tools available to them are too often oriented towards the idea of a singular structure. But any representation of a protein as a single conformation is a simplification — every protein exists in multiple structural states.

Trivially, we are aware that a given polypeptide chain can adopt a number of different conformations — the "folded state" of any given polypeptide chain covers only a tiny sliver of the possible conformational space. A protein that is "unfolded" occupies not a single, well-defined state but a vast multiplicity of states, and this kind of statement is not controversial because we tend to imagine unfoldedness as a messy chaotic jumble of conformations. The reality is less cut-and-dried: although unfolded proteins may have no regular structure, many still have a propensity to form particular secondary structures or interactions. The reality of denatured proteins is that they have a complex and varied energy landscape, not an array of possible structures that all have roughly equivalent energy. The flipside of the popular view is that the a protein's native state draws down to a sharp energy well, and this conception is also misguided.

The most dramatic counterexamples to the idea of a neat, punctate energy well come from proteins that adopt several different folds in the native state. One relevant case is lymphotactin, which freely interconverts between an α/β monomer and an all-β dimer under physiological conditions. Lymphotactin may be unusual, but the principal message from that study is one that ought be paid attention to in others, particularly when the protein in question has functional conformational diversity. Consider α-synuclein, a protein implicated in Parkinson's disease. In the presence of some detergent micelles this protein is known to take on an α-helical hairpin structure, with two helices laying down on the charged surface of the lipid headgroup. In solution, however, it seems to take on a number of different forms, and may interact with true lipid bilayers in a completely different way than it interacts with micelles. For proteins that interconvert between several different physiologically-relevant folds, one is never pursuing the structure, but rather a structure.

Of course, we don't expect most proteins or domains to regularly adopt alternate overall folds. However, reorientations of domains or monomers is a relatively common behavior, and one that poses a sticky challenge for structural biologists because incidental properties of a particular arrangement may bias our experiments towards observing it. A minor member of the ensemble, if it has favorable packing geometry, may exclusively populate a crystal. Similarly, NMR experiments to determine domain arrangement via residual dipolar couplings must always be undertaken with an eye to ensuring that interactions with the aligning media do not bias the results. No single structure of adenylate kinase can instruct us about its catalytic cycle, and structures of the unbound state do not capture the reality that the protein continues to open and close in the absence of ligand. Single structures do not capture motions of domains or monomers relative to each other and that often means an incomplete understanding of function.

Domain motions are also an overly dramatic example, because simpler rearrangements of the backbone take place in many proteins, even when regular secondary structures are evident. Fluctuations of the main chain play a functional role in several proteins — as, for instance, in the flaps of the HIV protease. Additionally, rearrangements of the backbone have a significant role in signaling, as in NtrC, which I'll talk about more in two weeks. Proteins where the main chain rearranges in response to ligand binding or post-translational modification generally cannot be described by a single structure.

Even if the backbone is rigid, every protein will have flexibility in the side chains of its amino acids. One of course expects to see this kind of behavior in side chains on the surface of a protein, where it is usually dismissed as irrelevant. However, we also know that side chains can rotate and move in the core of a protein, and that on some protein surfaces they can undergo coherent rearrangements. I'll talk a bit more about the functional relevance of side-chain motions next Thursday. For now, suffice to say that side chain rotations cannot be so easily ignored and sometimes have functional effects. Structural studies that do not capture these rotations may be missing something important.

My point here is not that single structures are stupid or useless. A structure can be very informative about about a protein's function, and often has great power to explain the effects of mutations and ligands. However, we should not mislead ourselves into thinking that any single structure will have all the answers, or indeed any of them. Every protein is a constantly interconverting ensemble of structures, and there are many layers of structural diversity within that ensemble, reaching from whole fold rearrangements to "mere" side-chain adjustments. Determining the structure of a protein is not a coherent goal for a research program. The successful structural biology study will characterize the conformation and energy of key, functionally-relevant members of the protein's structural ensemble and identify the pathways between them.

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Chosen at birth

Last post, I mentioned that the tendency to choose segregation as a means to solve problems was a feature of many societies in the world of Dragon Age. Another, related motif appearing in many Thedan societies is the existence of a rigidly-defined social order in which a person's status and even his occupation are set at the moment of birth. To varying degrees this kind of social rigidity appears in almost every social group in the game (except the elves). Through its dialogue and plot, Dragon Age: Origins repudiates these systems, but in its mechanics it supports them.

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Out of sight, out of mind

Playing Dragon Age gave me a relatively frequent sense of déjà vu. Although the game portrays a number of different nations and societies, there are recurrent features that speak to underlying ideas about the psychology of its inhabitants. One such motif is the tendency for its denizens to solve their problems through segregation. At several levels, the people of the continent of Thedas like to resolve issues by pushing problematic groups into isolated areas and pretending, as much as possible, that they no longer exist.

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A few things

I promise there will be actual content in essay form later this week, but I have to kick the week off with some housekeeping. Some of this is bad, and some of it is good (or I hope it's good).

Bad news first: I've been getting pretty regular comment spam on this blog for about a month now, at a rate of about one post every half day or so. I don't know why anyone's bothering — most of the posts affected are at least half a year old — but it's still pretty irritating to click back to the blog every day to delete internet pharmacy ads written in badly formatted bbcode. Blogger doesn't give me a lot of options for dealing with this, and I'm not in the mood to migrate to some other platform. So as a first step I am turning on moderation for all posts more than 90 days old. The only kind of comment I will refuse is an obvious advertisement, but comments will obviously show up slowly on those posts. My other options are to block anonymous comments, which I definitely will not do, or to use a CAPTCHA, which I find to be annoying and have evidence is only moderately effective. Hopefully this will resolve the issue.

On the good side, my friend Ben Abraham, proprietor of the SLRC blog and organizer of Critical Distance, is going to be visiting GDC. Michael Abbott and David Carlton have already gotten him a pass, but since Ben is in Australia and GDC is in America, there's a rather expensive plane ticket to deal with. On the right side of the blog you can see a little widget for donating money to help cover the cost of that flight. I'd appreciate it if you could spare a few bucks to give him a hand.

If giving money to Ben doesn't sound like something you want to do, then I hope you'll find space in your budget to give a few dollars to Penny Arcade's annual Child's Play charity drive. Child's Play buys toys, books, and games for children's hospitals throughout the world to help make the lives of children with serious illnesses much less miserable. I spent a lot of time in hospitals myself as a kid, and the only parts of those stays that didn't suck terribly was the time I got to spend getting my ass kicked by the original Legend of Zelda. You can donate money, plain and simple, or you can choose your local children's hospital and buy something from their Amazon wish list. There are items on those lists that cost as little as $10 and will make a big improvement in what is usually a horrible experience, so please donate something if you can.

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The uncertainty principle

Although it uses conventional horror tropes, Condemned: Criminal Origins takes an unusual approach for a survival horror game. Survival horror often generates tension through resource scarcity, limiting the player's ability to fight or heal himself, but because of its melee combat focus Condemned is necessarily limited in this respect. The relatively plentiful checkpoints mean that the player usually isn't punished excessively for failure. Yet Condemned is a frightening, tense game because it artfully keeps the player from knowing what is coming next.

Condemned has a significant advantage when it comes to generating fear because of its visceral combat. While a few guns can be found in almost every level of the game, its hero Ethan Thomas can't carry ammunition for any of them. As a result, he is forced to improvise melee weapons from the environment, and fight his enemies up close and personal. Moreover, he's not fighting fantastic creatures, but ordinary people who have taken on (in most cases) a subtly monstrous aspect. The close quarters and melee brutality make the combat intrinsically frightening, but if that were all that Condemned had going for it, it would not be a truly scary game.

Video games pose a special problem with regard to creating fear because any game is a system that can be mastered. If the designer wishes to evoke a fear response through an attack on the avatar, he must contend with the player's ability to manipulate a game's combat systems. If the player becomes proficient at fighting off the enemies, or lowers the difficulty to compensate for his incompetence, then the onset of a fight stops being frightening and starts becoming a problem to be solved. Similarly, if a cue or activity is known to precede a fight loses its power to develop fear. These moments cease to be tense and instead become a time where the player prepares himself for a battle he knows is coming. Knowing that a fight is about to happen lets the player feel in control of moments where he is meant to feel vulnerable. To be frightening, a game must re-weight the power relationship in favor of the designer, rather than the player.

Condemned presents just this kind of activity in the form of crime scene investigation tools that Ethan must use to track down the serial killer who has framed him. The player often must use the tools in order to find vital clues, but Ethan cannot hold one of these gadgets and a weapon simultaneously. Thus, every use of an evidence-collection tool leaves Ethan vulnerable to attack. In any number of games this would form the basis of a recurring sequence: see clue → collect evidence → get assaulted. The player would enter the state just described, in which starting to collect evidence would signify the onset of a combat sequence and allow the player to feel control. Instead, the developers of Condemned chose to attack the player during the course of evidence collection only occasionally. Sometimes Ethan gets attacked while he collects evidence, and sometimes he does not. The player cannot develop mastery because these sequences have ambiguous outcomes.

Condemned plays with this idea in other ways as well. Environmental sounds sometimes signify an imminent attack, and sometimes do not. In one level, some mannequins turn into enemies and others, often indistinguishable, don't. The levels occasionally rearrange themselves, without regular signifiers. Condemned avoids predictability, and this denies power to the player.

The finest articulation of the skillful design that went into Condemned comes in its penultimate chapter, in which Ethan must search the (apparently) empty house of the serial killer for clues. The killer has written trails of words that lead to important spots, but these can only be seen if the player uses Ethan's UV lamp. The whole house becomes an evidence collection routine, but the developers resist the urge to have enemies burst out of every door and window as Ethan searches. The occasional attacks they employ instead support the tension of the sequence without letting the player view it as a combat routine. This level is so memorable in part because of the exquisite suspense it generates.

The most interesting thing about choosing to attack at only some of these junctures is that it doesn't come at any cost. From studies of conditioning, we know that using a variable schedule of positive or negative reinforcement (i.e. not reinforcing every time) is no less effective at generating a desired response than continuous reinforcement. In fact, research has shown that conditioned responses created using variable reinforcement are more resistant to extinction than those created with continuous reinforcement. By associating the fear of a surprise attack with just some of these evidence-collection routines, the developers guarantee tension in all of them, without granting the player any feeling of power over the game.

Condemned ultimately falls down because of its last level, an uninteresting romp across a farm, beating down mutant hillbillies on your way to a dull and conventional final boss. Up to that point, however, Condemned cleverly steals power from the player by avoiding routine. Because the player can never be certain when an attack is coming, the intrinsic fright generated by the game's brutal combat is never diluted by the player's mastery. The player's uncertainty perpetuates his fear.

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Capsule: Nostalgia

Final Status: Story and most low-level side quests complete.

Put This on Your Box: At last, a game that ignores Britain's long history of brutal aboriginal repression!

Most Intriguing Idea: Evoking the 19th-century adventure novel in a game.

Best Design Decision: Near-ideal implementation of turn-based combat in the ground battles.

Worst Design Decision: Incredibly unbalanced and tedious airship battles.

Summary: Nostalgia unapologetically grabs for dated JRPG tropes, and for the most part succeeds in creating a pleasant experience. We have a chipper sword-wielding teen with spiky blond hair, a demure teenage girl with magic powers and amnesia, and two spunky orphans, all out to save the world from... well, it's a JRPG, so the story doesn't make much sense anyway. This time the hash is made with an inappropriate fusion of Norse and Greek mythologies, if you're keeping score. Battles proceed using a turn-based system similar to Final Fantasy X. The system is sharp, transparent, and perfectly executed (graphically appealing too: this is a quality game). On the ground, the difficulty curve is quite gentle and the battles are mostly easy. Even if you never take a single side-quest you will probably be perfectly prepared (or even over-leveled) when the end of the main story comes. The aerial battles are another matter: they are brutally difficult and often unwinnable. Players will frequently find themselves unable to either hit the enemy ship at all, or to escape. When victory can be achieved, it can sometimes take up to ten minutes to whittle down the massive life bars of enemy airships. Even at low altitudes, relatively frequent encounters with enemies that totally outclass the player's own Maverick airship occur well into the game, forcing frequent reloads. Nostalgia also appears to have the warm fuzzies for the British Imperium, but not a lot of time to spend on acknowledging what that meant for people who weren't white. To an extent this is excusable because the whole goal here is to evoke the feel of a Victorian adventure novel (the hero's parents have separate bedrooms), but it adds to the sense that this game is out of date — in this case, by at least a century.

If you can't say something nice... When it stays on the ground, Nostalgia is a fun little romp evoking pleasant memories of JRPGs past.

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Capsule: Mario & Luigi: Bowser's Inside Story

Final Status: Story finished.

Put this on your box: I HAVE CHORTLES!

Most intriguing idea: Leveling up your arch-nemesis to defeat your other arch-nemesis.

Best design decision: Constantly layering something new and interesting on the core mechanics.

Worst design decision: The unbalanced dodge mechanic and the tedious temporary invincibility in the boss fights.

Summary: Mario & Luigi: Bowser's Inside Story follows an established pattern for the Super Mario role-playing games. It combines light platforming action with turn-based battles that are supplemented with timing-based action to improve damage or defense. This has worked before and it works here, creating a perfectly competent RPG where Mario and Luigi roll around in Bowser's guts, powering up the big spiky turtle so he can take down the ludicrous Fawful, who has conquered both the Mushroom Kingdom and Bowser's domain. The gameplay is mostly solid, but the dodge mechanic is unbalanced. If you figure out the dodge for an attack, you'll never take a point of damage, but if you don't figure out what to do or the proper timing for it you will die constantly. Earlier RPGs in this series mostly used the dodge to reduce rather than eliminate damage, and that would have been a wiser choice here, as the ability to escape harm completely seems to have encouraged the designers to make enemy attacks very powerful. In addition, many of the bosses have some kind of invulnerability (Fawful, for instance, is effectively invincible for five turns at a time), which makes fighting them fairly tedious. On the plus side, every area inside of Bowser seems to have its own interesting twist, making sure the platforming never gets old, and powering him up uses some nifty rhythm games that I rather liked. The writing mostly works well, especially when you encounter the loopy dialogue of characters like Fawful, Sakon, or the doctor. Unfortunately, Bowser himself is pretty one-note, although his tough-love attitude towards his subordinates, and their adulation towards him, can be pretty funny. After a while, though, he wears thin, and there's so much of his dialogue in the game that it gets a bit tiresome. For me, the game overstayed its welcome a bit, but despite some minor design hiccups it's a high-quality experience that delivers both humor and gameplay depth.

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