Touch the void

In a curious way, maybe the climber stops living when he begins to climb. He steps out of the living world of anxiety into a world where there is no room, no time, for such distractions. All that concerns him is surviving the present... He leads a separate life of uncomplicated black and white decisions—stay warm, feed yourself, be careful, take proper rest, look after yourself and your partner, be aware. Be aware of everything until there is nothing but the present and there are no corrosive fears to eat away at confidence.

— Joe Simpson, This Game of Ghosts

Eric Simmons must climb a mountain. Not because it is there, not because it will bring him glory, but because up there in the snow and mist of Chomolonzo, something has gone terribly wrong. His brother Frank, sent to retrieve a sacred Buddhist terma from the peak, has gone missing. Angry and murderous ghosts have covered the slopes, somehow released from the bardo, a time between death and nirvana or rebirth. Equipped only with Buddhist implements he barely understands, Eric must reach the summit to find his brother and end the curse he unleashed.

Like climbing, Cursed Mountain requires the player to understand and manipulate the surrounding landscape. Survival horror games use atmosphere and resource scarcity to create tension and fear in the player. In Cursed Mountain, as in Resident Evil 4, the scarce resource is the space between Eric, whose best attacks require him to be motionless, and the enemies who are out to kill him. The awareness of space manifests in the player's tactical response to the ghosts: those that move rapidly or can harm you from across open ground will receive priority, because Eric moves so slowly and has no effective dodge capability. Spatial exploration and management are also key in the life-draining "ghost khorlo" segments, where the player must find and eliminate sigils while avoiding aggressive ghosts. Moreover, the healing incense sticks that can be used to replenish Eric's life are quite plentiful, but can only be used in specific places that are fairly rare. With even modest exploration of the game's world, Eric will always have plenty of healing items and unlimited "ammo"; what he lacks is open ground.

Although the areas the game requires you to traverse are very linear and generally require very little skill to cross, these features are disguised by their considerable verticality and mazelike design. There is only one way through, but that path is usually not obvious from a distance. Unfortunately the areas where Eric must actually climb the mountain are much less creative. Cursed Mountain is merely the best climbing game you could make based on walking. In the game, Eric uses his feet almost exclusively, except for some wall-climbing segments that differ little from those in Ocarina of Time. For the most part the mountain itself doesn't seem particularly devious: only a few spots require the player to do anything more than barrel straight ahead along an obvious linear path.

Even the strolling feels unsatisfactory. Eric's plodding pace works when you consider he's a man who's generally fit but who has built himself for considered motions in low oxygen environments rather than sprinting. Unfortunately, this characterization through motion falters because the walking animation stutters strangely on slopes and stairs, and Eric has considerable trouble stepping over tiny obstacles.

The gameplay also disagrees with the characterization when it comes to item discovery. Cursed Mountain depicts Eric as disbelieving, but respecting, the religion and culture of the Sherpas, as opposed to Frank who is openly contemptuous of them. Yet the game asks you very early on to smash pots to gain incense sticks, keys, and diaries that explain the events on Chomolonzo. This mechanic feels tired and stilted, of course, but more than that it is difficult to reconcile Eric's supposed uprightness with this degree of property destruction. The level design also fights against the character's central conceit: it's difficult to believe that an experienced, well-equipped mountain climber will be stopped by a relatively gentle slope or a low wall. Even the blocking potential of steep-walled chasms falters when Eric will be climbing sheer vertical rock faces in short order.

I feel such concern for the way the fiction plays because Cursed Mountain's narrative otherwise works quite well. It has rough and awkward moments, like any script, but the principal characters are convincingly drawn, and it efficiently juxtaposes several sets of interesting and compelling ideas. As a big brother myself, I felt empathy for Eric's conflicting feelings towards Frank and self-doubt of his own worth as a sibling. The script also sketches out a parallel between Bennett's quest for literal immortality through the terma with Frank's desire for figurative immortality through mountaineering history. A single, well-depicted near-erotic scene sets up a compelling and troubling connection between Frank's desire to summit the peak and sexual violence against the mountain, which the game's Sherpas see as a female goddess.

Yet the design constantly sells this narrative short, as best illustrated by its dull, prosaic depiction of the bardo as a darker rehash of an immediately preceding area, a sin compounded by the powerful explanation of this spiritual realm delivered immediately beforehand by a Lama. The level offers nothing resembling the test of enlightenment and understanding the Lama describes, just a simplistic prelude to a boss that doesn't seem to symbolize anything.

I cannot chalk this up to a general lack of ability, however, because some levels are masterful, including a maze of ice tunnels that can only be escaped with the help of radio direction from a man who may already be dead. Cursed Mountain shines when it uses isolation, a genuine uncertainty about where to go next, and hints delivered in ways that make you question whether Eric is even sane anymore. Part of the power of these sequences comes from the fact that Cursed Mountain merely makes you ask about sanity, and resists the temptation to provide an answer. Did you really hear Paul Ward? Did you ever really speak to Edward Bennett? Even the game's final moments concern an act of survival at once so implausible and believable you won't be sure of anything other than the scene's emotional authenticity.

Sadly, Cursed Mountain never embraces its core ideas fully enough to really succeed as a whole game. Greatness always seems just within its grasp, but the game is toppled from the heights by its unimaginative level design and mechanics, and its use of inappropriate gameplay tropes. In its best moments, however, Cursed Mountain truly inhabits the persona of a man whose entire existence relies on his understanding of space and distance, whose whole world is the howling wind and the biting cold and the lonely rock of a mountain that must be ascended, even if it means brushing up against the realm of the dead.

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The blunted blade

Japan's greatest swordsmiths, Masamune and Muramasa, were not contemporaries, but there are several legends involving both of them. One tells of a competition in which each one forged a sword and lowered the blade into a river. Masamune's sword seemed to be inert: fish swam up to it, the flowers floating in the river brushed by without harm. Muramasa's blade, in contrast, cut everything it touched. The fish were split in two, the flowers sliced to ribbons, and the very air hissed in pain as the weapon cleaved it. A passing monk saw the display and chided Muramasa, pointing out that Masamune's sword cut no undeserving thing, while Muramasa's blade killed indiscriminately. Time and legend have ascribed a bloodthirsty, evil aspect to Muramasa's swords, and it is this reputation that Muramasa: The Demon Blade aims to evoke.

Vanillaware, the creators of Muramasa, are probably best known for their 2007 role-playing game Odin Sphere, a game which was marked by a beautiful coherence of story, play, and symbolism. It would not be fair to expect another masterpiece of this quality, but I did expect the resemblance between the two games to be more than skin-deep. If you come to Muramasa looking for gorgeous 2D graphics and a fantastic score, you will not be disappointed. Unfortunately, the simplistic story and uneven design of Muramasa make it a hollow and repetitive experience.

Muramasa: The Demon Blade features two stories: that of the princess Momohime, possessed by the spirit of an evil swordsman, and that of the amnesiac ninja Kisuke. Unlike Odin Sphere, where the stories of the five main characters were intertwined in often surprising ways, these two tales have little besides backstory and a few shared characters connecting them. Moreover, the story is presented in very spare fashion, rarely offering anything more than a few lines of dialogue to explain why a particular boss must be fought or where to go next. There is no rich, textured narrative here, and as a result the game feels more like a quickie tour of Japanese folklore than a coherent story. The romance between Kisuke and Torahime is too understated, and Momohime's dedication to the reprehensible Yukinojyo is unmotivated and frankly sad.

The world of the game is, of course, feudal Japan, divided up into the provinces from the era of the Shogunate. These provinces appear in the game as a series of connected rooms (or zones, if you prefer), with some limited internal geography that must be traversed with light platforming. Sometimes these rooms are filled with enemies from the outset, in which case they can either be fought or bypassed. Random encounters also occur in these rooms, instead of in a separate battle stage, although when this happens the room no longer scrolls and the player must fight in the window onscreen at the moment of attack. Defeated enemies lose their souls, which can then be used to repair broken blades and forge new swords. Eating food (to provide "Spirit" to the character) is also necessary to forge additional weapons. The cooking element is very light, however, and most of the recipes rapidly become irrelevant because they have too small of a healing effect relative to the size of the life bar (which expands more than 50-fold over the course of the game).

The difficulty and complexity of combat, for the most part, depend entirely on the player's choice of "Muso" or "Shuro" play. In Muso mode, most battles can be won with sheer aggression, with the exception of some bosses and the random encounters I discussed in the previous post. In Shuro mode the fighting becomes more complex and tactical, although many enemies are still susceptible to a strategy of continual attack. Momohime and Kisuke have exactly the same controls and abilities, however, so the only real difference in gameplay between their respective stories is a few field enemies and the bosses. Even this disappears late in the game, when they gain the ability to fight each other's bosses. At that point the game reaches a repetitive nadir, but one that must be slogged through in order to level both of the characters up enough to wield the game's final sword and see the "true" ending. Leveling up the characters to defeat major enemies is generally unnecessary because your foes seem to scale with your level, particularly the bosses and the enemies leading up to them.

The troubling thing about these various components is that they all seem disconnected, or even opposed. The RPG elements, particularly the massive life bar, make the game's simplistic cooking system largely irrelevant. The scaling of enemies makes the leveling irrelevant, but at the same time, participation in the monotonous combat can only be justified by the need to level. The brilliant composers at Basiscape created a score that allows the battle music and field music to switch seamlessly between bars, and the battles take place without a screen swap, but the wonderful effect this has on the game's continuity is ruined by the scroll-lock and post-battle EXP screen. You run around collecting souls all game, but the story essentially ignores this grim task. Moreover, the need to do it has no real bearing on the gameplay — unless you consistently run from every battle you will have little trouble gaining the souls required to forge all the swords twenty times over by the end of the game. Muramasa is fundamentally incoherent.

I can't help but feel that Muramasa would have been better off if Vanillaware had embraced the brawler at the game's core and discarded the RPG elements (and Muso difficulty) entirely. The process of leveling up doesn't really add anything to the game, and the character development offers nothing for the player to do. Because the expanding life bar and experience screen notably interfere with other elements of the game, why not just get rid of them? Focusing the game on tactical combat, and the sword system on the selection of special attacks or abilities, would have made this a game about fighting smart rather than just about whacking things with a sword until you reach the next boss. Making Kisuke and Momohime play in noticeably different ways would also have made the game a more interesting experience.

Even those changes, though, would only somewhat ameliorate a fundamentally hollow experience. Muramasa: The Demon Blade reminds us of a famous swordsmith and his bloodthirsty blades, but the madness and viciousness thus evoked never really find their way into the gameplay or the thinly-presented story. Like its vast, fractured provinces, the various systems of the game never manage to form any coherent whole. Instead, the game comes across as a whole bunch of ideas thrown together willy-nilly and wrapped in as beautiful a package as possible. Muramasa has no secrets to reveal, no depths to plumb, no story to tell that equals its luxurious presentation. Like a ceremonial sword, it is glorious to behold, and better gazed upon than used.

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Muramasa goes to hell

Most of Muramasa: The Demon Blade is set in Japan, laid out on a map based on the provinces of its feudal period. In one act, however, the possessed princess Momohime jumps down a well in Kyoto (Yamashiro) and finds herself in a fiery underworld populated by demons. Although the game is suffused with elements from Japanese myth and folklore, this drop into a completely fantastic area represents a substantial break from the rest of the game's semi-historical motif. It represents a break in other ways as well, few of them good. Muramasa's episode in hell creates player frustration in almost every phase, to the detriment of one the game's most memorable sequences.

The stages set in hell involve a marked increase in difficulty. Most of the monsters encountered in this area are trivial little imps that have been seen all game and can be disposed of with a few strikes. Because they can launch projectile attacks if left alone, the best approach to deal with them is unbridled aggression. In the hell stages, however, these imps are accompanied by one or two hulking monsters that are very tough to kill. Not only do these oni take a great deal more damage, they also have an expanded arsenal of attacks that do significant damage to Momohime, and more importantly to her swords. Because a broken sword makes the player essentially incapable of offense or defense, these encounters can rapidly become very dangerous, and the player will spend much more time dodging attacks than is typical for a random encounter. Although the oni are vulnerable at every spot, fighting them requires a consideration for angle of attack and tactical retreat that goes beyond that needed for many of the game's bosses.

This is not an intrinsically bad thing, because the boss of this area, the Big Oni, also imposes tactical requirements that make it quite different from the preceding bosses in the game. It would have been clever to set the stage for this fight by making the oni enemies in the field have similar vulnerabilities and attacks to the big boss so that the player would be trained for the boss fight by the more ordinary encounters. Unfortunately, the oni from random encounters have only the slightest resemblance to the Big Oni in their attacks, and no similarities in vulnerability. So although they are in many ways much tougher to fight than the boss, the player takes nothing useful away from these encounters.

Irritatingly, the increased difficulty of these particular monsters does not seem to acknowledged in the experience yield from the battle. In Muramasa, the primary determinant of experience reward appears to be the monster level, which is in turn set by the player's level. This careless approach to the difficulty/reward dynamic is a characteristic flaw of Japanese RPGs, though not by any means confined to them. As Denis pointed out to me, the asymmetry between difficulty and reward appears in some Western RPGs as well. The foes introduced in Fallout 3's Broken Steel, for instance, give similar experience to their lower-level brethren, despite the steep increase in difficulty. At any rate, it's not unexpected to see this in Muramasa, though it is irritating, and it points to a larger issue I'll get into with my general critique later this week. Here, it merely accentuates the frustration of dealing with the more difficult enemies in the field, and helps turn the trip through hell, one of the least visually interesting areas in the game, into an annoying slog.

This means that the player arrives to the boss battle frustrated or even angry, which is a shame because the battle with the Big Oni is a really clever and interesting fight. The stage for combat is set by a pitch-perfect scene where Momohime convinces the Big Oni to swallow her whole, in classic trickster-hero style. The first phase of the battle then involves cutting your way out of the monster's belly, a classic epic trope that would in most other games have been played off as a cutscene. The main battle requires Momohime to attack the monster's horns, evoking the famous Japanese myth of Momotarō. Absent the cultural context, however, this resonance, and the accompanying hint about how to attack the Big Oni, are missing.

This again can easily become frustrating, because the target area on Big Oni is just large enough to be hit occasionally by attacking his body. Because every previous boss in the game is vulnerable at more or less every visible spot, the player might not even think this limitation on Big Oni is possible. Training the player to attack the horns with the randomly encountered oni would have been an ideal solution to this problem. Alternately, Vanillaware could have elected to have only the horns or head flash in response to damage, or to have shown the horns cracking and breaking as the battle progresses. However, the developers, being familiar with the myths about oni, simply might not have thought about how to convey that information to a Western audience. So, another possibility would be for the localization team to slip a hint into the dialogue boxes somewhere.

The journey into hell displays a set of outright mistakes and lost opportunities. Although the difficulty of this section is out of line with the rest of the game, the level of challenge is appropriate to the area being entered. However, because that challenge is not rewarded, what was merely tough becomes frustrating. The strategic requirements for dealing with the oni foes also break with the rest of the game, but it could have been used to prepare the player for a new wrinkle in the upcoming boss. Instead, the demons are dealt with using totally disjoint approaches: raw aggression for the smallest, careful dodging for their larger brethren, and targeted attack for the boss. That makes what could have been Muramasa's most striking and epic section into a frustrating and ultimately tedious slog.

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Capsule: Professor Layton and the Diabolical Box

Final Status: Story finished, most extra puzzles solved.

Put this on your box: A gentleman leaves no puzzle unsolved!

Most intriguing idea: You don't really need to make puzzles part of the story. You can just jam them in there any old way and people will still love it!

Best design decision: Moving the setting around to make each chapter's geography more varied.

Worst design decision: The tea set, a boring game of trial-and-error that doesn't fit the puzzle aesthetic or provide any entertainment.

Summary: Professor Layton and the Diabolical Box follows the same scheme as its predecessor — a mystery presents itself to the Professor, who then must solve puzzles to obtain clues, get around recalcitrant villagers, and just for fun. While some of the puzzles are directly integrated with story events, for the most part they are no more than tangentially related. Diabolical Box has a greater reliance on transfer and picture-scanning puzzles than Mysterious Village, in part because the latter form the basis for almost all the gameplay in the camera minigame. In addition there is a hamster minigame (cursed with appalling voice acting) that also has a puzzle component, in terms of arranging items in the hamster's play area to maximize the number of steps he will take. This pattern is broken with the tea minigame, in which the player must mix components to make particular teas. As mentioned above, the game would have been better without it. The story is little more than an excuse to tramp through environments looking for puzzles, with train-sized plot holes, but it does its job and is charmingly presented. The final twist is unintentionally hilarious, because it suggests that the Professor and Luke kept running into puzzles because they are puzzle maniacs. For the most part, it's more of the same, but when the original is so good, that's not really a problem.

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Chase fixing

Well, the dust has settled at Richmond, and my favorite driver is out of the hunt for the Sprint Cup. Of course, Clint Bowyer was definitively out of the Chase after his poor showing at Atlanta last week. It's been a tough, up and down season for Bowyer, who I like because he's talented, unassuming, and gets no respect, not even from his own employers. They rewarded his great showing the past two years by giving his team and owner points to somebody else (Casey Mears, of all people) and sticking Clint with a brand-new car and crew. The overall implosion of RCR has been a major disappointment this season, but Bowyer's performance, even with the greenest team, has been the best of a mediocre bunch. Still, with Bowyer definitively out of the Chase, and my beloved football starting up again, the question for NASCAR is: what's to keep me watching racing on Sunday afternoon?

Of course, Bowyer isn't the only driver I root for. I've always liked Stewart, and I'm also partial to the international contingent of Montoya and Ambrose (technically, the most Southern of all the drivers). If I'm going to go with the "no respect" theme, then I should also think highly of the Biff, which I do. And of course there are some drivers I root against. I don't like to see anybody wreck, of course, but I might let out a small cackle of glee when the Busch brothers, Hamlin, or "cousin Carl" turn in a poor effort. So I have some interest still in how the Chase plays out. The problem is that due to its structure the Chase is played out long before it ends. As I recall, the Homestead race has only had a real chance to decide the championship once, and the past few Chases have been effectively decided by the time the green flag waved in Phoenix. Most Chasers are eliminated from contention long before that: last year Kyle Busch was through after Dover, and Talladega dramatically thinned the list of possible champions.

The other problem, of course, is that Jimmy Johnson seems to always win this thing. It would be unfair to deliberately calibrate the Chase to specifically stop him, although not without precedent. After all, the Chase was basically invented to prevent Matt Kenseth (or a similarly consistent non-winner) from ever taking home the Cup again. Johnson just does too well on most of the Chase tracks for anyone to catch him. He's not magical, of course, but he's unflappable, and as I've mentioned before, almost fatally unmarketable.

One way to shake things up would be to alter the track selection, especially to thin out the intermediate tracks. Some of those certainly ought to be kept: a Chase without a stop in Texas or Charlotte would be a strange beast, and Kansas always seems to produce an interesting race somehow. New Hampshire's flat oval is just funky enough to stay. Still, there's no good reason to ever race at Fontana, and much less reason to go there in the Chase. If the series is going to hoof it out to Cali, then the Chase should visit Infineon. A road race would diversify the Chase tracks and make it a more complete test of the drivers. I think the racing is actually more entertaining at Watkins Glen (or even rainy Montreal), but the fall weather up north might be too much of an issue. Homestead is also a really uninspiring track, a race nobody would watch if the champion weren't crowned there, and seemingly selected for no better reason than the climate. I think the Chase ought to end at Nascar central in Charlotte, and Homestead's spot given to a more unique and interesting track. Since we're talking about my fantasy here, I'd put a Chase race in Darlington again.

Another way to address the Chase's boring side would be to alter the scoring. This might sound more radical, but the Chase already makes a pretty artificial change to the points. Moreover, the goal of the Chase is not necessarily the same as the season points, so there's no particular reason to keep that scoring system for the Chasers. With that in mind, here are three alternatives.

Option 1: Not enough pie

Chase scoring comes with a simple scale: 10 points for 1st place down to 1 point for 10th place, no points for lower. I would foresee two improvements from this approach. First, because the number of scoring spots is less than the number of racers in the chase, there will be a desperate fight for every position. Second, because the spread of points is so narrow and there's no more penalty for coming in 43rd than 11th, one or two bad races will not completely eliminate a contender. The compressed dynamic range should make it easier for those who are behind to catch up with a few good performances, keeping the Chase drama alive until the checkers wave in Miami. The Chase would be "seeded" by treating regular-season points as a race (so 11th and 12th place Chasers get no points) plus one point per regular-season win. The weakness of this approach is that it gives the non-Chasers a lot of spoiler power, and might de-emphasize Chase wins.

Option 2: "Winning isn't everything, it's the only thing."

Chasers are supposed to be champions. So in the Chase you get 1 point for winning, and 0 points for anything else. Regular-season wins and points, in that order, serve as a tie-breaker and nothing more, except that the regular-season points champion and the winner of the most regular-season races would get one Chase point each. Again, non-Chasers would have a lot of spoiler power, but that would fit in with the organizing scoring principle. More problematic, though, is that the drama could easily be sucked out of this system. In theory the only way to clinch a Championship would be with six wins in this system (maybe less if you started with a bonus), but in practice, and assuming relatively strong competition, three wins might be an insurmountable lead, with other drivers splitting up the other races. So, a strong showing early on could kill the drama.

Option 3: Only racing against each other

Use any points system you want, but in scoring the championship, only consider a driver's position relative to other Chasers. Again, this has the effect of compressing the dynamic range of scoring, meaning that the championship points race will be closer all the way up to the end. It would certainly mitigate the influence of a team being taken out of the championship due to somebody else's wrecks. On the other hand, a team's own errors would also become less damaging, and the whole thing smacks of grading on a curve.

The Chase has done a good job increasing the drama of the last couple of regular-season races and the first few Chase races. The problem continues to be that the system hasn't elevated the season's final few races above their traditional status as irrelevant epilogues to an essentially settled championship run. Shaking up the track selection to emphasize a broader range of skills, and compressing the points range so that the issue remains in doubt all the way to the end, might make the Chase more compelling and interesting to those who, like me (and all those Jr. fans), don't have their favorite driver in the mix.

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We didn't come for the rocks

I wonder if science fiction is really so easy to misunderstand. If sci-fi is just men in tight outfits scrambling across fake plaster rocks until the one in a red shirt gets killed, then of course the whole enterprise is ludicrous. But that's never been the draw. It's not "new stars, new gas giants" that the Enterprise seeks, but "new life, new civilizations". Kids might tune in for the guy in a plastic lizard suit, but adults stayed for the idea of a man without emotions. Could you really live like that? What would it be like? These are the real voyages of the Enterprise in particular and sci-fi in general: not out into space, but inward into the lives of the people out there. Nobody cares that Tatooine is an arid terrestrial with a surface rich in silicon oxides that suffers from persistent dust storms; they care about Luke Skywalker, Han Solo, and what they do in that vast desert. Surely the talented writers at BioWare understand this. Yet, though they set up an intriguing story about people in their sci-fi epic Mass Effect, they inexplicably delivered a game about the rocks.

Mass Effect, in its fiction and its science, clearly belongs to the same genre of space opera as Star Trek and Star Wars. Although the game gives somewhat more consideration to real physics than the other titles, its handling of biology rests at the same level. Most alien species, with the exception of the agreeably freaky hanar, look more or less like us, right down to the mammae, with various kinds of funky head designs to disguise the fact that they have relatably human faces and bodies. This isn't necessarily a bad thing. Although it is true that the medium of videogames frees the developer from the practical limitations that make such "aliens" necessary in live-action, actually implementing a radical design in a believable way (for instance, constructing a set of conversational mannerisms) would be incredibly difficult and would not be guaranteed to yield a creature human players could relate to. Anyway, an exploration of biological possibilities is every bit as much beside the point as the exploration of rocks. What the best sci-fi really explores is ideas about other ways of being — exploring people.

Mass Effect grabs hold of this possibility early on when we are introduced to the Citadel, an enormous space station of mysterious origin that serves as the seat of galactic politics and culture. Here dozens of races butt up against one another, and we are offered glimpses of intriguing new cultures: the libertarian militarism of the turians, the intensely political salarians, the religious hanar, and the slow-speaking elcor who preface every statement with a description of its emotional underpinning. Not all of these races are so interesting (the plight of the quarians quotes far too literally from Battlestar Galactica), but I found the possibilities exciting, especially given the relatively weak position of humanity in this milieu. I thought that the powerful conversation system and the generally well-written characters could be leveraged into an exploration of this rich society. Instead, I was given the 2183 equivalent of an ill-handling dune buggy and sent on the Johnny Shepard Good-Times Tour of the galaxy's most desolate shitholes. Bouncing across the tenth absurdly craggy moonscape to the fifteenth identical base, I wondered why I was still playing.

The answer should be the story, which is a very standard space opera, delivered with just enough cleverness and originality to stay interesting despite its scenery-chewing villains and reliance on played-out fantasy tropes. The player must track down the turncoat galactic agent Saren, who has forged an alliance with the Reapers, an serially-genocidal machine race from the depths of space. He and most of his organic allies have had their minds corrupted by Sovereign, an enormous ship which at first seems to be merely connected to the Reapers. At least in the order I played its missions, the game seemed to time its revelations well and develop them properly.

Yet I came to have three main problems with this tale. The first, a minor point, is that the Conduit spits its passengers out inside the Citadel structure. We are told that a mass relay works by nullifying the mass in a corridor between it and its terminus. The troubling implications of this process for the intervening matter — station, spaceships, and lovely Asari maidens — are not discussed, but I presume the sentient components of that matter would object. The second problem, more concerning, is that the Reapers' behavior is fundamentally inexplicable. The writers eventually give up on explaining their motivation, dismissing the question by telling us that evil metal gods work in mysterious ways.

The final problem, which might be fatal, is that there is no logical reason anything in the story should happen at all. Consider that Mass Effect carefully demonstrates that (1) the Citadel Council trusts Saren completely (Citadel mission), and (2) it is possible to sneak geth soldiers, tanks, and artillery emplacements past the galaxy's most advanced weapons scanners (Noveria mission). Would it not have been considerably easier for Saren to just import a couple thousand geth to the Citadel, walk into the control chamber, and let Sovereign in, relying on the Council's trust for the element of surprise? The only thing that prevents him from doing this is that he showed his hand in the player's first mission on Eden Prime — that is, Saren only needs the Conduit because he was trying to find the Conduit. Hiding the terminus in plain sight on the Citadel was a clever move by the writers, but it ends up being too clever by half, because it only delivers Saren to a place he could have easily reached anyway, were he and Sovereign even half as intelligent as the game wants us to believe.

Nonetheless, the writers managed to hold my interest up until this last, unsatisfying reveal, thanks in large part to solid characters underpinned by excellent voice acting. While some of them have one-note personalities, that single note is played very well, and others show unexpected depth. Codex entries and his own early actions prepared me to see Wrex portrayed as a low-rent Klingon clone, so the elaboration of his backstory as a thoughtful civic leader really worked. Sadly, the ultimate payoff for exploring these stories always seems to be yet another trip to yet another prefab base on yet another hunk of lifeless rock.

The strength of Mass Effect is not its larger plot or barren worlds but its characters and societies. The problem is that although the game's best mechanic is ideally suited to explore that angle, it's left hanging in the wind in favor of a standard power fantasy reliant on wobbly systems. As a shooter the game is merely passable, and the mechanics only get worse from there. The exploration and vehicular combat are irritating, the inventory and party management is trash, and the AI seems only marginally better than that used in DOOM. The hacking / surveying / finding artifacts minigame is a boring, mindless chore. In contrast, the conversation system and the cinematic presentation shine almost every time they appear, making for the most satisfying in-game discussions I've ever seen. Why not take that system and run with it? Why should the most interesting parts of the game be trapped in the completely non-interactive Codex?

I believe BioWare can get this right; they are halfway there already. They have created a universe which, like the best science fiction, is ripe for the exploration of new ideas and new ways of living. Unfortunately, all Mass Effect let us explore was the plaster rocks.

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Proteins stick together when it's crowded

One of the many ways that a living cell does not resemble a test tube is in the degree to which its internal environment is crowded. Cells are crammed full of massive protein complexes, vesicles, organelles, carbohydrates, peptidoglycan, and other assemblies that occupy a great deal of space. The tubes and cuvettes used for biochemical experiments, in contrast, typically contain nothing more than a few proteins and small molecules of interest along with a relatively dilute set of salts and buffering agents. Because many complexes appear to be stabilized by crowding, and excluded volume effects are known to favor compact, folded protein chains, there is considerable interest in estimating how protein behavior changes in the spatially-restricted environment of the cell. In some cases this is approached using NMR to study changes in dynamics and structure inside cells. In a recent issue of Biophysical Journal, groups from Florida State University and Rehovot University, in separate papers, address how crowding affects protein-protein interactions.

Both groups perform biochemical experiments in vitro to examine this question. In order to crowd the solution, they add reagents such as ficoll, polyethylene glycol (PEG), and dextran, and compare the changes in binding and kinetics to solutions that have merely been made more viscous (through the addition of glucose, for instance). Batra et al. study the association of two components of the E. coli DNA polymerase III and find that the presence of crowding agents slightly stabilizes the complex. However, as the size of the crowders is increased, this stabilization is diminished. Batra et al. develop a relatively simple mathematical model that suggests this observation results from the fact that larger particles pack less efficiently, leaving larger "holes" in which the protein complex sees something more like dilute solution.

Phillip et al. study several protein complexes. Similar to Batra's group, they find that crowding with dextran modestly increases the binding affinity of two of their protein pairs, but that this is not replicated for crowding with PEG. Particularly for PEG-1000, there was a clear decrease in affinity, although a non-crowding viscogen (ethylene glycol) had an even greater effect. Phillip et al. also measured the kinetics of binding, and found that the association rates were significantly lower in crowded solutions, as compared to buffer. However, when the rates were corrected for the effect of viscosity, it appeared that the crowding agents slightly increased the association rate. The authors attribute this to excluded volume effects in the binding transition state. The dissociation rate was also slightly reduced in crowded solutions, which the authors explain by the longer lifetime of the encounter complex (allowing a larger fraction of complexes to fall back to the lower-energy bound state).

Given that crowding appears to have a profound effect on the function of certain complexes, the relatively small effects observed in these studies might seem confusing. Batra et al. argue that although each individual binding interaction is only modestly stabilized, the effect should be cumulative. As a result, multi-subunit complexes will experience a greater effect than small heterodimers. Additionally, the most famous examples of crowding enhancement involve very large complexes — the ribosome, decameric assemblies, hemoglobin polymers, etc. In comparison, the complexes formed in these model studies are quite small. It may be that the stabilizing effect of crowding depends to some degree on the size of the complex to be formed. While similar size is difficult to achieve in strictly heterodimeric systems, it should be possible to monitor the assembly of large complexes like GroES/GroEL under crowded conditions. A study of the relationship between the size or number of components in a complex and crowding stabilization may prove instructive.

Batra, J., Xu, K., Qin, S., & Zhou, H. (2009). Effect of Macromolecular Crowding on Protein Binding Stability: Modest Stabilization and Significant Biological Consequences Biophysical Journal, 97 (3), 906-911 DOI: 10.1016/j.bpj.2009.05.032

Phillip, Y., Sherman, E., Haran, G., & Schreiber, G. (2009). Common Crowding Agents Have Only a Small Effect on Protein-Protein Interactions Biophysical Journal, 97 (3), 875-885 DOI: 10.1016/j.bpj.2009.05.026

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Take the shot!

Dimitri Rascalov runs for his life, as he must, because he has angered the wrong man. Niko Bellic, a former soldier and incredibly efficient killer, has sworn to avenge his cousin Roman's murder on Rascalov's orders, and has tracked his enemy to this dilapidated casino on the edge of town. Now Dimitri's only hope is to escape in his helicopter. Niko pursues him across the roof, sees him about to escape, then races halfway across the building to grab hold of the landing skid. Moments later, he is shaken off into the water next to a speedboat. Now there will be a spectacular chase, by boat and helicopter, across Liberty City, culminating in a confrontation at its most famous landmark. But it is a chase that should not be happening, because Niko Bellic, trained killer, had a rocket launcher, and a clear shot he should have taken. That he did not is a signpost to one of Grand Theft Auto IV's central flaws.

A video game, particularly of the kind GTA IV is, poses a challenge for the creator because the experience of the work that ultimately emerges depends strongly on the input of the player. In order to define the parameters of the experience the creator must exert authorship in a variety of ways. The display of cutscenes, the presentation of incidental dialogue, and the occurrence of scripted events are authorial influences familiar from other media. The developer also constrains the experience through the design of spaces, the placement of enemies, and the programming of artificial intelligence. Beyond this, the conditions for "victory" also define the player's experience because of his assumed desire to complete the game. The key is to use all these authoring steps so that they work coherently. It's not enough to make sure the characters behave consistently across all their cutscenes — every aspect of the game must be considered in light of the characters and their goals.

A reasonably intelligent criminal who knows he will be fleeing from police shortly would not, given any choice at all, take a slow and clumsy vehicle to escape from a crime scene. Nor would the player, for whom the victory condition involves escaping his pursuers. To this point, authorship has succeeded in bringing the player's perspective into line with the criminal's. The developer, desiring that the player/character should use an unwieldy vehicle anyway, has some options. One (good) option would be to arrange the mission in such a way that the heavy van is the only vehicle close to hand. An alternative approach is to make using the van a requirement of success for some obvious reason — say, something valuable is in the van and cannot be removed. A game starts to run into trouble if it requires the player to use the van, for no apparent reason, when a more suitable vehicle is immediately available. This final approach breaks the fiction, using an arbitrary, externally imposed requirement to force the player and characters to act in ways at odds with their goals and personalities.

GTA IV uses this approach all the time, both in and out of actual missions. At one point, Niko receives a call from Packie McCreary, telling him to go visit Packie's brother Gerry in the penitentiary in a remote corner of the city. It is arguably a problem to even mention prison in a game where you can kill a dozen cops, get arrested, and undergo no greater inconvenience than the trip to the police station and the loss of your considerable arsenal of assault weapons. It is certainly a problem to have Niko Bellic — a wanted illegal immigrant, drug runner, and prolific murderer — walk into that prison. And it simply implodes the fiction to have him go to that prison so that Gerry can tell him to call Packie, who — you will recall — set this whole trek into motion with a phone call in the first place. The long, credulity-stretching detour to the prison leads the story precisely nowhere, and what is most troubling about this is that it's not the worst use of authorship in the game.

Consider, for example, the mission "A Long Way to Fall", in which Niko, in a cutscene, walks up to a door and leans his head towards it, allowing a drug dealer on the other side to bang the door into Niko's face. For Niko, ostensibly a hardened soldier, to approach a door this way under these circumstances would be problematic for any kind of narrative. Of course, if Niko had walked peacefully into the building, accepting fresh-baked cookies from little old ladies, the scene would be a reasonable continuation of the fiction. But the immediately preceding gameplay emphatically warned against carelessness. Prior to encountering this door, the player has climbed through a housing complex full of the dealer's hostile posse. Because of the game's difficulty, this is only possible by advancing cautiosly, with judicious use of cover. Moreover, when the player first enters the apartment, he must use a doorway as cover against the well-armed thugs waiting in the main room. The importance of approaching a door carefully will therefore be at the front of the player's (and ostensibly Niko's) mind. In order to serve their dramatic end, the developers use a cutscene to force Niko to act in ways counter not only to the way the character should act, but also to the way the game has been encouraging the player to behave.

For me, this is the defining flaw of GTA IV — so many of the missions, cutscenes, and incidental moments actively undermine the propositions the game is trying to sell you on. Niko is an unstoppable trained killer until the developers' plan for a mission requires him to act like a man who has never fought inside a building, or to choose an obviously inappropriate vehicle, or not to kill a man until he reaches Alderney. Rascalov is a nervous wimp constantly concerned about playing by the "rules" until the developers' plot requires him to kill all his business partners and allies. The characters act not from the internal motivations arising in a fully-imagined personality, but from the external motivation of the developers' desire to move along to the next set-piece.

GTA IV has been hailed as a new gold standard in video-game storytelling, and if that is true then we should double-check the purity of our ingots. The character who is always precisely as smart or as foolish, as competent or helpless as the plot requires at a given moment is a consistent staple of poorly-written action flicks and romantic comedies. GTA IV operates entirely on that same wavelength, except that the authoring of mission objectives and cutscenes means that Rockstar attempts to make the player be as smart or stupid as their dramatic purpose requires. But Rockstar can only constrain action, not thought. They can make me chase Rascalov all around the city, but they can't make me forget Niko Bellic's rocket launcher, or the shot he should have taken.

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What's the word, Slim?

Tuesday saw the widely-expected unveiling of Sony's new hardware model: a slimmer, lighter, and less energy-hungry Playstation 3. They also debuted a new, $299 price, meant to make the PS3 more competitive with the XBox 360 and Wii. At the same time, my PS2 has started to sputter; it had a tough time chewing through Persona 4, and since then has only gotten more feeble. This would be a great time for Sony to sell me a brand-new PS3 with a reduced form factor and lower energy consumption, but Sony has chosen not to include backwards compatibility with PS2 games in the feature set of their new hardware. In the end, that turns out to be a minor problem for me, and perhaps a larger one for Sony.

Don't get me wrong, I'm not a Sony "hater". Obviously I have and love a PS2, and much of my home theater is Sony electronics. And I'm not going to say that the PS3 Slim is a great huge terrible waste of time because it doesn't have backwards compatibility. Nothing could be further from the truth — the Slim answers most of the demands of the average consumer and the game developers Sony needs to keep happy. I personally think this particular change is too little, too late to fundamentally change the balance of power. Nonetheless, the lower price, in combination with Blu-Ray, makes the PS3 a much more solid choice, and arguably the best choice, for anyone buying his or her first HD console.

But I'm not buying my first HD console. I have an XBox 360, and that means I already have access to nearly all of the games I'm interested in. I don't need a Blu-Ray player, though I might use it if I had one, and while I'm fascinated by some of the games I've seen available for the PS3, I don't need those either. Not even for the new, low price of $299. What I do need is a way to keep playing my PS2 games, and Sony is still selling new PS2s for $100. Sales of the PS2, though they've declined dramatically, have still been bringing in much-needed money for Sony, and their pronouncements that backwards compatibility is never coming are probably meant to prop those sales up. But it seems to me that continuing to sell the PS2 is a dead end.

If I buy one, the executives at Sony will think they gained $100, but they're wrong. The sale of a PS2 is a loss of at least $199, because it's a lost opportunity to sell a PS3. Sure, the profit margin on the PS2 is higher, but the guy who buys a PS2 is going to use it to play PS2 games he already owns, or to play used games, and none of this will earn Sony a dime. These players won't be playing PS3 games that earn royalties for Sony, or supporting the Blu-Ray market that Sony is invested in. That means that the sale of a PS2 is a lost opportunity, not a net benefit. What Sony should be striving to do is find a way to turn any would-be PS2 buyers into PS3 buyers. Now that the PS3 Slim offers a more competitive price, they should leverage their existing, unbelievably massive PS2 install base into new buyers for PS3 by presenting that console as an upgrade that possesses all the capabilities of the console those users have already invested in (some substantially), plus exciting new capabilities and games.

Why hasn't Sony done this? Some initial offerings of the PS3 (back when it cost too much for me) had a hardware solution, but this seems to have been uneconomical, and anyway would probably be at odds with their goal of shrinking things down for the PS3 Slim. The answer, then, is software emulation, which seems to have been pretty difficult to implement for even some titles, and given the size of the PS2 library, would represent a QA nightmare. I understand that problem, and if Sony feels that the investment required to make emulation possible on their sole SKU going forward is not worth the (considerable) reward, then I'll accept that, though I feel their cost-benefit analysis has been suspect in the past (e.g. Home).

But what I would like to see is all the cards laid out on the table. Those recent comments from Sony reps that backwards compatibility is never coming carry as much weight for me as the ranting of some random fanboy, because Sony's communication with their customers this console generation has been an endless cycle of dissembling and outright lying (in which they are admittedly not alone). They did this with rumble functionality, and with the Slim itself, so why wouldn't they do it with backwards compatibility? There's no reason to trust what they say, especially when it takes the form of some slick marketing director spewing nonsense about surveys. The simple fact is that used backwards-compatible PS3s are selling for more than the new console price right now, a sure sign of continued interest and demand. What Sony should do is come out with the tech guys and say, "Look, here's the problem, here's what we've tried to do, here's why it hasn't worked, here's how much it would cost to make it work, and that's why we're not doing it." Or, if they really do have plans to try, come out and say that's happening, but they don't know how it will turn out. That may not do much for the corporate ego, but it would go a long way towards establishing some credibility.

As it stands, I feel like I'll get burned whatever I choose to do. If I buy a PS3 in hopes that BC is coming, and it doesn't, then all I'm really getting out of those $300 is access to a set of exclusives that mostly don't excite me. I could see myself paying up to $50 to play Flower, but not more than $300. I'm willing to opt for the PS2 and maybe eventually get a PS3 when its case is strong enough, but I'm not so wealthy that the $100 means nothing to me. If I replace my PS2, will Sony screw me by introducing backwards compatibility shortly thereafter? I don't know, and until Sony chooses forthrightness over the ability to make a big splash by holding back secrets, I can't possibly trust what they'll say. So until my PS2 gives up the ghost permanently, what I'm buying from Sony is nothing, which from their perspective should be the worst outcome of all.

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Filling the donut hole in dynamics

This is the last of my series of posts about the dynamics-focused topical issue of JBNMR. There are plenty of other excellent papers in it, and I encourage you to at least glance over all of them, especially if you're an NMR person.

Standard NMR dynamics experiments on isotropically tumbling proteins cover a wide, but not comprehensive, swath of fluctuation timescales. Limited information about motions that take milliseconds or more can be obtained from hydrogen exchange data; the AMORE-HX experiment is meant to obtain this kind of information. Fluctuations with time constants in the range of μs-ms can be measured by relaxation-dispersion experiments, as were used in the Peng lab's paper. Motions that are faster than the rotational correlation time of the protein can be characterized using dipolar relaxation data of the kind I collected for the field-cycling experiment. There are additional kinds of data that also cover these areas, but the glaring hole lies between the correlation time of the protein (several ns) and the low end of the chemical exchange regime (several μs). Several teams, including that of Christian Griesinger, propose to fill this gap using data derived from residual dipolar couplings (RDCs). In the topical dynamics issue of the Journal of Biomolecular NMR, his group demonstrates the use of this technique in measuring the dynamics of side chains in the small protein ubiquitin. The article is open access, so feel free to open it up and read along.

The strength of the dipolar coupling between two nuclei depends on their magnetic properties (specifically their gyromagnetic ratio), the distance between them, and the angle between the internuclear vector and the vector describing the external magnetic field. For solution NMR this last component is typically not important because most proteins tumble randomly with respect to the magnetic field, causing this interaction to be averaged away. However, if you were to somehow introduce a tiny amount of bias into the tumbling, very slightly aligning the protein parallel or perpendicular to the magnetic field, a residual portion of this coupling could be recovered. The effect is considerable: a net alignment of less than a fraction of a percent generates couplings on the order of 30 Hz or more.

There are many ways of inducing this alignment. Large charged particles such as phage or DNA nanorods have been used, as have assemblies such as charged or polar lipid bicelles. In addition, proteins can be labeled with paramagnetic metals to induce fractional orientation. Even mechanically manipulated media, such as acrylamide gels, can be used to achieve alignment if they are stretched or compressed along the field axis. When a new method of alignment is introduced, it is typically tried out on a small, abundant protein with good relaxation characteristics, most often the regulatory protein ubiquitin. The upshot of this is that there is a fantastic amount of RDC data on this protein, and Griesinger's group uses this data to model the motions of its methyl-bearing side-chains.

This may sound strange, because RDCs are typically employed for structure determination. The angle defined by the measured coupling results from the overall tumbling bias of the protein (an alignment tensor) that is the same for each coupled pair, and their angle within that frame of reference, which can be used to uniquely define a structure. However, the dipolar coupling cannot be measured in an instantaneous fashion. It must evolve over time, just like chemical shift or a J coupling. As such, the dipolar coupling reflects an averaged orientation over the evolution period. In principle, the degree of averaging can be modeled as some kind of order parameter, similar to the S² of the Lipari-Szabo system, reflecting all these motions. This should encompass not only the fast dynamics that determine dipolar relaxation rates, but also motions slower than the global correlation time, up to near the ms range.

In order to derive dynamics data from their set of experiments in 13 different alignment media, the authors first scaled the RDCs from C-H methyl bonds. The reason they did this is that the three hydrogens in a methyl group rotate constantly around an axis passing through the adjacent C-C bond (cyan in the isoleucine side chain depicted at right). Because this rotation is typically very fast, simple to model mathematically, and pretty uninteresting, it can be deconvoluted from the dynamics data to give us what we're really interested in, the behavior of the C-C bond. Using an alignment tensor derived from a separate dataset of N-H RDCs in 36 different alignment media, the calculated C-C RDCs are combined into a matrix, from which simplified parameters describing motion can be derived. Fig. 1 depicts this schematically (note, the legend has the variables m and i reversed in meaning).

The order parameter (S²_rdc) reflects the rigidity of the bond and ranges from 0 (highly flexible) to 1 (perfectly rigid). The values measured for the methyl groups of ubiquitin cover almost this entire range (Fig. 2a), which is typical of side chains which tend to have less constrained motions than the backbone. It's also evident from this figure that S²_rdc is roughly anticorrelated with the number of dihedral angles between a given methyl group and the peptide backbone. The anisotropy of motion (η_rdc) is generally low, and appears to be roughly correlated with the number of intervening dihedrals. Both of these observations agree with previous data from other dynamics experiments, as well as reasonable expectations about the movements of these groups.

Farès et al. compare their S²_rdc to order parameters determined using other approaches. As one would expect, for most methyls the S²_rdc, which encompasses motions from a wide array of timescales, is lower than the S² determined using a Lipari-Szabo model-free approach that is only sensitive to ps-ns motions. The most notable exceptions are three residues for which the RDC method fit order parameters greater than 1. Probably these represent some unanticipated failure of the model, although these violations occur at groups that are expected to be rigid (two are alanines) and which have high S² from the Lipari-Szabo model. One potential culprit is the previously discussed scaling, which may need to be adjusted if the axial rotation is unusually slow. The best agreement with existing data comes from an approach that combined J-coupling data with less-specific RDC information, but only when those data are corrected for very fast motions using backbone order parameters.

Because the RDC fundamentally contains data about the orientation of a given bond, it should be possible, with a minimal degree of modeling, to extract specific information about the kinds of motions being made, data that cannot be obtained from methods such as the Lipari-Szabo model-free interpretation. The authors modeled side-chain motions around the dihedral angles χ₁ and χ₂ using the MFA data. The agreement between these results and the existing ensembles is not particularly good, but it's not completely obvious where the fault for this lies. Similarly there was limited agreement between these order parameters and those derived from existing ensembles. Agreement with the EROS ensemble structure determined last year was good, but it is difficult to judge what this means, as that ensemble was calculated using the same or similar data to what was used in this paper.

It's fair to ask whether this sort of data analysis will be possible in systems with less comprehensive data. Even this extremely rich dataset proved problematic, for instance in the cases of the alanines and the disagreement concerning rotameric states. It remains to be seen whether this approach will be practical in cases where significantly fewer alignment media can be used. However, it is also true that methyl groups have the best relaxation properties of any group in a protein, and that the experiments for the determination of RDCs are simple and sensitive. This means that if this approach can be made to work, it can provide important dynamic data even in the largest proteins studied by NMR. Farès et al. have performed an impressively comprehensive analysis of dynamics in a time regime that NMR has had difficulty accessing. Hopefully they will next bend their considerable talents towards reproducing as much of this analysis as possible in a more difficult system with sparser data.

Farès, C., Lakomek, N., Walter, K., Frank, B., Meiler, J., Becker, S., & Griesinger, C. (2009). Accessing ns–μs side chain dynamics in ubiquitin with methyl RDCs Journal of Biomolecular NMR, 45 (1-2), 23-44 DOI: 10.1007/s10858-009-9354-7 OPEN ACCESS

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Apparently, the moon hit their eyes like a big pizza pie

This post continues my series about selected articles from the dynamics-focused topical issue of JBNMR.

It is helpful, in examining some NMR articles, to understand that NMR spectroscopists have a long and resilient tradition of giving their pulse sequences silly names. You can think of it as the biophysical equivalent of fly geneticist behavior. From the basic COSY and NOESY experiments (pronounced "cozy" and "nosy") to the INEPT spin-echo train, to more complicated pulse trains such as AMNESIA and DIPSI (which, I am not making this up, is used in an experiment sometimes called the HOHAHA), the field is just littered with ludicrous acronyms (look upon our words, ye mighty, and despair). A team from Josh Wand's lab now joins this club by developing a multiple optimization for radially enhanced NMR-based hydrogen exchange (AMORE-HX) approach. The name is ridiculous, but the experiment fills an important role and illustrates a very active area of technical development in NMR.

The experiment they developed is intended to measure the rate of hydrogen/deuterium exchange at amide groups on the backbone of the protein. This sort of exchange reaction proceeds pretty quickly for most residue types, and can be either acid- or base- catalyzed. For it to happen, however, two things must be true. One of them is that the amide proton must not be in a hydrogen bond already. Also, the site of the reaction must be accessible to water. These requirements should indicate to you that HX measures the rate of local unfolding and can therefore be interpreted as a measure of fold stability at each NH group on the backbone. This data is of obvious interest to researchers studying protein folding. In addition, because some structural transitions are proposed to involve an unfolded state this may have explanatory power for protein interactions and regulation.

A typical HX experiment involves taking your protein, switching it rapidly into >75% D₂O buffer, then placing it in the magnet and taking a series of HSQC or HMQC spectra that separate signals from backbone NH groups by the proton and nitrogen chemical shift. These spectra can be taken with very high time resolution (<2 min each), and the rate of exchange can then be measured by the decay of peak intensity as hydrogen is replaced by deuterium. Assuming that the chemical step occurs significantly faster than the rate of local unfolding and refolding, this decay can be directly interpreted as a local unfolding rate. This works quite well, but as proteins get larger there is a significant likelihood of signal overlap. It would be nice, with these large proteins, to separate the hydrogen signals using an additional chemical shift — say, that of the adjacent carbonyl. Unfortunately, taking these decay curves using 3-dimensional spectra like the HNCO turns out to be impossible because of the way these experiments are collected.

Multidimensional NMR spectra rely on a series of internal delays during which a coherence acquires the frequency characteristics of a particular nucleus. In a typical experiment, the delays are multiples of a set dwell time, the length of which is determined by the frequency range one wishes to examine. Typically the collection proceeds linearly through the array, so for m y dwell times and n z dwell times you would collect 1D spectra with the delays:

0,0 0,y 0,2y 0,3y ... 0,my

then

z,0 z,y z,2y z,3y ... z,my

and so on until

nz,0 nz,y nz,2y nz,3y ... nz,my

This is called Cartesian sampling, and it has some advantages. The numerous data points typically do a good job of specifying resonance frequencies, and processing this data is a fairly straightforward proposition. The glaringly obvious disadvantage is time, of which a great deal is required. Completely sampling either one of these dimensions separately can take less than 30 minutes, but sampling both can push a triple-resonance experiment into the 60 hour range. Most annoyingly, because triple-resonance spectra can be really rather sparse, this extremely long experiment often over-specifies the resonance frequencies. That is, much of this time is spent collecting data you don't need.

Because spectrometer availability and sample stability are not infinite, there is considerable interest in making this process more efficient. One of the methods for doing so is called radial sampling. In this approach, the spectrum is built up from a series of "diagonal" spectra that lie along a certain defined angle with respect to the two time domains (imagine the above array as a rectangle with sides of my and nz to get a rough idea of what this means). If these angles are judiciously chosen, the spectrum can then be rebuilt from just a few of them with only modest losses in resolution. Gledhill et al. apply this approach as a means of addressing their time-resolution problem. Guided by a selection algorithm, they use just four angles (at 500 MHz) to resolve more than 90% of the peaks possible in myelin basic protein. As a result, they were able to collect HNCO-based HX data with 15-minute resolution. This isn't enough to catch the fastest-exchanging peaks, but it's more than sufficient to catch core residues.

Gledhill et al. used some additional tricks to gain extra speed in the experiment, however. Using band-selective excitation, they cut down the experiment's relaxation delay to 0.6 s, which is important because this delay is a considerable portion of the duration of each transient. Having done this, they started to get really clever. Because this experiment is being used to measure the intensities of known frequencies, it is possible to significantly reduce the amount of processing required by employing the 2D-FT only for those regions that contained actual peak intensity. Moreover, they could extract peak intensities from each individual angle plane. Because they did not interleave the collection, this enabled them to substantially increase the time-resolution when necessary.

For peaks that exchanged quickly Gledhill et al. took relaxation data from the individual angle spectra, to maximize the time-sensitivity of the data. For slowly-exchanging peaks, they averaged the data from the angle spectra to maximize the signal-to-noise ratio. The resulting intensity curve seems a bit noisy, but this is an acceptable price to access new peaks. More importantly, the precision of the overall rate (as opposed to the instantaneous intensity) appears to be on par with simpler methods of measuring HX.

Successful use of the AMORE-HX experiment will depend on a wise selection of acquisition angles, a process that may benefit from further optimization. Because the HNCO has relatively good dispersion, the pulse sequence should enable HX measurements for just about any protein that is suitable for NMR. This would allow for a direct assessment of large enzymes and complexes, as well as a measurement of local stabilities in domain-domain interfaces.

Gledhill, J., Walters, B., & Wand, A. (2009). AMORE-HX: a multidimensional optimization of radial enhanced NMR-sampled hydrogen exchange Journal of Biomolecular NMR DOI: 10.1007/s10858-009-9357-4

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A step towards incorporating dynamics data into drug design

My field-cycling article (previous post) is part of a dynamics-focused topical issue of JBNMR. In my next few science posts I'll describe some of the other contributions.

Research into the interplay between protein structural dynamics and function is a window into important fundamental knowledge about biochemistry, but the general justification for public funding of these studies by medical agencies is that they will have the ultimate effect of improving our ability to design and optimize drugs. However, even though our ability to characterize macromolecular dynamics has increased dramatically in the past few decades, there are few, if any, cases in which this knowledge has been applied successfully to the design of therapeutic agents. In part this is because incorporating data on fluctuations into the design algorithms poses a significant challenge. It's also true, though, that we understand only part of each system, i.e. the dynamics of the protein target, not the small molecules it binds. If dynamics studies are to make the maximum possible contribution to pharmaceutical sciences, the motions of the ligand must be characterized. In their article in Journal of Biomolecular NMR, Jeffrey Peng and students from Notre Dame attempt to address this shortcoming in the case of a substrate for the phosphorylation-directed prolyl isomerase Pin1.

Pin1 is implicated in a number of regulatory and signaling pathways, which seems strange because it doesn't possess any intrinsic transcriptional regulation ability, nor does it covalently add or remove phosphate groups. Instead, Pin1 has an enzymatic activity that accelerates, generally without altering the relative populations, the conversion of prolines from their cis- to trans- state and vice versa. This activity is specifically targeted to prolines that are adjacent to phosphorylated serines or threonines. In addition to the catalytic domain that does this work, Pin1 possesses a WW domain that has identical specificity. Because Pin1 does not alter the balance between cis- and trans- Pro, only the rate at which one changes to the other, its role in signaling has been difficult to ascertain, although there is intense interest in this area.

You don't need to understand an entire pathway to design an effective inhibitor. What you do need to understand is the relationship between specific chemical groups and binding affinity. Getting that knowledge can be very difficult if the proposed drug is flexible. In that case, refinement methods that focus only on the particular chemical groups rather than their dynamic properties could go badly astray. Unfortunately, the dynamics of protein-bound drug molecules are difficult to measure. Their proton signals are likely to be swamped by the protein, and small molecules are often difficult to label with isotopes convenient for NMR. Peng et al. propose to address this by studying ¹³C relaxation at natural abundance.

A little less than 99% of the world's carbon is in the form of NMR-inactive ¹²C, which is a problem for NMR because carbon is very abundant in proteins and drugs. Of the rest, most (about 1% of all carbon) is dipolar, NMR-detectable ¹³C, which is usually not enough to accomplish anything in terms of protein NMR. As a result, NMR researchers typically adopt the strategy of expressing their proteins using bacteria grown in media containing ¹³C₆ D-glucose. Such enrichment of drug molecules probably could not be carried out for pharmaceutical research due to the cost and the limited availability of properly labeled reagents. Fortunately, advances such as magnets stronger than 17 T and cryoprobes make sensitive detection of natural-abundance ¹³C a plausible approach. Because natural-abundance measurements also simplify the experiments and analysis considerably, Peng et al. adopt this approach in their study.

Peng et al. measure μs-ms fluctuations in a 10-residue peptide in the presence and absence of Pin1. Keeping in mind that such motions can only be detected when they are associated with a change of chemical shift, it is reasonable that no such motions are detected when the peptide is all by itself. In the presence of Pin1, however, methyl groups on phospho-Thr 5 and Val 7 experience some kind of chemical exchange process on the order of several 100 /s (at 278 K), as does a methylene group in Pro 6.

Peng et al. rationalize their observations with reference to a previously-determined structure of the Pin1 WW domain in complex with this peptide (explore it at the PDB). As you can see from the lowest-energy member of this NMR ensemble (left), the residues where they detect these fluctuations in the methyls and methylenes are those that are most involved in the binding interaction. The WW domain is represented as blue ribbons, while the peptide is shown as sticks down at the bottom. That the Pro and pThr form part of the interface is unsurprising, as they constitute the specific binding sequence, while the Val side chain appears to be in position to make some hydrophobic contacts. Everything makes sense, but that doesn't mean it's telling us what we want to know.

The structure above shows us the interaction of the peptide with the WW domain, while what we're really interested in getting at is the catalytic domain. Using an exchange spectroscopy experiment, Peng et al. determined that the ms dynamics they were observing probably reflected the binding of the peptide to the WW domain. To avoid this interaction, they created an artificial Pin1 that contained only the catalytic domain, and found that this also caused chemical exchange in the methyls and methylenes. Cross-checking against the exchange spectroscopy rates suggested that the ms dynamics in this case reflect the result of Pin1 catalytic activity, namely the interconversion from cis to trans and vice versa.

Unfortunately, this experiment did not report the most desired data, i.e. the dynamics of the ligand on the enzyme. On-enzyme fluctuations certainly contribute to the exchange experienced by the ligand, but because the on-enzyme population is so small (at most 2.5% of ligand) this would only be detectable in the case of an extremely large change in chemical shift. In principle one could deconvolute the dynamics from a partial-occupancy system where 50% or more of the ligand is bound to enzyme, but reliably fitting all the parameters for a two-state chemical exchange system from CPMG data is an already-dicey proposition. Fitting a four-state process from data like these is unlikely to be practical. So, in order to observe on-enzyme dynamics the drug of interest will need be saturated with its target protein, which would require millimolar protein concentrations for most ligands. Under those conditions, the spectra will also contain significant signal from the protein. The 70% deuteration used in this experiment, combined with ¹³C depletion, will probably be enough to suppress this, although these isotopes will increase the cost of the technique (and diminish protein yields).

Nevertheless, this paper establishes that the natural-abundance approach to measuring ligand dynamics on the µs-ms timescale is feasible. Because methylenes and methyls are common moieties in drugs and small molecules this technique may have broad applicability. Investigating the motions of small molecules bound to large proteins poses a unique problem because these systems don't have the advantages of either small molecules (low R₂) or proteins (exotic labeling schemes). The ongoing work of Peng et al. suggests that this problem is tractable, which may have positive consequences for our ability to design and optimize drugs.

Peng, J., Wilson, B., & Namanja, A. (2009). Mapping the dynamics of ligand reorganization via ¹³CH₃ and ¹³CH₂ relaxation dispersion at natural abundance Journal of Biomolecular NMR DOI: 10.1007/s10858-009-9349-4

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