Laptop advice requested

It's becoming increasingly clear to me that an emerging requirement for scientists is that they possess a laptop computer in addition to any desktop. It just seems to be an essential feature for giving talks at conferences and (crossing fingers) for jobs. For whatever reason it seems like you just can't ever trust that your presentation will ever work on somebody else's computer. Plus, on the rare occasions that I travel it might just be convenient to have one. So, I've decided to purchase a laptop, and would like some advice based on personal experience or whatnot.

The requirements I have are pretty basic. I don't plan to do any heavy computational lifting or gaming with this machine. I need it to be capable of word processing, running Powerpoint (or equivalent), displaying images, and accessing the web. It also needs to work smoothly with external projectors (this used to be a problem with Macs; is it still?), have good battery life, and be hardy enough to get knocked around in a plane or in my car and still function well. I would strongly prefer that it not put out enough heat to neuter me if I have the misfortune to actually use it in my lap. Obviously, since this will be a secondary computer, I want it to be relatively inexpensive, not much more than my rebate check if I can help it.

So, any recommendations? Are there any Macs that fit the bill, or are they all in the way too expensive zone? Are the Dells (Vostro, Inspiron) a suitable option, or are they also too pricey? Does HP make any laptops that won't burn your pants right off? Can laptops running Linux deal with LCD projectors?

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Hunting water in the lipocalin drylands

Although interactions with water play a significant role in determining protein structure and function, the precise behavior of water within any protein structural ensemble can be quite difficult to pin down. Despite their importance, these interactions are often transient, making them difficult to pin down. NMR and crystallography have had some success in addressing structural waters, but even these techniques can have substantial difficulty proving that the data genuinely represent proximal interactions with water rather than long-range polarization transfer or some other artifact. An indirect approach is often required in order to resolve these kinds of questions.

Consider the case of β-lactoglobulin (BLG), a member of the lipocalin family. An image of the protein displaying the solvent-accessible surface area, shamelessly stolen from (1), is at right (explore this structure at the PDB). BLG binds nonpolar ligands in that long cavity in the middle (the calyx), which the crystal structure identified as containing several water molecules in a small change (blue spheres). In addition, there were two long-resident structural waters present in other cavities in the protein (red spheres). Qvist et al. wish to establish whether these water molecules are, in fact, present in the calyx in solution.

To do this they use a kind of relaxation-dispersion experiment in which they measure the longitudinal relaxation rate (R₁) of various atoms in water. Dispersion is achieved by using different external fields. Across the frequency range used in this study, the R₁ profile of bulk water is flat due to its very short rotational correlation time. Adding the protein alters the dispersion profile because much of the water becomes part of a "hydration shell" for the protein molecules. In addition, the observed profile will be altered by any waters that are internally bound to the protein. As a result, one would expect that the addition of palmitate, which binds in the calyx (and thus displaces those 5 structural waters) would significantly alter this profile.

In fact this is not what happens. There are slight differences between the profiles when palmitate is added (the authors use NMR to ensure that the palmitate was actually bound), but the results rule out the possibility of water in the pocket with a residence time >10^-8 s. This is inconsistent with the presence of 5 ordered waters in the pocket. In order to support this result, the authors perform molecular dynamics simulations and free energy calculations. From these it is evident that the observed differences in relaxation dispersion could not be due to organized water in the calyx. The authors calculate that burying the waters in the nonpolar calyx would come at a cost of 71 kcal/mol, too much to make such a thing believable even if they were off by an order of magnitude.

So, what was in the calyx in the structure? The authors suggest that some kind of small organic molecule or mixture of such molecules might have been responsible for the observed electron density. This is not an indictment of the structure generally, however: the present study finds solid evidence for the presence of the two structural waters identified elsewhere in the protein. Additionally, Qvist et al. found evidence in the dispersion profiles for additional structural waters, which they thought might be located at the dimer interface. This is consistent with the predictions of a crystal structure of the BLG dimer.

All of this might sound hopelessly arcane, but because it usually must be displaced, the presence of water in a binding pocket has significant consequences for the energetics and kinetics of ligand binding. Understanding how proteins arrange these kinds of binding sites, and what kind of moieties can penetrate them, will be important in designing new proteins, and developing drugs to alter the binding properties of proteins found in nature.

1. Qvist, J., Davidovic, M., Hamelberg, D., Halle, B. (2008). A dry ligand-binding cavity in a solvated protein. Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0709844105

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It's ALIIIIIVE!

Our 500, that is. As I related in previous posts, our 500 MHz spectrometer was hamstrung by the signal from a local HDTV broadcast and subsequently had to be moved to a space in the basement of one of our research buildings. This process took about a year, because that space first had to be renovated, and in fact the last upgrades to the space won't be finished for several days yet. But the 500 has moved into its new digs, (hopefully) finished quenching, and is ready to work. So yesterday I finally was able to collect an HSQC on it for the first time in many months.

So, here's the spectrum I got:


For comparison, here is the last HSQC taken before we stopped using it. Obviously, this is of a different protein, but both spectra were taken using the same probe on samples with the same concentration, at the same temperature. I countoured them identically. The only difference here is that the protein in the spectrum below has much better relaxation characteristics than that in the spectrum above. In theory, this should produce much better signal to noise. Oh, and also that when I took this spectrum I was also receiving data about cubic zirconium.


This is really good news for a couple of reasons. The first is simply that we have another magnet, and that allows us more flexibility in scheduling. No more fighting when people want 9 days of NMR time next week. The second thing is that, for a variety of reasons, some experiments just work better on a lower-field magnet. In particular, I have some experiments that are sensitive to chemical shift anisotropy and R_EX; using a lower-field magnet attenuates both these problems.

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Is in-cell NMR feasible?

As I mentioned in my last post, it is sometimes difficult to tell if a protein structural model matches the physiologically-active form. One way to address this problem would be to take structural data under physiological conditions, i.e. in the cell. While this obviously isn't a viable approach for crystallography, it seems possible with other techniques such as FRET or NMR. Existing NMR structures are solved under solution conditions, and the cytoplasm is a kind of solution. So we would expect to be able to solve structures, or at least get spectra, in vivo. However, the success of NMR strongly depends on what kind of solution you have, and a forthcoming paper in JACS suggests that the bacterial cytoplasm may not be amenable to NMR.

This paper has a bit of history behind it. A few years ago, Gary Pielak's lab attempted to determine the dynamic behavior of chymotrypsin inhibitor 2 (CI2) and apocytochrome-b5 in living E. coli (1),(2). They found in these experiments that the dynamics of these proteins did not differ significantly between in vivo and dilute solution conditions. A later experiment, however, showed that most or all of the signals that had been observed arose from protein that had leaked out of the cells and into the surrounding medium in which they were suspended. This wasn't true for all proteins: cells expressing α-synuclein (αSN) and FlgM did not leak protein. Subsequently the first two papers were retracted (3), while Gary and his folks tried to figure out just what was going on.

Li et al. encapsulated E. coli cells that were producing either CI2 or αSN in alginate microcapsules to prevent protein leakage and took some basic NMR spectra. Samples of the surrounding fluid proved that neither protein was leaking into the surrounding medium. Nonetheless, although normal spectra of αSN were observed, the cells expressing CI2 did not produce any CI2 spectral signals at all (normal metabolite signals were observed). Experiments showed that these cells did contain CI2 (4). What happened to the signal?

Li et al. hypothesize that this is an effect of the viscosity of the cytoplasm. The intensity of an NMR signal depends on the concentration of the atoms giving rise to the signal, and also the relaxation properties of these atoms, i.e. the rate at which the polarization induced by radio-frequency pulses returns to equilibrium levels. For larger molecules and viscous solutions, the rate at which detectable polarization decays (R₂) is higher, and the rate at which polarization returns to equilibrium (R₁) is lower. The behavior of R₂ is one of the chief obstacles to performing NMR on proteins or complexes that have high molecular weight.

In order to establish that this cytoplasmic viscosity is a reasonable cause of the invisibility of CI2, Li et al. measure the NMR relaxation of αSN and CI2 in a solution doped with poly(vinylpyrrolidone) (PVP), which increases the viscosity significantly. They find that the R₁ of αSN is essentially unchanged, while that of CI2 decreases significantly. The R₂ of αSN resonances increases 2-6 fold relative to dilute solution, but for CI2 this increase is much more significant, up to 40-fold. You can see these features in the graph at right. These results indicate that the relaxation behavior of resonances in a stably folded protein like CI2 is dominated by the global rotational diffusion, while the relaxation of resonances in a disordered protein like αSN is dominated by the significant local fluctuations. Because these latter motions involve less surface area and hence less drag (as compared to the folded protein), the viscosity of the solution has relatively less of an effect. Thus, the vanishing CI2 peaks could conceivably be the result of differential relaxation responses to viscosity.

However, other groups have managed to acquire NMR spectra of folded proteins in E. coli before. These experiments may have experienced the same leakage problem that affected the previous CI2 results, and some control experiments addressing this possibility are likely to be forthcoming. At the same time, the PVP solutions here are supposedly 5 times as viscous as E. coli cytoplasm, and yet CI2 resonances are apparently still visible. This suggests that the complete obliteration of these resonances in cells cannot be chalked up entirely to viscosity. One possibility is that CI2 overexpresses so significantly that it massively increases the effective viscosity of the cell. It may also be possible that the vast quantities of CI2 expressed in these experiments are crammed into a small volume somehow, perhaps as relatively loose inclusions in the cytoplasm or loaded in the periplasmic space, where interactions with peptidoglycan may rotationally lock them. It is also possible that PVP does not capture some important aspect of cellular crowding. For instance, the washing-out of the CI2 signal may be an effect of transient protein-protein interactions. Fluorescence and electron microscopy experiments may be able to address this concern.

If the observations of CI2 are really a result of some unique localization or a consequence of the significant overexpression then it is possible that NMR of different proteins on somewhat less active promoters may yet be viable. In addition, in vivo studies of proteins in systems such as X. laevis oocytes or cultured eukaryotic cells may not be subject to the same difficulties, due to differences in cytoplasmic viscosity. The major implication of this work, however, is that in vivo NMR results cannot be taken at face value. Careful controls will be required in order to ensure that the experiments actually measure protein within the cell, and in the absence of such controls the results should be interpreted cautiously.

1. Bryant, J., Lecomte, J., Lee, A., Young, G., Pielak, G. (2005). Protein Dynamics in Living Cells. Biochemistry, 44(26), 9275-9279. DOI: 10.1021/bi050786j

2. Bryant, J., Lecomte, J., Lee, A., Young, G., Pielak, G. (2006). Cytosol has a small effect on protein dynamics. Biochemistry, 45(33), 10085-10091. DOI: 10.1021/bi060547b

3. Bryant, J., Lecomte, J., Lee, A., Young, G., Pielak, G. (2007). Retraction. Biochemistry, 46(27), 8206-8206. DOI: 10.1021/bi700744h

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

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The dimer interface of PhoQ

Relating a protein structure to its function is often not as straightforward as we would like. As any protein crystallographer knows, many biomolecules will only form crystals in specific, limited conditions, with particular buffers, protectants, and co-solutes. Sometimes even the water must be replaced with, say, isopropanol in order to form a crystal. History has shown that despite this, most protein crystals imply models that match with biochemical data. Sometimes, however, crystal structures of a single protein or two highly homologous proteins disagree substantially. In that case, it is up to the biochemists to determine which structure (if any) correctly represents the physiologically relevant ensemble.

That is just what happened in a study soon to be released in the Journal of Molecular Biology from the lab of Bill DeGrado. Goldberg, Soto et al. wish to resolve a question about the dimerization of the periplasmic domain of PhoQ, part of a two-component signaling system in bacteria. These systems sense an external stimulus (in this case divalent cations) and activate a set of genes. PhoQ deals in the first half of the equation, and exists as a dimer (pair of bound proteins) that stretches from the periplasmic space (where it senses the signal) across the inner membrane and into the cytoplasm of the bacterium. Because it is in general very difficult to crystallize membrane proteins in their entirety, the isolated pieces (domains) of this protein were crystallized independently. Unfortunately, the sensor domain does not dimerize strongly in solution, and the crystal structure of the E. coli PhoQ (explore this structure at the PDB):
does not match up particularly well with the structure determined for the homologous protein in S. enterica (explore this structure at the PDB):

Goldberg et al. note that the differences in these structures are unlikely to reflect actual differences between the proteins, because the amino acid sequences are 85% identical. That doesn't guarantee that the structures will be the same (see this post), but as a rule nearly-identical structures are what we would expect. Goldberg et al. set out to determine which structure represents the physiological form, and they decided to do it in vivo, with the full-length protein.

In order to do this, they engineered several mutants of PhoQ, each of which had a cysteine at a particular position in the N-terminal helix of the sensor domain (visible side chains in above figures). Cysteines have the property that they can form disulfide bonds with other cysteines when the chemical environment is right. Consequently, if the cysteine of one dimer is in close proximity to the cysteine from the other, they will stick together or cross-link. Once these proteins are expressed in E. coli and the cells are broken open, the concentration of this cross-linked species will be proportional to the frequency with which the relevant side-chains encounter one another. As you can see, the two structures imply very different predictions about the patterns of cross-linking that will be observed. If the first structure is correct, the successful disulfide bonds should be distributed evenly along the helix. By contrast, if the second structure is correct, the mutations that produce cross-links will all be located at one end.

Goldberg et al. observe the first of these patterns. The side chains that are visible in the figures above are the ones where mutating in a cysteine produced a significant amount of cross-linking. Obviously they are distributed along the entire length of the N-terminal helix. When the researchers ran a simulation that attempted to predict a structure on the basis of the cross-linking data, they also came up with a structure that looked very similar to the first crystal structure above. While not all of the cross-links observed appear to be compatible with the structure, this may just be a result of a few additional conformations in the ensemble.

In this case, the experiment was performed on the E. coli protein, so it is still possible that the structure for the S. enterica protein reflects actual differences arising from the 15% difference in homology. Obviously the next step would be to perform the similar set of experiments on the S. enterica protein; doubtless these experiments are already underway. As the authors note, it is also possible that the two structures represent the active and inactive forms of the protein. Repeating the experiment in minimal media and adjusting the levels of magnesium may address this possibility.

1. Goldberg, S., Soto, C., Waldburger, C., DeGrado, W. (2008). Determination of the physiological dimer interface of the PhoQ sensor domain. Journal of Molecular Biology DOI: 10.1016/j.jmb.2008.04.023

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Rapid evolution of lizards in the Adriatic

Enough about Ben Stein and his lies about evolution. Let's talk some truth about evolution, namely scientific truth. The fine folks at Zooillogix brought my attention to a paper that flew under my radar at a time I may (perhaps) have been more focused on basketball. In it, researchers from Harvard and Amherst studied a population of lizards on a very small islet in the Mediterranean. In less than 40 years, this population of lizards has evolved to have significantly different morphology from its parent population, a new set of endosymbionts, and novel anatomical features rarely in related lizard species. It's an interesting case of evolution in action.

Back in 1971 10 lizards of the species Podarcis sicula were transplanted from one small islet in the South Adriatic to a nearby, somewhat smaller hunk of rock. Over a three year period from 2004-2006, Herrel et al. returned to these small islands to see what became of the lizards (1). What they found is that the transplanted lizards had taken over the second islet. The lizards from the second island were still genetically very similar to those on the originating island (see their supplementary figure 5). However, there were pronounced differences in the diet. Whereas the population on the island of origin ate very little plant matter (<10% of the total diet), the transplanted lizards appeared to subsist mostly on plant matter, with some seasonal variations up and down from 50%.

The change in diet appears to have prompted some substantial changes in morphology as well. The size and mass of the lizards on the second island is significantly greater, and the head dimensions are altered. For the smaller female lizards, this translates directly into an increase in bite force. For the male lizards, however, the differences in head size alone are not sufficient to account for the observed increase in bite force, implying that there are additional adaptations of some kind. Herrel et al. speculate that the increased bite force of the lizards helps them to eat and digest leaves. This is reinforced by the observation that structural features related to the opening of the jaw are largely unchanged.

There are additional adaptations internally. For instance, the lizards on the second island have a structure called a cecal valve (and additional anatomical changes to the cecum) that are believed to aid in the digestion of plant matter such as leaves. This is particularly interesting because this valve structure does not appear in the originating population, and is rare among related species of lizards (the suborder scleroglossa). Moreover, the hindgut of these lizards contained nematodes that are absent from the parent population, suggesting the development of a novel symbiosis. The authors note that the new morphological characteristics are present in juveniles as well as adults, suggesting that the changes involved are genetic, though further experiment is required. The authors also note some interesting changes in population dynamics and behavior that appear to have resulted from the altered eating habits of these lizards.

That these new features appeared within less than 40 years is especially striking. In less than a human lifetime this population of lizards evolved adaptations such as altered jaw morphology, as well as an apparently novel internal feature. While small populations and constrained locations can accelerate the process of evolution, it is still instructive to consider this result when discussing the "likelihood" of evolution. Significant morphological adaptations can evolve very rapidly. Imagine what the power of evolution could do given hundreds of millions of years in which to work.

Oh, wait... you don't have to.

1. Herrel, A., Huyghe, K., Vanhooydonck, B., Backeljau, T., Breugelmans, K., Grbac, I., Van Damme, R., Irschick, D.J. (2008). Rapid large-scale evolutionary divergence in morphology and performance associated with exploitation of a different dietary resource. Proceedings of the National Academy of Sciences, 105(12), 4792-4795. DOI: 10.1073/pnas.0711998105

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Expose Expelled

Maybe you know someone who has been suckered in by the numerous distortions, outright lies, and malevolent accusations of the "documentary" Expelled, starring famous bore and Nixon speechwriter Ben Stein. Perhaps even you have been convinced by its "startling" array of important "facts" about the "controversy" between the scientifically useless design conjecture and staggeringly successful evolutionary theory. If so, I urge you to direct your credulous friend (or yourself) to Expelled Exposed, a website devoted to debunking the lies of this appalling propaganda flick. Even if you favor a creationist viewpoint I think you will find it highly disturbing how freely the creators of this film distort the truth to favor their views.

In addition, for general knowledge on debunking the claims of creationists you should check out The Panda's Thumb, TalkOrigins, TalkDesign, TalkReason, and the National Center for Science Education.

Also, New Scientist has an excellent article up featuring 24 misconceptions about evolution. Read it.

Also check out some other articles about the film and its marketing:
Biologist PZ Myers, interviewed in the film, is expelled from a screening of Expelled. But they let Dawkins in.

An animation in Expelled was ripped off from Harvard University and XVIVO. It should be noted that famous cdesign proponentsist William Dembski was for a long time in the habit of stealing this film to display in paid lectures.

Expelled tries to draw a line between belief in evolution and anti-Semitism. Interestingly, one of the creationist scientists they interview is the anti-Semite Maciej Giertych.

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Creative focus in game and technology development

Tynan Sylvester has a really incisive opinion piece up at Gamasutra illustrating a fundamental disjunction between what developers look at in a game and what players look at. The gist of his piece is that developers often focus on "scaffolding": the mechanics and code that underlay the game world. Meanwhile, the players consider mainly the "masonry", that is, the experience of the game world itself. The not-so-subtle point here is that many of the minds behind games have put their creative focus on the wrong thing.

The developers of Crysis and like games, as well as the graphical powerhouse consoles, succumbed to the tyranny of precision, the mindset dominance of quantifiable fidelity. The simple fact is that the graphical capabilities of hardware and software are easily measured, in terms of resolution and framerates and numbers of polygons on screen at a time, and it's easier to improve something if you know how to measure it. Unfortunately, creation of these super high-definition visuals takes a great deal of effort and expense, and ultimately it doesn't help that much. What players really want is an experience that feels real, not an image that looks real. Experiential fidelity, unfortunately, can't be quantified, and it's difficult to plan hardware (or even software) that creates it.

The success of the Wii and DS, however, suggest that good feel can be, if not created by hardware, at least encouraged by it. It's too simplistic to try to explain this success by saying that these platforms "tapped into another market". If that's so, then we must ask why they tapped into that other market. The answer is that these systems create an experience that the user can relate to, despite their shortcomings in terms of graphical excellence. The remote and stylus are very intuitive control devices. As a result, the player can interact with the game worlds easily, and often exactly in the way he expects to, rather than pressing buttons on some bizarre alien boomerang. Although the Wii and DS may not be great scaffolds, they allow for some great masonry.

I don't mean to say that graphics don't mean anything. However, though they are the most easily measured aspect of a game's performance, they do not define what makes a great game. It is the experience that matters. The graphics help create the experience, but even the best graphics in history cannot rescue a game that has lousy audio, an idiotic story, or nonsensical mechanics. Nor am I trying to sell coding short: lousy coding can ruin (or almost ruin) an experience easily. The point is that the creative focus of hardware and software developers should always be on the experience to be created. Everything else must flow from that.

You should check out Sylvester's website, by the way. He's got some really interesting stuff up there. I particularly liked his piece on Hitman and his article on substance and style in game design, although I disagree with some conclusions of the latter.

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Congratulations Doctor Labeikovsky! (Now with photographic evidence)

That cool breeze you felt this morning blew in from the icy recesses of the underworld, the result of a sudden climactic change that occurred because labmate and occasional commenter Wladimir Labeikovsky successfully defended his thesis on the interaction of tau with prolyl isomerase Pin 1 yesterday. He now has his Ph.D., a sure ticket to worldwide fame and fabulous wealth. Congratulations to him on bringing a successful conclusion to a long graduate career, and looking good in a suit. Hit the jump for pictures.

Wlad cleans up nice, doesn't he?

Wlad still laughs at Dorothee's jokes.

Little known fact: as part of your training for grant applications, you must successfully break a piñata in order to receive a Ph.D. in biophysics. The piñata in this case is full of candy and Skyy vodka sample bottles.

Here's a view of the party afterwards. Nothing says "science" like a bunch of folks standing around drinking.

Wlad attempts to murder a committee member with a champagne cork.

Here we see Wlad's adorable godchildren during the brief time that they were not covered in frosting.

Enough said.

By the way, I would appreciate it if whoever received my worldwide fame and fabulous wealth in error would forward them to me promptly.

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Does flexibility increase protein stability?

Since my favorite physicist of the blogosphere put up a great post on the basics of NMR in preparation for a geeky post on the subject, I figure I'll add in a geeky post of my own. As Chad mentions in his post, the dependence of the resonance frequency on local chemical structure allows us to get a great deal of information about covalent bonds and their conformation from NMR spectra. In addition, certain NMR experiments can provide us with detailed information about the motions of atoms with respect to one another. Although the various approaches differ in the level of detail they offer, the sum of NMR dynamics experiments fairly effectively cover the range from picosecond motions to month-long fluctuations. My particular interest in these approaches is in their use to dissect intramolecular signaling in proteins.

The major part of my graduate studies at UNC under Andrew Lee was devoted to understanding the pathways by which changes in dynamics are propagated in eglin c (explore this protein at the PDB). Eglin c is an excellent subject for this kind of study because it has very good spectroscopic properties, and because it has no discernable allosteric properties at all. This means we can do a lot of different experiments on it, and also that our observations about long-range dynamic interactions are likely to be generalizable to any protein that has a hydrophobic core, not just allosteric ones.

My work with eglin c involved making mutations to the protein and determining what dynamic changes resulted. In the course of this I discovered several mutants that caused significant changes in the motions of amino acid side chains on the timescale of picoseconds to nanoseconds (1,2). This is the range covered by the Lipari-Szabo model-free formalism (3). One mutation in particular, V54A (read: valine 54 to alanine), caused almost universal rigidification of the core of eglin c on this timescale. However, the protein was significantly less stable.

This is curious because a lack of stability is known to correlate with an increased frequency of localized unfolding of the backbone. In elements of secondary structure, hydrogens attached to amide nitrogens tend to be caught in hydrogen bonds. When placed in a solution of deuterated water, these hydrogens can only be replaced by deuterium when the hydrogen bond breaks (this is called hydrogen-deuterium exchange or HX). This process can be monitored by NMR (and less specifically by mass spectrometry), and is known to speed up when the structural elements are less stable. So the V54A mutant is more flexible on this slower timescale, and less flexible with respect to some faster processes.

The significant caveat here is that we only looked at some groups in the protein. For reasons that are a bit too complex to discuss in this post, it was only feasible to observe the dynamics of methyl groups. This is a problem because the core of eglin c has a substantial number of aromatic side chains in it. Any picture of dynamic behavior would be incomplete without examining these side chains and quantifying the changes in HX behavior, which I did not do.

In an upcoming article in Biochemistry, Josh Boyer and Andrew Lee address these deficiencies (4). In order to look at the aromatic side chains, they employ a clever biosynthetic labeling scheme devised by Akke's group (5). While this only allowed them to look directly at δ-carbons of these residues, the general rigidity of aromatic rings meant that they could generalize the observations to essentially the whole side chain. They found using this approach that the dynamic response of aromatic side chains to the V54A mutation was more heterogeneous than that of the methyl side chains. As you can see in the figure I have shamelessly stolen from their paper, the central portion of the core appears to have rigidified across all residue types, while the edges of the core and the outward-facing residues of the protein all appear to have become somewhat more flexible. The location of V54 is shown here in black.

This heterogeneity of the dynamic response was also observed when the HX results were taken into account. The distribution of responses, however, is very unexpected. The top portion of the figure at left shows rigidified side chains as a transparent surface, and destabilized backbone amides (as measured by HX) as solid surfaces. Note that the two regions of destabilized amides are linked by contiguous side chains. The lower figure shows side chains that have become more flexible as transparent pink surfaces. The solid backbone surface indicates a region of the protein that has become more stable. You will notice that this stabilized region lies a significant distance from the mutation site (though as the figure above shows it is connected to it by a network of contiguous side chains), and that it corresponds with a bundle of aromatic sidechains that have become more flexible. A significant portion of the protein is clearly destabilized, as was expected from the previous results on V54A. However, the co-localization of side-chain rigidity and backbone instability (and vice-versa) doesn't jive with our expectations.

Our physical intuitions suggest that rigid things are stable and flexible things are not, and in many cases these intuitions have been borne out—in studies of backbone dynamics of proteins from thermophilic organisms, for example. However, the present results can be rationalized if one supposes that conformational entropy makes a significant contribution to stability. Indeed, because the structure of V54A is known to be essentially unchanged from wild-type (2), the enthalpic contributions (hydrogen bonds, charge-charge interactions, etc.) will not be altered. Entropic contributions will therefore dominated the observed changes in stability.

Rigid regions of a protein may have no place to disperse thermal energy other than localized unfolding, while flexible regions may have the option to dump some of that energy into side-chain fluctuations. When mutations increase the available space for those fluctuations, therefore, stability may be expected to increase, and vice versa. Obviously, the benefits of flexibility to ordered backbone structure will diminish as the fluctuations approach a magnitude that permits solvent to permeate the site.

Boyer and Lee's results, though probably expected by those who have followed previous research in eglin c, do not mesh with our macroscopic understanding of the relationship between rigidity and stability. However, they are not so outlandish that they can't be rationalized in terms of what we already know about proteins. An investigation of this anticorrelation in other eglin c mutants (promised in the paper) would be welcome. In addition, it will be interesting to see if this phenomenon is observed in other proteins (especially thermophilic proteins). As new labeling techniques and experimental approaches expand the range of molecules that can be effectively investigated using NMR, we may learn significantly more about the role of conformational entropy in the stabilization of protein folds.

1. Clarkson, M.W., Lee, A. (2004). Long-Range Dynamic Effects of Mutations Propagate Through Side Chains in the Serine Protease Inhibitor Eglin C. Biochemistry, 43(39), 12448-12458. DOI: 10.1021/bi0494424

2. Clarkson, M., Gilmore, S., Edgell, M., Lee, A. (2006). Dynamic Coupling and Allosteric Behavior in a Non-Allosteric Protein. Biochemistry, 45(25), 7693-7699. DOI: 10.1021/bi060652l

3. Lipari, G., Szabo, A. (1982). Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. Journal of the American Chemical Society, 104(17), 4546-4559. DOI: 10.1021/ja00381a009

4. Boyer, J.A., Lee, A.L. (2008). Monitoring Aromatic Picosecond to Nanosecond Dynamics in Proteins via 13C Relaxation: Expanding Perturbation Mapping of the Rigidifying Core Mutation, V54A, in Eglin c. Biochemistry DOI: 10.1021/bi702330t

5. Teilum, K., Brath, U., Lundstrom, P., Akke, M. (2006). Biosynthetic ¹³C Labeling of Aromatic Side Chains in Proteins for NMR Relaxation Measurements. Journal of the American Chemical Society, 128(8), 2506-2507. DOI: 10.1021/ja055660o

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Odin Sphere gameplay notes

So I came to a compromise on the review issue. I'm not going to do much discussion of the aspects of a video game that don't relate directly to the theme or the atmosphere, which are the subjects I want to focus on in the critiques. At the same time, there are going to be some instances, like this one, where a game really deserves some additional criticism or discussion for some of the design aspects. In some cases this will be because of exceptionally good choices that were made in the design process, but in others it will be because of the flaws in the process. And the simple fact is that Odin Sphere had several flaws, some of which nearly made me toss the controller down and take the disc out.

Odin Sphere is an example of a rare case in which the coding, rather than the artistic direction or the writing, was the weakest link in the game. Music, graphics, story, and design create the experience for the player. Coding can't create a good experience if these elements are trash. It can, however, work against them by pushing a player's mind out of the world of the game. It's a thankless task to be a programmer, I'm sure. The designers, artists, and musicians get all the print. But it's the people in the trenches writing the game engine that make all the other aspects work together to create art. In this case, unfortunately, the programming (and perhaps the hardware) wasn't equal to the task that the experiential elements demanded.

Although it is graphically beautiful, Odin Sphere doesn't seem to run on very efficient code. Anytime a large number of moving elements are present on screen the framerate drops, sometimes significantly. This isn't just a problem with too many enemies—the frequent slowdown in the Netherworld stages is associated with background elements. Even stages with relatively few enemies can suffer significant drops if too many phozons are left floating in the air. Because several of the boss battles are very complex, with lots of moving elements, they can be especially afflicted. The battles against Odette and Belial get to be very frustrating because of the framerate failures. The feeling that you could punch in a combo and go make a sandwich before the animation stopped really removes you from a fight.

Speaking of going to make a sandwich, Odin Sphere features some truly amazing load times, especially when going between the Pooka Village and the restaurants. No number of charming little animations will hide the time it takes to move between screens and stages. Often there is substantial loading after you enter a stage as well. Slowdown and load screens kill a player's engagement in a game.

Aside from the technical base, a few design elements were notably off. The controls were a bit muddy, and made to feel more so by the fact that cancels were impossible. Once you start a combo your only option is to see it through or stop acting entirely, which was something of a problem in the more complex boss fights. The inventory system also could have been much improved—it seemed like half the time you weren't fighting was spent managing the tightly constrained inventory. Shrinking that down so much didn't feel like it served much of a purpose; there's no need to be "realistic" about the inventory size in a game where each level ends with a gigantic treasure chest falling from the sky. The inventory management was a needless distraction from the game's better elements.

Another flaw was that the revelation of recipes for alchemy and the restaurants didn't match up with the modular nature of the story. You find a recipe for a moderate healing potion in the first book of the story, but the stronger healing potion isn't found until the fourth book. In the meantime, however, you have brought several characters to a point where the "H" potion isn't of any benefit. There was no gameplay or story reason to hold the Elixir back, and doing so forces the players to replay books in order to give their characters a meaningful healing potion for the final stages. Revealing the recipes piecemeal would have been more acceptable if the player was free to experiment and find things on his own. However, you aren't allowed to make any alchemy mix for which you don't have the recipe.

As for the restaurant, the fact that the story is told very much out of order makes its recipe system seem extremely strange. Why can Velvet buy "egg on toast" right away, when in the linear timeline that recipe won't be found until much time has passed? This complaint can also be leveled at the alchemy recipes. In short, the recipe system was illogical and needlessly forced a replay of previous books.

The reason the replay of previous books was a problem was that you literally had to replay them. That is, once you finished all five main books and wanted to buff your characters for the final battle, you had to start each of those books from the beginning, rather than entering each one at a point later on where you had access to all or most of its levels. As a result, you had to play rather a long time to reach a point where you could buy the bags you needed for inventory or get the raw materials necessary to make elixirs. It seems like this could have been avoided.

For the most part, however, Odin Sphere features reasonable design choices. The diversity of fighting styles of the main characters means that the repetition of levels and bosses never gets boring. The character development system is intuitive and still manages to feature an intelligent trade-off. And for the most part, the game is very forgiving about providing you with materials to produce the potions you need in the areas where they're needed (the notable exception being Titania). While most battles allow you to get by just by whacking at your enemies and using special powers, you can usually find additional strategies or tricks for each particular fight that makes it easier. The game is balanced, has a relatively even difficulty progression, and only one really unreasonable enemy (the slimes). The choices are solid but not spectacular; for the most part this is a game where the mechanics try to make themselves as unobtrusive as possible. That's why the smaller failings seemed so glaring.

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Eleventy-one!

Discount Thoughts turned eleventy-one with that last post, which I think is an excellent time to shake up the structure of this annoying little site. I've decided to kiss the keywords list goodbye and revamp the links. I've replaced it with a non-exhaustive list of keywords and the most popular posts (relative to my other posts) from the last month or so. The list of papers (formerly headed as "Shameless Self Promotion") has been removed and will now exist only in this post. Let me know what you think of the changes.

"Solution Structure of Polymerase μ's BRCT Domain Reveals an Element Essential for Its Role in Nonhomologous End Joining"

"Dynamic Coupling and Allosteric Behavior in a Non-Allosteric Protein"

"Role of Structural Plasticity in Signal Transduction by the Cryptochrome Blue-Light Photoreceptor"

"Long-Range Dynamic Effects of Point Mutations Propagate through Side Chains in the Serine Protease Inhibitor Eglin c"

"Increased Rigidity of Eglin c at Acidic pH: Evidence from NMR Spin Relaxation and MD Simulations"

And some contact information, rendered in my native language:
Email : clarkson dawt emm double yew at geemail dawt cawm
Speak it to learn my address.

Also in this post: the Discount Thoughts lexicon!

bafmodad (baf mō dad) — In a video game, an object which is not of any intrinsic use but which must be obtained in order to advance the plot. The Medallions from Ocarina of Time are a classic example. The bafmodad is conceptually related to the cinematic MacGuffin. Term invented by Jerry Holkins and Mike Krahulik as a corruption of the name of an actual item from Starfox Adventures.

cameralepsy — The repeated recrossing of a boundary between two camera fields of view. Generally occurs in games which have fixed cameras and camera-relative controls. If the smallest angle between the cameras is > 120 degrees, there is a high probability of a cameraleptic seizure, which can only be cured by ceasing to move or (in games like Grim Fandango) changing the control scheme.

disingenue — A person, often famous, who falsely claims that his or her bad deeds are due to the influence of poorly chosen friends.

magibabble — The fantasy equivalent of Treknobabble, magibabble is pseudoscientific, jargon-filled dialogue concerning technicalities of magical physics that will either cause the destruction of the world, allow the heroes to save the world, or both. Excessive use of magibabble is a particularly common sin of fantasy RPGs. The emphasis on jargon is a critical feature of magibabble. Ordinary myths and legends often hinge on some technicality of magic, but they are not examples of magibabble because the rules are explained in plain language. Magibabble is typically used to disguise plot holes or laziness.

tatropmr (tä trōp mər) — The Abstract Transcendental Realm Of Perfect Mathematical Relationships, a nonexistent place which is frightfully dull, rather difficult to comprehend, and not inhabited by natural laws. All those who fundamentally misunderstand the process and meaning of science are eternally condemned there. First identified by Paul Davies, also the first to be publicly damned to it.

the turtles — A key problem with many arguments for creationism or intelligent design is the turtles: specifically, the fact that they go all the way down.

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