Interfacial Digressions

A Note on "Pure" Chemistry

2012-02-12T09:50:00.000-05:00

Over at the Curious Wavefunction, a recent post touched upon the idea of "pure" chemistry not being well-recognized as of late by the Nobel Committee. I of course find the entire notion to be rather silly - if one can't see the chemistry in those Prizes, then you have my condolences - but I thought it would be interesting to do the following exercise.

The American Chemical Society has a yearly "Award in Pure Chemistry" that is intended to recognize fundamental research in chemistry by a young researcher. Just to see what the ACS has considered "pure chemistry" since the 1990s, shall we?

Hmmm. This is a bit unusual. It seems that amongst the fairly obvious "traditional" chemistry researchers that are being recognized, there are a bunch of interdisciplinary scientists who are undermining the sanctity of chemistry! And some of them - dear Odin! - even have biological interests. Even if you go further back, I recognize a number of names who have become rather renowned for their interdisciplinary research.

Clearly, there is a mismatch between what the chemical community officially considers "pure" chemistry versus what the unofficial position tends to be, if one considers the chemical blogosphere to be representative. Hmmmm.

Random Interfacial Griping

2012-02-08T15:41:00.007-05:00

To no one in particular.....

1.) We teach a lot of very handwavy material in introductory general chemistry at the university level here in the US. It does not mean that such material never gets a proper treatment. One thing to keep in mind is that such a course is generally intended to appeal to a wide audience, frequently to no one's complete satisfaction. (Especially the premeds.)

2.) Who the frak wrote this Wikipedia entry? I emphasize the following:

Nitrogen-15 is frequently used in nuclear magnetic resonance spectroscopy (NMR), because unlike the more abundant splinless nitrogen-14, it has a fractional nuclear spin of one-half, which makes it observable by NMR.

Just so we are all clear - you can do ¹⁴N NMR. See, for example, the excellent webpage here for a bunch of references on exactly that. It's just that ¹⁴N is a quadrupolar nucleus (I = 1), which makes life a little more interesting, but is not everyone's cup of tea.

3.) Inverse problems can be challenging. Obviously.

And with that, I'm done. At least until next time!

A Modest Update

2012-01-18T08:29:00.001-05:00

Point the first -

- It was remarked to me that my occasional overindulgence for parenthetical remarks in my writing suggests that I am secretly a Lisp programmer at heart. I snickered. It does look like an interesting language to learn, though…

Point the second -

- I was reminded of a thoroughly snarky point I had made to some friends a while back. String theory (or whatever your preferred scheme for quantizing gravity and unifying the fundamental forces) can explain physics. But the really interesting stuff will require using physics to explain the laundry list of actual phenomenona that we have on our platter. So learn your statistical mechanics, as you can then do everything from analyze (to one degree or another) magnetic samples, ion channel clusters in membranes, and socioeconomic behavior.

So love your stat mech and cherish the partition function!

Point the third -

- A recent blog post over at Just Like Cooking sparked a comment, harkening back to a brief digression elsewhere last year. I was of course led astray for a bit, and dug up some earlier papers on the topic. Not that my to-do list is by any means feeling underweight, but I feel it could be edifying (and potentially interesting) to try and work through the idea in a manner that would be fairly accessible to a (more) general audience. So less "death by Hamiltonian," and more "spiced up with Hamiltonians," if one will permit that metaphor.

You Shall Pass!

2012-01-08T09:32:00.000-05:00

I saw this paper, and it was just asking be blogged about here. I figured I’d give it a shot.

Disclaimer – Not my work, never met any of the authors (although I’m sure they’re all within six degrees of me scientifically). The paper is open access, which I think is a good policy for me to adhere to in any future efforts along these lines.

Citation: L.A. Clifton, et al. “Low Resolution Structure and Dynamics of a Colicin-Receptor Complex Determined by Neutron Scattering.” The Journal of Biological Chemistry. Vol. 287, No. 1, pp. 337-346; January 2, 2012.

Among the many things that bacteria can do, one of them is knocking off other bacteria. There are a number of ways to go about this critical task, not surprisingly, and one of them involves proteins known as bacteriocins. These are proteins that the bacterium uses to kill off potential competitors, as they typically go after closely related bacteria. In this paper, the authors are focusing on Colicin N (ColN), a bacteriocin produced by E. coli. ColN depolarizes the inner membrane of Gram-negative bacteria by forming pores in the inner membrane, resulting in cell death.

The question the authors address is a fundamental one – how does ColN get past the lipopolysaccarhide-decorated outer membrane of a bacterium? It is ~ 40 kDa in size - so, clearly, not going to be able to easily masquerade as an ion or small molecule and pass unhindered through a pore in the outer membrane. The authors note that past research on ColN demonstrated that it is dependent on the presence of an outer membrane protein, OmpF (or related porins), to be effective. Cells that are OmpF-deficient will not be killed off by ColN. I should note that OmpF is a trimeric porin that permits the passage of ions and small molecules through the outer membrane. It was suggested that ColN could pass through the OmpF pore, but would need to be completely unfolded to do so. So there is clearly something going on here that is interesting.

The paper describes a multipronged approach to this question – the authors integrate microscopy, neutron reflectivity, and small angle neutron scattering (SANS). The authors step through their case – they first present the thin film imaging (Brewster’s angle microscopy) and neutron reflection data for their model of the OmpF/phospholipid monolayer. The microscopy suggests similar stability for the OmpF/phospholipid monolayer, although different topography and compression behavior (the formation of domains appears less evenly distributed in the OmpF/phospholipid monolayer, and there are “kinks” in the isotherm for the phospholipid-only monolayer compared to the OmpF-containing one). The neutron reflection data also seems to support the existence of an OmpF/phospholipid bilayer, despite Fig. 3B being mislabeled by my eye. Normally the neutron “refractive index” - neutron scattering length density, aka nSLD – is plotted as a distance away from some reference (e.g., an easily determined interface or a metal layer on which your sample is ultimately deposited). It seems that is what they intended to write (the x-axis seems to be labeled as such) but is mislabeled with the “Q/A^-1” tag.

In any case, much of biologically-oriented neutron scattering is dependent on the existence of contrast variation in the nSLD. You can purchase deuterated compounds (such as lipids), prepare buffers in deuterium oxide, and even express & purify deuterated proteins. You then mix and match your deuterated and protonated components to see what each component looks like when in complex with everything else. It is a low-resolution means of doing so, but the benefits can outweigh the disadvantages.

The authors move onto the ColN portion of their work, showing the microscopy and neutron reflection data for ColN interacting with the OmpF/lipid monolayer. The time-lapse microscopy of ColN with the pure lipid monolayer and the OmpF/lipid monolayer shows increased image intensity, but appears to “smear” homogeneously with the pure lipid monolayer while forming larger, brighter spots with the OmpF/lipid monolayer. Their analysis of the neutron reflectivity data indicates the presence of the ColN in the same layer with the OmpF, and not just interacting with its surface, as they see in the ColN + pure lipid monolayer sample. Given the contrast variation matching, they state that they are able to see ColN extend as it inserts into the lipid region, suggesting that it is unfolding to some extent. The increase in surface pressure would suggest that it is not going through the OmpF pore but is, instead, inserting into the lipid region next to the OmpF. If it was inserting through the pore channel, the surface pressure might be expected to level off and not keep increasing.

The SANS data round out the story – they’re looking at the ColN/OmpF complex in detergent. (I know, I know.) Anyway, their data-derived model has one of the ColN domains slithering down between the cleft between OmpF monomers, while the remainder of ColN remains protruding outward. If you look at Fig. 6C, the blue distance distribution (where you are only looking at scattering from ColN) has two peaks, one that overlaps with the red trace (where one is only looking at OmpF) and a separate peak. So this at least makes sense. They do discuss the potential for translocation via the pore, and some recent literature on that possibility.

Mostly, I thought that this was a really interesting bit of research – while there is the obligatory mention of potential application to antibiotic development, it’s pretty obvious that the fundamental scientific question of “how does a largish protein get across a cell membrane where the cell has no interest in letting it inside?” I think that the experiments were reasonable, were carefully done, and did not set off too many massive alarms in my brain while reading. I would like to think that you could use something like nanodiscs or bicelles for the SANS studies so you could at least approximate a native membrane environment – clearly, sample homogeneity is a concern, as scattering methods can be notoriously sensitive. (Did I ever tell you about the time I spent a good afternoon into evening washing banjo cells for SANS experiments since said cells were just disgusting?) I haven’t worked with nanodiscs – although I’ve heard and read more than I can shake a stick at - and my experience with bicelles hasn’t been quite so detail-oriented, so maybe it would require sublime experimental mastery beyond the typical.*

Anyway. That was kind of fun. Also, how many of you saw Ohm’s Law Survives to the Atomic Scale? I imagine people will want to confirm this, as it is definitely seems really cool. Clearly, it was custom-made by “hand” (well, scanning tunneling microscope), so no immediate applications to large-scale mass production any week soon, but that isn’t why we do science.

Now, off to think about thermodynamics for a while. I need to come up with a reasonable explanation of some data today…..

The End Draws Near....

2011-12-31T10:55:00.000-05:00

....for 2011, at least.

I really don’t have much to add about the recent charges in the UCLA lab safety case, especially given the posts elsewhere (most of which are collected at The Safety Zone here: 1; 2; 3 ). As I can easily count the small molecule syntheses I’ve done since my undergraduate days on my hands with room to spare, I am definitely not someone who can offer hard-earned advice on safety in synthetic chemistry labs based on extensive personal experience.

I will note that – obviously due to my biological inclinations – that lab safety can be just as much as insulating your experiment from you as protecting you from any hazards. Which, given one’s perspective on human nature, might be a more effective means of motivating compliance with lab safety standards.

Onto cheerier subjects….

My plan to bring up Helmholtz when Gibbs is mentioned did not quite pan out this year. I don’t think it’s going to happen. I’ll have to be contrary in some other manner in the future.

I did get back to blogging and commenting a bit this year, and I intend to keep it up next year. While the notion of doing substantive ResearchBlogging is a reasonable one, it would entail winding back the semi-regular sarcasm a bit for thoughtful commentary. I’m not sure if my system could endure the shock. It might happen, though. Having said that, if one has any substantive questions where my thoughts might be of interest, ask away.

Best wishes to all for a happy, healthy, and productive New Year!

Trust but verify.

2011-12-22T11:15:00.001-05:00

The question of how much to trust computational methods is brought up here at Chemiotics II. My answer is that it depends on what one is looking for in the first place.

If one is looking for some sort of completely accurate and precise way to have all biological phenomenona fall out of "first principles," well, I wouldn't hold my breath. Of course, I don't think anyone is really waiting for that. At least I hope not. I believe my feelings on these sorts of issues are best described by personal experiences I've had with computational methods.

In grad school, I had an interest in this one mid-sized protein (somewhere between 40 to 60 kDa) that was known to bind this particular ligand. There was a crystal structure of the protein with and without ligand, although of course it was hardly the entire story (which is why it was the subject of my research attentions). In any case, collaborators did some MD simulations, and it was consistent with what we had found and was known. In their next bit of work, they mentioned that they found something new regarding the mechanism of ligand binding. This was going on the same time as I was doing some work, and as it turned out, my data did not rule it out. And so new research was inspired for those who took up the project after I left.

Currently, I am embroiled in a sordid and complex tale of transmembrane signaling involving the receptor and varying amounts of soluble cytoplasmic proteins that propagate that signal. There was a fairly recent paper detailing MD studies of the signaling process. Well, part of it, I suppose - huge chunks on either end of the transmembrane receptor were not included, and none of the cytoplasmic proteins that bind and are modified by the receptor were included in the study. Certainly a daring attempt, but it's hard to get too worked up over it when it doesn't resemble anything that I actually work with on a daily basis.

In short....I think properly used, it can be a useful way to bridge what is measured experimentally with the metaphors we use to describe processes. (For example - people love using descriptions involving simple machines, but what is actually measured are thermodynamic or spectroscopic quantities. Of course, "force spectroscopy" looks to change this, but when you yank apart a protein, you are no longer just gently playing around at kT or sub-kT conditions to see what kinds of deformations you get naturally or as a response to some stimulus. Anyway....) Certainly, for small enough systems, I am inclined to give them a proper reading, and in cases where the system might be larger but is somewhat well characterized, the same applies. In giant systems where they toss out a number of critical components or oversimplify to the point of absurdity, I am generally far more skeptical.

Merry Christmas to those who celebrate, Happy Hanukkah to those who celebrate, and a delightful winter holiday season to the rest.

Chemists, controls, and computing.

2011-12-10T20:37:00.000-05:00

I have no experience with drug discovery, so I suggest one reads the excellent commentary offered over at The Curious Wavefunction and In the Pipeline inspired by a recent article on the role of computer simulation in pharmaceutical research, presuming that they haven’t already done so. What I thought was interesting enough to post about in response is in Wavefunction’s blog post.

They are reluctant to carry out the kind of basic measurements … which would be enormously valuable in benchmarking modeling techniques.

Methods development research can be difficult to support. Even obtaining modest funding can be difficult. It’s one reason why it can usually seem incremental in nature, as it’s easier to scrounge a few small devices or specialty materials to use with existing research infrastructure. This one is near and dear to my heart, as I have two such projects going on at the moment, and a third which is still in the planning stages. Unfortunately, it’s not the kind of stuff one could convince people it needs to be funded and generously at that. That was really more just me griping. But that is par for the course for me here at my blog…..

Unlike chemists, engineers are usually more naturally inclined to learn programming and mathematical modeling. Most engineers I know know at least some programming. Even if they don't extensively write code they can still use Matlab or Mathematica, and this is independent of their specialty (mechanical, civil, electrical etc.). …The lesson to be drawn here is that programming, simulation and better mathematical grounding need to be more widely integrated in the traditional education of chemists of all stripes, especially those inclined toward the life sciences.

I of course agree, but am inclined to mention a few things. This may be an artifact from my recollection/experience and is no longer the case, but I’ve seen a tendency for computational methods & applications courses intended for chemists to be heavy on the typical computational chemistry aspects (basic electronic structure calculations, a dash of MD, some molecular mechanics) along with a fair bit of introductory programming. Not that there’s anything wrong with that….but wait, actually, it is problematic.

I would think a more useful course might still contain some introductory programming and some of the typical computational chemistry, but I’d like to think that one could also take the time to introduce the students to chemo/bioinformatics as well as a module on proper data fitting. Of course, it might be claimed that it’s better suited for an upper-division chemistry laboratory, which would be fine. The important thing is to get people weaned from MS Excel and to actually start fitting data, not algebraically torturing your data until it’s in a format that can be linearly plotted and then fit with Excel.

Also, given that I have this notion of this course being something that all students will probably find useful in the future, the programming and software elements should be those that will easily lend themselves to a broad range of applications and uses in the future. I would imagine that introducing students to something like Origin or Igor Pro would be useful, as well as (re)introducing them to Mathematica, Matlab, Maple, or other comparable software. While the power of Fortran is well established for the numerical-heavy applications in computational sciences, I feel it would be better to have students introduced to something like Python. You can leave the Fortran for those who want to do the computationally intensive theoretical chemistry, while I’m sure the majority can use Python as a useful tool in their work.

This above is clearly influenced by personal biases (I'm a bio/physical chemist who is in the process of adding "systems biologist" if he keeps it up for much longer), but I think that sort of mix in a "computers & chemistry" course would serve a good cross-section of the chemical community. Any and all commentary, feedback, suggestions, and brutal eviscerations of my points are welcomed.

Cackling in Glee.

2011-10-05T11:11:00.001-04:00

I actually can't muster up any of my lazy man's wit for this year's chemistry Prize - is it physics? Physical chemistry? Materials science? Just sublimely wonderful and scoffs at the narrow cognitive categories that spring up on occasion. It also emphasizes that as chemists, we have fellow travelers in numerous allied pursuits - remember, if we want to continue blaring the "we are the central science" mantra, we have to recognize chemistry of all sorts whatever its ostensible classification.

The comments at ChemBark brought up two questions in my mind -

(1) - what do we consider chemistry?

and

(2) - why did it seemingly not catch anyone's attention as a candidate for the Chemistry Prize?

I've mentioned - in the vein of Roald Hoffmann - that chemistry stands on the pillars of structure, reactivity, and synthesis. Anything that causes us to reevaluate our understanding of even one of those pillars is noteworthy, as quasicrystals surely did in terms of understanding structure. That they can also occur naturally would indicate that our understanding of geochemistry can stand some fleshing out.

Now, if you had asked me about quasicrystals yesterday, I'd have thought that they'd be a Physics Prize one of these years, given that most of what I had heard about them was through physics seminars I'd attended over the years. But it used to be that the gap between physics and chemistry was far smaller - Rutherford (he of the physics vs. stamp collecting joke) picked up his Nobel in Chemistry way back when, and van der Waals was a Physics laureate. This year's Prize is a nice throwback in that regard. I think there might also be something to the comments on ChemBark that solid state chemistry is something of an underexposed topic in undergraduate curricula here in the US, and many of us just don't have that proper background in the field (which is certainly my case - most of what I know is because I stumbled into having learn something about the field in grad school). There might also be an echo chamber effect going on in the chemistry blogosphere. ;)

Now to start preparing for next year's betting pool!

Nobel Notes

2011-10-04T12:03:00.000-04:00

Thumbs up to the Nobel Foundation for their decision regarding Ralph Steinman's laureate status. I always felt that the rule was to make sure nominations of those who have passed on were not submitted in the first place. Now, whether or not one agrees with this is a separate issue.

Given that my professional interests are not very immunological or astrophysical, I don't have any particularly incisive commentary about the Physiology/Medicine or Physics Prizes.

A followup to a comment elsewhere - Tom Wainwright passed away in 2007, so unfortunately he would be ineligible for a Nobel. Given that Aneesur Rahman and George Vineyard have also passed on, Alder is really the only "founding father" of MD who would be a possibility.

On the off chance it is a magnetic resonance Chemistry Prize this year, I will not be sarcastic and post "but it's just applied physics! Why are applied physicists winning Nobel Prizes?" I'll actually just write a short blurb on what was so cool about the new laureates' research. (All other fields of physical chemistry being recognized are fair game for such commentary, though.)

Surely you jest!

2011-09-27T16:46:00.000-04:00

So, I strongly recommend everyone checks out this paper -

Accessing protein conformational ensembles using room-temperature X-ray crystallography

- which was just published in PNAS this week. The paper cites a 2004 paper by Bertil Halle (which I mentioned a while back) on the potential consequences of flash freezing and cryocrystallography.

Enjoy!

2011-09-20T18:36:00.000-04:00

No, I am not going to talk about the recent paper on the success of Foldit. Mostly since if you can even get a crystal structure for something, it's probably not agonizingly painful enough for me to work on - as I've said before, give me your disordered, your poorly soluble, your aggregated masses yearning to be analyzed.

Anyway, I wanted to mention this interesting-looking paper:

Binding Leverage as a Molecular Basis for Allosteric Regulation. I haven't had a chance to really dig into the paper, but the idea itself is simple enough - ligand binding can couple to various collective motions in proteins to varying extents, due to which we observe allosteric modulation of enzyme function. There are obvious oversights (one example that they mention in the paper - the lack of attention paid to proteins that aren't enzymes such as signaling proteins of various types), and I'd want to pore through which structures they used in the PDB (e.g., how did they deal with the family of structures that are generated by NMR if applicable). Then again, I usually consider thought-provoking ideas worth the publication, even if a judiciously skeptical outlook may make them seem a little less lustrous.

2011-09-09T22:39:00.000-04:00

As it’s that time of the year again to start speculating about potential Nobel laureates for 2011, I’ve already chimed in at The Curious Wavefunction and left a short note over at ChemBark.

While I’d be pleased to see another magnetic resonance prize (or five), there is a huge name which I’ve neglected, mostly since I was afraid that his time to be recognized had passed, but as he was the Welch Award recipient this year – John Waugh from MIT. Of course, if it were up to me, I bet I could come up with at least half a dozen trios of deserving recipients for magnetic resonance. But anyway…..

I’ve wondered about this earlier and I might as well bring it up again – what about the Kavli Prizes? Currently, they’re awarded for astrophysics, nanoscience, and neuroscience – will see ever see a dual Kavli and Nobel laureate? Or will being recognized with one put you out of the running for the other? Having said that, I know people were predicting someone getting a Nobel for semiconductor nanocrystals, so perhaps if Lou Brus is recognized by the Nobel committee, we’ll see one this year.

Quantitative biochemistry is not giving me quite as much of a headache. Although I'm hardly done with it just yet. I do envision there being an extremely dense biochemistry publication in my future. Speaking of which, back to working up data....

A few quick thoughts.

2011-08-27T19:52:00.000-04:00

I am still trying to unenviably navigate an n-dimensional parameter space, attempting to optimize the biochemistry for the present bane of my existence in order to get to some proper structural & biophysical studies. It is further complicated that whenever I do seem to devise a plan, something odd crops up in my data in amidst the general experimental madness (remember, if you work with n components, you need to vary one and keep n-1 constant : easier said than done!).

In any case, I stumbled across this interesting paper. Given my innate worrying about structural data obtained under cryogenic conditions, this was right up my alley – utilizing mesoporous materials to confine proteins and their hydration waters, and then using your interrogation method of choice across a range of temperatures without having to worry about the effects of bulk water. I can envision that this would be an excellent way to more explicitly bridge the gap between cryocrystallography and dynamic/functional studies done under more physiologically relevant conditions.

There was a very long back-and-forth over at The Curious Wavefunction this past week. I basically have the opinion that expecting physics to “explain” chemistry and biology is perhaps a bit overly demanding. I mean, it’s not as if all physicists are just waiting to wrap up high-energy/elementary particle physics and then retire, after all! There are still a number of unresolved questions in physics, and as a number of them involve many-body systems, it would only seem reasonable that those are the ones that would likely be of the most immediate application to chemistry and biology.

Now to finish preparing for this inclement weather…..

2011-07-07T09:11:00.000-04:00

As has been often noted about a number of topics, one’s biases will always skew one’s perspective.

On the one hand, we have this very interesting paper where the authors suggest correlated motions in ubiquitin over a distance of ~ 15 Angstroms (1.5 nm) based on further squeezing information from previously acquired NMR data with the help of computational methods. On the other hand, there is this other very intriguing paper where the authors put forth using gadolinium tags as a way to obtain structural constraints in proteins on the order of ~ 6 nm (60 Angstroms) via ESR/EPR techniques.

In the former, we’re looking at proposed long-distance correlations based on a bunch of relatively weak, short-range interactions (NOEs and RDCs), while in the latter we have nanometer-scale distance constraints being derived from a technique that is well matched to determining distances at the nanoscale. I figure the astute reader can figure where I stand on each given my tone.

Suffice it to say, it’s the reason why I’ve recently developed an interest in ¹⁹F NMR (oh, to work with a nucleus that has a decent gyromagnetic ratio and isn’t as common as protons in biological materials!), for one, as well as metal binding tags for paramagnetic relaxation enhancement studies.

In other news, my resolution for the second half of 2011 is to always try and work in a mention of the Helmholtz free energy into each discussion I am involved in that touches upon thermodynamics, as I think far too many chemists have gotten comfortable in their Gibbsian-oriented world.

I suppose this is one of those “it was bound to happen” things – one of the summer undergraduates who is in my lab at my current institution mentioned that the undergrad biochem lab uses a hexahistidine-tagged protein for overexpression & purification. I of course remember having to prep and subsequently grind up giant amounts of animal muscle to extract a protein in my undergrad biochem lab. I feel slightly dusty.

With that observation, I will call this blog post to an end.

SAXS And Promiscuity - Or, What Your Biochemistry Text Doesn't Cover.

2011-05-19T23:37:00.000-04:00

Glycolysis is one of those things you learn about as an undergraduate (in high school as well, to be fair, but in a good bit more detail in an undergraduate biochemistry course) and – at least in my experience – it was presented as a topic that had already been well-explored and thoroughly annotated. After all, if it hadn’t been, would they have put it down for posterity in a textbook?

Snickering aside, I was pretty intrigued to see the following paper the other day for more than just being another entry in my “clearly, plenty of mechanistic detail was glossed over in my biochem text” list. Basically, the research team utilized a combination of crystallography, small-angle x-ray scattering (SAXS), and computational model to develop a scheme for the mechanism of phosphoglycerate kinase. They propose that the enzyme has a preferred “open” conformation where substrates (1,3-bisphosphoglycerate and ADP) can bind (separated by over 15 angstroms), and then a “closed” conformation, where the domains “fold in” on one another, bringing the substrates together for chemistry and which exposes a hydrophobic patch, which they suggest drives the preference for the “open” conformation. In the supplemental info, they do have some movies for download which make for fun viewing.

It is a nice example of what some in the structural biology field have been pulling for, an integration of high-resolution methods with lower-resolution methods that can provide additional insight into dynamics at the domain scale and above. Just as a representative example of this thinking is the SIBYLS beamline at Lawrence Berkeley Lab (SIBYLS – Structurally Integrated Biology for Life Sciences, where they possess the ability to do both crystallography and SAXS at the same station). They’ve also got a fairly lengthy review linked to on that page that describes the interplay between crystallography, SAXS, and computational methods.

In the spirit of Wavefunction’s link post the other week, at least, I stumbled across this recent paper on stochastic ensembles, conformationally adaptive teamwork, and enzymatic detoxification today. I am still working through the paper, but – given that one of the authors has written rather extensively on atypical (non-Michaelis-Menten) kinetics in enzymes – he is putting forth a new set of organizing thoughts for understanding the unusual substrate binding and catalytic properties of detoxification enzymes (which frequently have multiple isoforms differentially expressed in tissues). These enzymes are not only promiscuous in terms of the substrates they’ll work with, but are also involved in multiple metabolic processes. So it's hardly as straightforward as biochem texts are fond of portraying with those nice, neat flow charts. I have occasionally considered this as a possible reason for all those secondary metabolites in plants that no one can figure out why they're present in the first place - you have a bunch of enzymes floating around in the cells and given enough time, stuff happens. But that is perhaps another post for another day.

A modest update.

2011-05-14T17:44:00.000-04:00

So, it appears that efforts in semi-quantitative biochemistry are approaching reproducibility, which is always interesting to observe. Of course, I have yet to surmount the final barrier of putting quantitative boundaries on both axes of my graphs (so far, there’s only the one axis that has actual numbers on it), so I suspect a couple more replications will be adequate to get it to a state where I will not dread being laughed at in public.

I suppose it will be something of a recurring theme in the future, but the crux of my interest in discussing protein dynamics is just how much territory that phrase covers, as what it implies varies from person to person, frequently corresponding to their own scientific interests. You have everything from the famed “protein dynamical transition” at around 180 K to research examining HD exchange in proteins, to studying the motion of various motifs in proteins as a function of a certain parameter, and extending all the way up to processes occurring at cell biology scales.

My current distraction (from reading things that are actually a bit more germane to my own work) is found here - a set of reviews on ion selectivity. It’s one of those topics I’ve found perpetually fascinating, although haven’t really worked in to any extent. Off to downloading!

2011-04-28T11:45:00.000-04:00

I feel like I should note this given my last post – the expositions of NMR that I can recall sitting through over the last decade have focused on the effects of judiciously applied B1 fields have on nuclear spin magnetic moments, not absorption/emission of electromagnetic quanta. I suppose the "absorption notion" – even if only intended in a handwavy pedagogical manner – is one that can feel fairly natural and not too extraordinary (given that spectroscopic methods that do involve actual absorption/emission of quanta are ubiquitous). But onto what I really wanted to discuss today.

The perils of quantitative biochemistry.

I have returned to contending with my old nemesis, sedimentation assays. Back in the day, I was interested in the interaction of a protein with a polymer, in particular the stoichiometry of said interaction (e.g., how many monomer units needed to bind one protein). While I eventually managed to get a reasonable-seeming estimate, it took a few tries to really pin down the optimal way to do it in a clear and reproducible manner. Nowadays, I am interested in the formation of a protein complex on the surface of a vesicle.

Once again, my latest attempt at quantification of a particular interaction was doomed to “no one with two neurons to rub together would trust anything on this gel.” Lesson learned yet again to not just double-check everything, but quadruple-think every step and every sample that is loaded, to say nothing of any assumptions about the entire process. In the vein of the old adage, one has to pick two of the following – quickly, easily, properly – to do their biochemistry. Being crunched for time, I naturally figured the first two would be best (as my brief attempt at doing this same basic type of measurement - same system, albeit with some modest differences) worked out somewhat well a few months ago).

One of the major issues is that reasonable-enough precautions (a particular wash step) one might take to improve the quality of said measurements is not feasible in this system since said precaution will cause unwanted (and functionality-inhibiting) aggregation. Alas. The major issue is that there are a number of little things that need to be done just right in order to ensure gloriously clear results and measurement-to-measurement reproducibility.

Odds and Ends –

1.) A good chunk of my tax refund this year is going for my chronic science habit. Software, books, and single malt Scotch. Well, OK, the last might not properly qualify.
2.) I find the entire International Year of Chemistry thing to be charming. The efforts being made by various organizations is vaguely reminiscent of someone thinking that as long as they make an effort on their partner’s birthday and Valentine’s Day, things will work out. Your mileage may vary, but that's the feeling I get in the back of my mind. We should view the IYC as a beginning, not merely a window of opportunity, to educate, enlighten, and entertain those around us.
3.) I have this urge to discuss protein dynamics. Future posts, I suppose.

And with that, I’ll be off.

You spin me right round baby.....

2011-04-26T14:20:00.000-04:00

I saw this come up in the comments here, and figured that it would make for a cute post. I will start off with the mundane, though.

One is frequently asked to picture electromagnetic radiation as an oscillating wave, with the electric and magnetic fields orthogonal to one another as it propagates. This, I imagine, does not come as a surprise to anyone reading this.

As is propagated in the above link, the NMR experiment is presented as utilizing radiofrequency (RF) waves to tickle the nuclear spin magnetic moments. Of course, that leads to the question presented in that post – how does an RF wave (with a wavelength on the order of meters) get absorbed at the scale of a single nucleus? One might also ask an analogous question on the other end of the experiment when one is recording a signal on your nearest friendly NMR spectrometer.

Now, for two related things to think about –

The first is the oft-neglected sibling in the magnetic resonance community, electron magnetic resonance (EMR, also known as ESR or EPR depending on who you speak to). One of the fun little things that you can shovel a sample into is a flat sample cell. This is exactly what it sounds like – your sample is basically sandwiched between two planes of quartz. It is helpful since you can position your sample (typically aqueous in this case, as they’re notoriously lossy) at a point of maximum magnetic field (high B1) and minimal electric field (low E1), which keeps the resonator Q-factor high as well as keeping your sample from heating up, which can make for sad spectroscopic pandas.

The second is the development of so-called “Low-E” probes for the biological solid state NMR community. Given that they are not infrequently studying aqueous samples with some amount of salts (aka lossy as hell), and the traditional need for high power decoupling to get adequately resolved spectra, minimizing sample heating has been a major focus of effort within the community. The result here is a probe that minimizes heating from the electric field, actually using some insights from the EMR/EPR/ESR community.

Now, if we think about what’s going on here….they’re trying to minimize the influence of the electric field (E1) by either judicious sample placement or probe design. We know from basic electromagnetism that an EM wave is composed of both electric and magnetic field components. It would seem that the absorption of EM radiation in magnetic resonance is not necessary for a successful experiment. It would, in fact, appear to be the case that what is important is the magnetic field that is generated by an appropriate EM source (RF for NMR/MRI, microwave for EMR) for the magnetic resonance experiment. The electric field appears to simply be a source of woe and frustration.

Of course, as noted in the comments to the above link, this is not new thinking. Hoult and his collaborators have been working on this topic in various ways and manners for over two decades now. There’s also that fun paper by Hanson regarding the necessity of quantum mechanics for understanding magnetic resonance. And more recently, there’s been a rather lengthy (and somewhat dense – I have a copy printed out, haven’t had a chance to really dig into it just yet) article on virtual photons in magnetic resonance, following up on Hoult’s suggestion from a while back.

Alright, back to things....

2011-04-14T20:48:00.000-04:00

There's a discussion here on a variety of topics, and in the comments, the issue of "homegrown" versus "bought" talent comes up. Of course, as may be par for the course, I suspect I see schools somewhat differently than the majority of the commenters there (I think of how awesome their NMR people are, naturally, followed by their biophysical chemists), so I was like, "How can someone say School X has no homegrown superstars - Professor Y went from fresh assistant professor to NAS electee in like 15 years?" But anyway.

I am feeling uncommonly pleasant and motivated this evening, though. Small victories in lab will do that to a person. I suppose I should power on through and finally finish up my taxes.

Return?

2010-11-18T21:07:00.000-05:00

Still alive. Still doing science (albeit it elsewhere, as my first postdoctoral position went sideways). Back to doing NMR on large biological systems.

I may or may not get around to posting/commenting again regularly. We will have to see.

P.S. - A very well-deserved award of the chemistry Nobel this year. Then again, isn't it always? ;)

A Modest Response

2009-10-07T09:49:00.000-04:00

Yet another round of science Nobels have been given out. And once again, the chemistry committee decided to award it to scientists who studied polymer synthesis by a multicomponent macromolecular assembly in a crowded, aqueous environment. They even had the gall to use crystallographic methods to study it!

This is definitely not chemistry. No way at all. I mean, synthesis, macromolecules, polymers, crystallography, people wanting to understand macromolecular structure and function at an atomic and molecular level? How can this be chemistry? Someone explain that to me, will you?

Unless, of course, it *is* chemistry. But that couldn't be. Could it? It's all quite troubling.

P.S. - Still alive, looking for a new position, staying on my toes.

Some random thoughts....

2009-02-08T13:48:00.000-05:00

Or, Structures, Struggles, and Teaching People How to Make Nuclei Dance.

So, a few issues of interest and/or importance to yours truly. This is going to be short.

1.) Vaults! I've suddenly become very entranced with both their structural (39-fold dihedral symmetry for the win!) and functional (no one really knows what they do!) beauty. The structure of rat liver vault at 3.5 Angstroms was recently published (see here) – they were able to clearly assign the major vault protein (MVP) to the electron density, while the other proteins and ribonucleic acid is still up in the air. The internal dimensions of approximately 620 Angstroms in length and 400 Angstroms in diameter is enough to encapsulate most entities within the cell. There's some mention that they might interact with lipid rafts, given some sequence homology considerations. Amusingly, since the number of coordinates and structure factors exceeded what could be put in a PDB file, it's been deposited under three separate accession codes.

2.) I mentioned not too long ago that I'm having some issues with our resident BIAcore (surface plasmon resonance) system. Let me describe to you a representative issue with the current chip and setup: The baseline for the control surface after being coated with buffer and blocking agent is significantly lower than the original baseline. It is possible we have a bad chip, or a bad instrument (some sort of drift in the optical components), or something else. So while troubleshooting and such is going on, it brings to mind a major difference between my current work and former work in terms of nature and aspect. With the SPR setup, it's a unit that is, for lack of a better phrase, a single entity. Sure, we can remove and insert sensor chips, and it's controlled by the adjacent computer, but it's basically a large self-contained box. I can't really go peeking inside, checking things out for myself since it's a shared facility instrument. Back when I was boldly going where no one had gone before in the world of NMR, it was all modular and accessible. I had a oscilloscope checking on the forward and reverse power going to and coming from the probe (remember, in ssNMR, one works with higher RF powers than in solution, typically), I could break out the network analyzer to check the performance of the probe or an amplifier/frequency generator, I could open up the probe to see what chaos might have befallen us, I could always quickly toss in a standard sample (adamantane, KBr, glycine) to quickly assess how far off-course we might have strayed.... I could change filters/attenuators in an appropriate fashion to see where misery was striking at the heart of my experiment. And there was a sense of general, simple assessibility – I could use any network analyzer that I could borrow from someone in the department to check what I might see with my lab's. I could always repack a chemical standard with new material from scratch without it being a big deal or expenditure of time. I could examine the raw data to check and see if the FID was starting at a maximum or minimum (there's a way to check the angle in the SPR software, but it doesn't seem to be something most people do), for instance.

I suppose it's a matter of personal experience – my graduate experience was analogous to the laboratory where I worked as an undergraduate (where we did primarily EPR and time-resolved optical spectroscopy). I – as a general rule of thumb – like to know what's going on at a certain foundational level. I am not a fan of being told, “Yeah, you need to call the company and ask.” I expect with time I will get more used to such commercially available instrumental setups, but until then, I shall lament this state of affairs.

3.) I have a wild bug up my nose about the issue of how to best teach NMR to people. I think most people familiar with magnetic resonance first become acquainted with it via the module in introductory organic chemistry classes, with maybe a mention of it in the introductory physics sequence. Now, I don't really think that there's anything wrong with this – when I was taking organic chemistry, I had the inspired thought, “Hey, protons are spin-1/2 particles, but so are electrons....what about coupling those spin-1/2 particles with nuclei that are spin-1/2 particles? Could we correlate nuclei to electrons? This could be useful for metal-containing systems, since you've got unpaired electrons!” I did some math, and then I found out who George Feher was, and then my moment of insight became bittersweet. I had a few extra drinks that weekend to get over the disappointment, and now that I've told my “how I independently rediscovered ENDOR” story, I won't be tempted to tell it again for a while. However, back to the question - what about the next step? It seems that most people get a “structural determination via spectroscopic/physical methods” sort of class, where they also get into additional methods such as IR, UV/Vis, mass spec, and the like. But, let's say that I become science czar, and I mandate that NMR be removed from this class and be included in a one semester (or equivalent) course in NMR. What else should we teach in this course? How broadband should its audience be – should we let biochemists who have a thing for structural biology expect to get something out of it as well? What about physics majors who are plotting on becoming chemical/molecular physicists and possibly develop the next generation of NMR experiments and applications? How much “death by operator algebra” should we have in this class? What kinds of experiments should be include in the lab component? I have my own ill-formed opinions at this point in time, but it's been fun to mull over on my own and I thought I should share.

And just like that, I'm gone....

It's Cold In Here

2009-01-28T20:03:00.000-05:00

Or, One More Reason to Take Protein-Ligand Crystal Structures with A Grain of Salt

This is going to be a really short post. Sorry, folks.

Ashutosh pointed out the other week to be wary of ligands in the Protein Data Bank (PDB) – see here. This is of course good advice, and everyone should read that post on the off chance they haven't already done so. Done? Good!

One other thing that I think is underappreciated by those who aren't protein crystallographers/structural biologists/biophysical BAMFs is that, for the most part, modern (synchrotron) protein crystallography is done under cryogenic conditions. Now, this typically is a bit warmer than the boiling point of liquid nitrogen, so you've got a protein crystal wallowing around somewhere above 90 K but not too far above 150 K. I'm not going to say that this is an entirely bad thing – it minimizes radiation damage, allowing for the higher-flux radiation sources to do their job. This of course can lead to higher-resolution structures and most of us generally appreciate that. However, here's the question – does it accurately represent the protein-ligand complex under physiological conditions?

A really interesting analysis of this question was addressed a few years back in 2004 by Bertil Halle from Lund University. I would recommend reading the paper, but if you're interested in the abbreviated conclusions, to wit:

1.)Flash-cooling of protein crystals – and subsequent cryocrystallography – is capable of retaining the general backbone fold and positioning of the protein, but

2.)The quenching of the solvent/ligand/ion degrees of freedom are not necessarily an accurate representation of the complex under physiological conditions and are probably more indicative of the system at the glass transition.

So the next time you look at a crystal structure and wonder, “How in Hades did that ligand end up there?” think about this. For a macroscopic analogy, consider the following structure:

Now, if it were 50 degrees colder, while the overall positioning would be similar, there's no reason to expect that every finger, strand of hair, and toe would be in the same position.

Expect more thoughtful posting one of these days, but don't hold your breath....

Clusters of Spins (AKA the Promised NMR Lit Post)

2009-01-21T22:47:00.000-05:00

So, here's the citation-heavy post about some interesting work done on systems of primarily – but not exclusively – biological interest via magnetic resonance spectroscopy since the turn of the century (I've always wanted to say that).

I mentioned CERM at the University of Florence in my last post for good reason – it's something of a place where, sooner or later, all paths seem to cross there in one way or another. It's either having read one of the texts that's come out of the faculty there, collaboration, or you just find inspiration in what they're doing. Because I have a certain unavoidable bias towards ssNMR out of (most likely) residual Stockholm Syndrome, I will mention this paper as an example of extending the work done at Florence in using NMR to understand paramagnetic metalloproteins in solution to the solid state.

Lyndon Emsley and coworkers at ENS-Lyon are pursuing a number of really interesting projects in magnetic resonance, both of basic importance to NMR (see, for instance, this paper) as well as to problems in chemistry, metabonomics, biology, and paramagnetic systems.

Paul Ellis & coworkers at PNNL have done some really stunning work with Zn-67 ssNMR and Mg-25 ssNMR. Zinc is one of the most common metal cofactors found in biology, and they've managed to do direct measurements of zinc in proteins via solid state NMR (see here and here for examples). Given that it's a quadrupolar nucleus, it's even more awesome. (While comments noting that they do this sort of work at really low temperature are accurate, it does not diminish its luster.) Magnesium-25 is another nucleus that this group has focused on as of late. See this paper for some Mg-25 ssNMR. The applications to half-integer quadrupolar nuclei in general, as well as their efforts in developing low-temperature ssNMR methods, are of interest in general and not just for biological systems.

Dieter Suter and his group at Dortmund have done some really neat things with optically-detected magnetic resonance, including metalloprotein research and their work on NMR of quantum wells. You can read more at their webpage here Fortunately for all of us, you can find a number of publications here. Suter was also involved in the Pines' group work on geometric phases in the late 1980s, so if you love it when interesting theory and experiment comes together, it should bring a smile to your face.

The Jaroniec group at Ohio State has been doing a variety of interesting things in solid state NMR, but I will mention their work with spin-labeled proteins here since it most tickles my fancy. You can read about it here, where they used spin-labeled proteins to obtain long-range structural constraints. Another advantage of spin-labeling is in facilitating assignment of congested NMR spectra – since you know where you introduce your spin label, you could potentially “blank out” certain residues that crop up in these congested regions to simplify assignments.

This is by no means a comprehensive list or thorough assessment of the field, more just intended to whet one's appetite. I would also like to point out the lecture notes from a solid state NMR workshop for graduate students and postdocs here. It may be a bit much to take in all at once, but some of you may find it makes for interesting reading despite not having a chance to hear the actual lecture. (FYI – no, I did not attend this workshop.) The list of speakers is a lineup of some of the major players in (biological) solid state NMR in the United States. If one's curiosity is still rampant, and you wish to expand your geographical purview, here is a list of lecture notes from a European ssNMR school (which, in case you're trying to pin me down, I did not attend) taught by a number of the major names in ssNMR in Europe. While I've long since downloaded these files and saved them, I can't guarantee that all lecture notes are still functional.

FYI - If I didn't mention you or your advisor or past advisor, I really didn't mean to do so. I would be pretty sure you've done/are doing awesome work, it's just that this is not a detailed journal club-style post. I will take recommendations, though, for any possible "journal club"-style posts, where the focus is on a single article or group of related articles. Tell me what totally BAMF science you've done, I'd love to hear it!

Assorted Awesomeness.

2009-01-17T09:55:00.000-05:00

So, I was going to do a post on general bad-assery and things I've found fascinating in magnetic resonance this time around, but it's taking a bit longer due to real-life laboratory fun. I will probably rework it as a really long bibliographic post, and perhaps have some discussion about it here and there. Onto the real-life laboratory fun, with apologies to Gossip Girl....

So, I've been aware of surface plasmon resonance (SPR) as a method for investigating kinetics and thermodynamics for a while now, and generally thought it was an interesting application. You can hardly do any reading in the biochemical literature and not eventually stumble across a paper that uses it. However, it was one of those things which I had never actually done until starting my current position.

Well, in order to further preserve some ostensible anonymity, I will be avoiding explicit details of my current work. The particular interaction I've been thinking about a lot as of late has been between this relatively small (~ 40 kDa) protein which I will call Blair, and this larger oligomeric protein that I will call Serena. Now, Serena prefers to relax in an appropriately tailored buffer, as is not surprising. So while I lovingly prepared Serena in such a buffer, I figured that for the purposes of the SPR experiment, I could just squeeze her into a relatively analogous buffer (minimal change in pH, no dramatic differences in overall ionic strength) that was a standard for doing SPR. I didn't think she'd mind, after all, she can retain her hotness under many conditions, or so I was led to believe.

Well, suffice it to say, after three rounds of SPR, I was mistaken. Now, I've learned how to finally run the instrument like a pro, I know that I need to go above and beyond the minimum for filtering my solutions and centrifuging out the dust in my samples, and can rewrite the scripting code half-drunk (not that I have, just that I can, it's really easy). There were other likely culprits (some potential issues with my controls, some concerns about coating densities) which I eliminated from the list of suspects the second and third times I ran the experiments, so I was making progress there. I just need to tweak the buffer conditions so Serena doesn't get all broken-down and sad on me. Because a hot blonde with great legs deserves better, metaphorically speaking.

Because, after all, how can you not want to see this?

Metaphorically speaking, of course.

One minor MR-related thing that will make my next post all about the spectroscopy – I find the idea of using nuclei other than ¹H in MRI to be endlessly fascinating. I'm very intrigued by the work people have been doing with, in particular, ²³Na – everything from cardiovascular physiology to neurological imaging to muscloskeletal studies. Not to say that I don't spread my love around for all NMR-active nuclei – certainly I'm not the only one to check out this absolutely fantastic website by Pascal Man periodically just to see what's new in the world of quadrupolar NMR – but ²³Na MRI seems to be a bit ahead of the pack, just from my anecdotal observations, in terms of development and rate of progress at the moment. If any MRI pros are out there, I'd love to hear from you, as I am positive I am missing out on some really neat things.

Well, that will be my obliged post for the week. I am going to strive for two posts next week. But don't hold your breath.....

Summations

2009-01-10T11:29:00.000-05:00

Another week, another post. So, in my last post, there was that interesting looking H_hyperfine term that describes the interaction between the nuclear spins and any unpaired electrons in the environment. I will make a few points about paramagnetic NMR and something I've been reading up on as of late in the biochemistry/biophysics field. And a random comment or two, of course, at the end.

NMR of paramagnetic species, while a topic of growing interest and fruitful research over the past couple of decades, still tends to get people not involved in the field to look at you and think, “But what about the enhanced relaxation, broadened lines, and paramagnetic shifts? It just sounds really hard!” To anyone who might be thinking that as they read this, I would like to note that a number of the metals involved in biological systems are, in fact, quadrupolar nuclei. Also, something like one-third of all elements can exhibit paramagnetism. So, you can pick one challenge or another, it appears. Life may not be easy, but think about it like this. Since there hasn't been a whole lot of work done compared to other areas, it's a growth field with plenty of opportunity. Besides, as the bioinorganic chemistry folks like to remind us, the number of metalloproteins in nature probably hovers around at least 20 to 25%, if not a bit higher. I hardly think neglecting all of the proteins out there is a good idea just because you're terrified of some hard work. To say nothing of potential applications in materials science and chemistry!

One thing to keep in mind is that the effects aren't necessarily the same in every direction. If you have a paramagnetic center which isn't isotropic, and there's a distinct anisotropy, one could have two marker peaks that are the same distance from the metal but only detect one because of the anisotropy. If you've ever done any EPR, or have any familiarity with it, one can – in principle – resolve the components of the g-tensor. (Determining the angles between the g-tensor and the molecular frame is far more challenging from my understanding, though, and not something I've ever done.) Sometimes there will be axial symmetry (where two of the components are equal to one another), other times all three components of the g-tensor will be different. In any case, while there is practically always the averaging of the electron magnetic moment, distance matters when dealing with paramagnetic systems. If you're close up, you'll not only experience more of the effect, but it's more sensitive to any anisotropy. If you're further out, less effect, and it looks more isotropic.

So, experimentally speaking, one can decompose an observed chemical shift from a paramagnetic sample, δ _obs, as follows:

δ _obs = δ_diamagnetic + δ_contact + δ _dipolar.

The nomenclature here can get a bit confused, so step-by-step, and hopefully not too confusing, here we go. δ_diamagnetic is the “base” diamagnetic chemical shift, what you would get if there was no metal there. δ_contact is the so-called contact shift (or Fermi contact shift) – this is what one gets from the average delocalized electron density on the nucleus interacting with the nucleus's nuclear spin magnetic moment. δ _dipolar is also known as the pseudocontact or dipolar shift – this is what you probably think it is, just a through-space dipolar interaction between the nuclear spin and the electron spin. Now, there are all sorts of neat computer programs that can incorporate paramagnetic restraints for structure calculations/refinements, determine magnetic susceptibility tensors from NMR data, and other related pursuits.

If any of this sounds interesting to you, please let me know. I can pump out a lit post with a number of citations without much difficulty during the week, it would be my pleasure. Or if you'd rather receive an email, let me know. However, for a starting point, I would strongly recommend a look at the work done by Ivano Bertini, Claudio Luchinat, and coworkers at the University of Florence here. That should keep you busy for a while.....

I've gotten interested in the entire “intrinsically disordered proteins” topic as of late. It's interesting for a couple of reasons, some related to my current research, others simply since it's a neat topic. It makes one more careful in one's thinking that proteins just fold to some structure and stay there – proteins actually have functions, which are connected to other macromolecules and their functions. A protein can bind a ligand/substrate, where it may adopt a restricted ensemble of conformations. That protein's function may be modulated by interacting with another ligand or with another protein (for instance, an electron transfer protein), which could alter its structure. It may finally need to adopt another conformation to release the ligand or product. So whenever someone says, “Well, I've determined the structure of a protein,” what you should really be thinking is, “That person has determined the structure of a protein at a particular point along its functional pathway.” It also serves as a handy thing to bring up in the inevitable protein folding discussions that crop up every so often offline and online. Not every protein comes away from translation and adopts its active conformation right away, after all. As a short and hopefully accessible example, if we'll remember our blood clotting biochemistry, there are a whole bunch of zymogens (or proenzymes, if you will) involved in the process. They're activated upon trauma in healthy individuals – there's no need for them to be active when you're not bleeding, after all. There's a somewhat recent paper I'm still digesting here on a model involving intrinsic disorder with coupling allostery between binding sites. It's an interesting topic, mostly since I've been focused on shorter-range interactions earlier on in my education/research, and am now becoming more intrigued on what I will facetiously call “structural systems biology” - what is the structural and dynamic underpinnings of all these neat things that happen in cell biology and physiology from a physical chemistry/biophysics perspective?

I'm always intrigued by the interplay between solution and solid state NMR, and how one tends to delve into the other in order to get information that is typically denied to them in their home territory. You have the solution NMR folk introducing residual dipolar couplings, you have the solid state NMR people trying to minimize the effect of the geometric/angular effects in measuring certain parameters, it's all kinds of fun. I will probably comment on this here and there in the future, I think it's endlessly fascinating despite no longer actively in the magnetic resonance community.

There was an interesting post on equipment (the abundance or lack thereof, and of varying quality) at In the Pipeline the other day here. I've since moved onto a postdoc in an honest-to-Buddha biochemistry/biophysics lab since my graduate school days in a (biphysical/NMR) chemistry lab, and have had use of pre-made kits on occasion. I think it depends on one's perspective and background – it can be a crutch to use them, but I don't plan on doing a great deal of molecular biology while I'm here. Is it really the best use of my time or the lab's money to pour my own gels (as I did in grad school) or prepare my own DNA plasmid purification materials, especially if I'm not going to be doing it week in and week out for the next 2 to 3 years?

The one thing I did miss in graduate school, compared to my undergraduate institution, was the presence of a professional research-grade electronics shop. I especially missed it in my last two years of graduate school, when I seemed to get problems with instrumentation about every three to four months. While we managed to get a good amount of support from our department's NMR facility, it's not quite the same as having a facility capable of not only repairing instruments but also helping one devise potential work-arounds or new solutions. Of course, it is a character-building process, taking apart an NMR probe, diagnosing the problem, and then fixing it (at least until it fluxes up again).

In any case, a substantial enough second post, I think. Best wishes to all!

Disclaimer - Any arguable/unrigorous point in the above post is 99.9% me being lazy about it.

Perspectives on Interfaces

2009-01-01T18:24:00.002-05:00

I remember being asked once in graduate school by an undergraduate if I had instant recall of all of the detailed information on proton chemical shifts for all of the various chemical environments out there. While I didn't laugh – to be honest, while I remembered the general trends, there was no way I could rattle anything else off at the drop of a hat⁰ – it reminded me of the disconnect that I occasionally felt while in graduate school for chemistry. I never felt like much of a chemist – the aspect to chemistry that really distinguishes it from the other natural sciences¹, synthesis, was something I did sparingly once I finished my undergraduate organic chemistry sequence. Grad school was focused on the area of solid state nuclear magnetic resonance (NMR) spectroscopy – a topic which gets minimal mention, if any, in most undergraduate curricula in the natural sciences. Worst of all², I worked on proteins while in grad school – given the occasional bit of heat that can get generated come October depending on who's gotten the call from Stockholm, having any connection to biochemistry can make one become the token biochemist who tries to humbly explain why the Nobel went to someone who may actually have done some pretty decent work and is not, in fact, an unwashed lump of pirate scum.

However, being firmly ensconced between biology, chemistry, and physics is actually a really great place to be in my books. The biggest advantage³ is that I can lament the biological understanding of chemists and physicists, the physics blindness that chemists and biologists exhibit, and the appalling lack of chemical intuition that biologists and physicists demonstrate. However, I try not to do this too often, as I find it obnoxious and mostly only of use while at happy hour tweaking people's noses about this sort of thing. I of course hear the “jack of all trades, master of none” refrain, so I figure it all balances out in the end. While in grad school, in addition to playing with a couple of proteins via solid state NMR, I also had the opportunity to dip my toes into the uses of solid state NMR in investigating polymorphism in organic compounds, coordination chemistry complexes, a few discussions on possibly utilizing it for various materials, and the standard hijinks with cryogenic liquids that are part and parcel of every magnetic resonance laboratory (LN₂ chilled vodka has a certain ambience to it).

So, given that NMR is a wonderfully interdisciplinary technique, and because I feel like rambling on about certain applications thereof given my past research interests (temporarily on hold while I get accustomed to things in the new laboratory where I make my home), I feel compelled to start what I suspect will be an infrequent but persistent habit of discussing various aspects of NMR dear to my heart. Now, when I think of NMR, I think of many things⁴, but among the first is the Hamiltonian which describes the plethora of interactions possible during an NMR experiment:

H_total = H_Zeeman + H_RF + H_dipolar + H_{chemical shift} + H_quadrupolar + H_hyperfine + H_scalar

The first term, H_Zeeman, is the interaction between the nuclear spins and the static applied magnetic field of the magnet iself, while the second term, H_RF, is the interaction between the nuclear spins and the varying applied magnetic fields – aka the radiofrequency pulses – that compose the NMR experiment. The next term, H_dipolar, is composed of the various dipolar interactions that are at play in between the nuclear spins. This can be divided into the homonuclear and heteronuclear dipolar interactions. The chemical shift component, H_{chemical shift}, appears next, describing the shielding the nuclear spin experiences from its local chemical environment. The quadrupolar interaction, H_quadrupolar, crops up at this point, describing the interaction of the nuclear spins with the local electric field gradient for nuclei whose spins are equal to or greater than 1. The following term is the hyperfine interactions, H_hyperfine, where the nuclear spin is interacting with an unpaired electron. The last term is the scalar coupling (or J-coupling), H_scalar, which is the indirect through-bond interaction between two nuclear spins. Now, one can rewrite and manipulate this Hamiltonian's form as one sees fit or finds convenient – one could include the scalar couplings in the dipolar interaction term, for instance. Here, the off-diagonal matrix elements of the tensor describing the dipolar interaction would account for the scalar coupling and other effects.

Depending on what one does, these interactions may be nonexistent, attenuated, or averaged out. Sometimes you want to reintroduce these interactions (the excellent work on residual dipolar couplings is an example of this), other times you want to quench them (for instance, the interest in developing heteronuclear decoupling methods in solid state NMR of organic solids, including proteins and other biological samples). Obviously, if one never works with paramagnetic samples, fretting about the hyperfine interaction term is likely not the best use of one's time. And, well, if you do MRI, you may not have to think too much about chemical shifts in certain cases.⁵

Anyway, now that I've bored everyone with some elementary ramblings on NMR in anticipation of more interesting posts later on, I think I'll end this section. I do intend to keep writing about things of general scientific interest, my occasional thoughts about other things that cross my mind in related veins, and hopefully tweaking a few noses. I will eventually get the blog list and layout figured out in due time, but don't hold your breath. Any recommendations or suggestions - whether it be blogging technicalities or about the content - will be appreciated (although not necessarily followed up on). Hope everyone who's reading this had a delightful holiday season and New Year's!

Notes:
0.) I do have pretty good recall when it comes to the ¹³C chemical shifts of interest for amino acids, and a lesser level of recall when it comes to the ¹⁵N chemical shifts. Of course, that's from years of interpreting NMR spectra, not because I made any specific effort to stash them in my memory.

1.) At least in my opinion. Your perspective may differ on this, of course. I've made the point elsewhere that chemistry is all about structure, reactivity, and synthesis (see here ) and that instead of having grad students synthesize something interesting, biochemistry has model organisms synthesize the sample of interest (GFP, for instance) to allow the biochemist to focus on something more than just preparing the sample. This is a far longer, more subtle, and more complicated argument than I am giving justice to here – perhaps I will bring it up in the future.

2.) Just kidding.

3.) There are also challenges to being stuck in the middle.

4.) Not least the numerous NMR jokes. Here's one I'm sure you've heard - “How many NMR spectroscopists does it take to prepare a sample?” “Zero – they get it from a collaborator.” For those of you who are only familiar with solution state NMR, the tradition of coming up with inventive names for pulse sequences also exists in solid state NMR – HORROR, MELODRAMA, CRAMPS, and WISE, among others.

5.) This comment is based on a postdoc candidate talk I gave at an MRI lab - one of the students remarked that he hadn't thought much about chemical shifts once he finished up his magnetic resonance class, as his dissertation research primarily focused on relaxation phenomenona.

Disclaimer - Any arguable/unrigorous point in the above post is 99.9% me being lazy about it.