A neat bit of work.

From the people who brought you a structure of an unengineered full-length GPCR in phospholipid bilayers comes a structure of an unengineered full-length mercury transporter in phospholipid bilayers.  Gotta say that this is pretty neat.....

While new technical difficulties are otherwise occupying my efforts, I have enough things in the air to keep me busy.  Speaking of which, back to it! Read more!

Trifling Thoughts.

1.) I really was hoping to go for a couple of more years before having to deal with a poorly behaving high-power RF amplifier.

2.) Whenever someone says that quantum mechanics should be understood at an intuitive level, I wonder if they never had to occasionally take solace in the mathematics earlier on in their education.  While it was fleeting during intro E&M at university, the intermediate E&M class did have me routinely finding some sanity in mathematics.  Perhaps my intuition is just exceptionally poor, of course. 

3.)  Given all the hoo-ha about AFM recently, I thought this was interesting and timely.

And there's a good ending point for today......


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Angry Birds: Academia

The title is just what I think would be a thoroughly hilarious game for one's smartphone.  However, as I am a cranky postdoc (at least until early next year), I have found the franchise thoroughly helpful for those moments where I need to kill a minute or four in lab waiting for something to incubate/boil/equilibrate.

While I am tremendously interested in chemical microscopy of all sorts, various online discussions of a recent Science paper regarding AFM studies of single molecules of reactant and products reminded me of the somewhat odd excitement over "seeing" something.  Your eyes are limited spectrometers that can only deal with photons, and many don't appreciate the signal processing involved between your eye and your brain.  Now, if we could do electrons and neutrons as well, I'd be less worried about our restricted optical window.  Heh.

I keep meaning to post long overwrought thoughts about NMR, lipid bilayers, and the scurrilous treatment of valence bond theory by some people, but don't hold your breath.  Maybe once I clear my to-do list further, I'll have the cognitive energy to do so.

Question for any who might be reading - is there an opposite of "minimal publishable unit"?  When you have a ~ 15 page article in a solid journal along with another ~ 15 pages of supplementary information, maybe you want to see about splitting that work up.  Especially if you're citing the SI in the main article. 

I'll go topple over some structures now.....






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A bit overdue, I suppose.

It seems that I have neglected you, dear blog.  I would express my regret, but, well, I've been busy.  I do hope that any occasional visitors have been having themselves a healthy, enjoyable, and productive 2013.

In any case, research is progressing to the point to where critical experiments will be run within the next few months, and presuming that my calculations are not too far off, we should obtain a reasonable amount of interesting data that will be published not long thereafter.   Which should help with the entire "what to do next?" conundrum.

However, before I meander to other topics, an observation - the one thing that keeps biochemistry challenging is that your samples are never entirely happy.  You can purify it to some vague approximation of homogenity, put it in a buffer it likes, add cryoprotectant, and flash freeze in countless little aliquots for storage at -80 degrees Celsius.  And when someone takes out an untouched aliquot 3 years later to check its viabiity, they find it's.....reluctantly alive.  You compare it to a sample made of protein you prepared a week ago, it's clearly working nowhere near its potential.  Now, I had the occasion to stop by my old grad school lab the other year, and the one student working on my old project is still using the small molecule ligands that was synthesized by myself and another student ~ 10 years ago. And that sample was not aliquoted out in tiny volumes, each one to only be used once, rest assured. 

Onto said other topics......

- I have to say I am extremely tickled and intrigued by the idea of approaching quantum mechanics as a certain generalization of probability theory.  It's probably a good thing I have no intentions of being a faculty member anywhere, as I'd probably try to pull off a presentation like this to students. 

- I sort of postponed my entire relativistic quantum chemistry self-study program.  I will get back to it.  One of these days.

- I am finding myself in the position where I need to start doing some serious practical and theoretical NMR teaching to a very small audience, though.   The whiteboards in lab are going to get messy, I suspect.

- Should probably start microblogging more often, to boot. 

Anyway, going to try and chime in more regularly here over the upcoming weeks and months. 




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Chiming in with a #ChemCoach entry

Here's my contribution to the ChemCoach Carnival, for what it's worth.


Your current job.

I am a postdoc in an actual chemistry department on the East Coast (USA) doing a mix of physical chemistry, biochemistry, and spectroscopy, along with dashes of molecular biology, computation, and misery. 

What you do in a standard "work day."

No such thing as a standard work day for me -  I'm presently slogging through heaps of molecular biology in order to validate a protocol for producing uniformly 13C/15N labeled protein (mostly since my PI isn't yet comfortable with how we do things in the 21st century).  Earlier this year, for example, I was doing everything from NMR on inorganic solids (after effecting some probe repairs on my own) as well as some p. chem. of lipid mixtures, in addition to protein NMR (solution and solid state). 

What kind of schooling / training / experience helped you get there?

 I was a biochem/chem undergrad (did my undergrad research in a biophysics lab - lasers and magnets, what more could one wish for?) and did my Ph.D. in an honest-to-Buddha chemistry department, doing biophysical chemistry.  I was briefly diverted into a year at a cancer research institute doing biophysics/soft matter-oriented work (that was a trip, let me tell you), but returned to my chemical roots. 

How does chemistry inform your work?

Just the other day I was trying to get this horrendous mix of inorganic chemicals to go into and stay in solution, actually - and people say there is no chemistry in biochemistry!  Heh.   My perspective on many of my scientific interests is rooted in my chemical background - for example, signal transduction could be translated as essentially controlling the rates of chemical reactions across interfaces, after all.  It's a rather essential component of what I do on a daily basis.

Finally, a unique, interesting, or funny anecdote about your career

I was at a conference where I spoke to the colleague of this scientist who had written a paper I had read and digested multiple times, as it was extremely germane to my interests.  He noted that said scientist had never followed up on this particular aspect since they found that obtaining reproducible data was far too tricky.  I should have realized then that my project might have been a bit ambitious, but I persevered and did manage to get reproducible data.  It just took another 2.5 years on top of the 2 years I had spent working on said project.....  Read more!

Some minor notes.

The reason they're making your son take chemistry?  Because they're mean.  Or perhaps because they hold to some archaic notion that education is about broadening one's horizons and tempering the intellect, not just about preparing one for any particular career path.  I haven't decided. 

Small-molecule synthetic chemistry in the NY Times?   A pleasant surprise to see in the national newspaper of record, at least last I checked. 

Microbial physiology via NMR.  So cool.   Also, kind of jealous.  I really need to get some awesome stuff done sooner rather than later.

I think that will be all for the evening.


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Nobel Notes (Pt. II)

Upon request.....

Molecular Dynamics

So if we skip over the entire Monte Carlo simulation work, the first proper MD work was Berni Alder and Tom Wainwright in the late 1950s on hard-sphere systems.  Wainwright, as I noted last year, passed away a few years ago.  George Vineyard (Brookhaven National Lab) used MD to study radiation damage right around 1959/1960, and Aneesur Rahman did the first MD simulations of an actual liquid (for the pedants - yes, I know it was argon) in the mid 1960s.  Unfortunately, neither Vineyard and Rahman are still with us.

Here it gets more complicated, as the move to more "chemical" systems involved a number of people (David Chandler and Bruce Berne among others).  And then there's developments like ab initio MD (Car & Parrinello in the mid-1980s) which have been more on the fundamental side, in terms of bridging MD to DFT in this case, and has found plenty of application (Car & Parrinello were jointly awarded the APS's Rahman Prize for Computational Physics a while back).  


Magnetic Resonance/NMR

So, I really do think solid state NMR has a prize with its name written on it.  It's obviously been a while in the making, but it is finding great use in multiple areas of application (chemistry, structural biology, polymers & materials, and various subfields and intersections branching off from these).  My general feeling is that it would be tricky to award a Nobel for solids NMR without including Alex Pines (Berkeley), as he's had his hand in the development of cross polarization (which basically everyone uses, whether you're doing static solids NMR or magic angle spinning solids NMR), as well as contributing to multiple-quantum NMR spectroscopy, quadrupolar NMR methods, and various other proofs of principle and applications (investigations of the Berry phase by NMR to his more recent efforts in combining optical pumping and hyperpolarization for imaging purposes).  There are other names here, but it gets tricky.  I suppose Pines' former graduate advisor, John Waugh, could be here as well.  And there's always the risk I'm forgetting someone, since I'm sure there's some paper from 1975 that I haven't read (or whenever). 

I think I've brought up Harden McConnell before, not only for his contributions to NMR but also for his work in EPR and its applications to understanding biomembranes, including early work in spin labels.  Of course, I'm sure people will gripe about the fact that his more recent work was biochemistry and immunology-oriented.  Heh.

I wouldn't object to Ad Bax being included, but I can see where it might be hard to convey the Nobel-quality novelty after Wuthrich's prize 10 years ago or so now.   I know he's tremendously well-cited - heck, I've cited him! - and always places very highly on those h-index rankings, but I could see this being an uphill battle to some extent.

So that's that, at least for now, I'd say.




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Lit Post.

A few interesting-looking papers that I have stumbled across lately:

- Cellular solid-state nuclear magnetic resonance spectroscopy. Clearly, analyzing membrane proteins that are still in the native intact cell membrane is what we'd all like to see.

- Red wine, iron telluride, and superconductivity. Sadly, I do not think that soaking any of my biological samples in a preferred alcoholic beverage will facilitate noteworthy results. Although perhaps I should try, just in case.....

- The Next Big(ger) Thing. Actually not a research article, it's a news item on mesoscale science.

As I've had to suppress a fair amount of sarcasm while writing this post, I will end things here. Read more!

Random Interfacial Griping

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! Read more!

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. Read more!

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. Read more!

You spin me right round baby.....

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.... Read more!

Some random thoughts....

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....

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Clusters of Spins (AKA the Promised NMR Lit Post)

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!
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Assorted Awesomeness.

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.....

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Summations

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. Read more!

Perspectives on Interfaces

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. Read more!