A modest update.

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! 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!

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. Read more!

Return?

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? ;) Read more!

A Modest Response

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. 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....

Read more!