The Magic Angle

Invasion of the Brain Snatchers

2005-02-04T15:05:00.000-05:00

Next week, my fair institution of higher learning will be visited by the world renowned mathematician and philosopher, Dr. William Dembski.

*mops up sarcasm that was dripping and is now flooding the floor*

See here for more information. It should of course be noted that it is not a department that is sponsoring him, but rather a group of various religious organizations (and the Philosophy Club). Rest assured, I am sure the media hacks who back ID will be spouting off, "But Dr. William Dembski gave a talk at Columbia University in New York, and that's one of them fancy Ivy League schools, blah blah, blah" as soon as the curtain calls on the session.

I'm contemplating attending the entire debacle (because I'm sure it will be one), but I wonder if I want to kill off my brain cells in that regard. It would be far more enjoyable to down a six pack to be honest.

It would be nice....

2005-01-05T16:25:00.000-05:00

In Praise of the Mad Scientist by Samantha Hunt (Village Voice)

There's a certain romance that I see in Hunt's essay - the idea of being able to pursue one's scientific interests without needing to slave over a lengthy grant proposal to the NSF or NIH; without requiring one to be independently wealthy; going after that which you think is important regardless of whether or not you can make a profit off of it. Sure, it would be nice to have a fully loaded lab all to myself. I would imagine that many lives would be greatly simplified if they could pursue important goals without needing lots of personal capital, or perhaps spared the effort of writing up grant proposals for submission to a government bureaucracy.

There is a strange disconnect I noticed right away in the argument - is Hunt after inventors who will revolutionize society or scientists who will add to society's knowledge of the world around us? While I am again going to be obscure about what it is that I do, my work has interesting relationships to issues in both environmental science and drug discovery. I personally don't have much interest in either field - I am far more intrigued by the fundamental physics and chemistry that underlies the problem. Scientists are not necessarily out to invent anything - it may come up that in the course of their work that something needs to be done that has never been done before, and therefore they invent. It also occurs that things come to be by luck - as I recall, Viagra was originally studied in relation to cardiovascular disease, not erectile dysfunction.

I may or may not come back to this in the days ahead (why, yes, I've left this blog in cobwebs for a few months now), but what really strikes me is that Hunt seems to have a very strong opinion of not only how science should be done but also what science should be done. It's actually kind of amusing when she's spent some effort in extolling the virtues of originality and independence.

P.S. - In response to a comment left about two months ago, I do not worship Darwin. That was a bad attempt at humor with the "honest-to-Darwin" comment.

P.P.S. - For everyone who's done a Google or Yahoo search linking to this page looking for information on solid state and magic angle spinning (MAS) NMR, Rob Schurko's solid state NMR pagehas a nice set of introductory notes. You may also want to check out this webpage by Klaus Eichele at the Universitat Tuebingen. For quadrupolar NMR, I'd suggest Pascal Man's very engrossing webpage .

Adventures in Babysitting (Or, Watch Michael Try to Grow Protein Crystals)

2004-09-03T23:35:00.000-04:00

Most of us probably have somewhat fond memories of growing simple salt crystals when younger, either in school or out of the classroom. It's fun, it's easy, and it's actually kind of pretty after a fashion. For those of us who went on to take more science classes at the university level, you may have fond (OK, probably not-so-fond) memories of trying to crystallize a compound you synthesized in a laboratory course. Crystallization is an important step for many investigations of chemical structure and function for at least two reasons - it is generally a sign that what you have is reasonably pure (although not necessarily what you're looking for), and is also suitable for analytical methods where a solid is required. This, of course, is what is needed for x-ray crystallography. This method depends upon the fact that x-rays will diffract in a comprehensible way from a regular, periodic arrangement of atoms and/or molecules in a crystal. The sorts of salts we used to play with as children and organic compounds that we may have played with as young adults are not that typically large - perhaps equivalent to a few hundred daltons (where one dalton is equivalent to the mass of a single hydrogen). They are typically pretty anhydrous (one can heat them up, driving away any stray water molecules, without doing much damage - although heating it up too much will melt the salt, and that's a different story) and, for the most part, quick to prepare.

Proteins, however, are a different story. And here things get interesting. Protein crystals can have lots of associated waters. It's been found that some protein crystals are, in fact, mostly water that's bound to the protein in one way or another. Some protein crystals can take up to a month to grow properly. As some of you are probably saying to yourselves right now, proteins are generally far larger than a few hundred daltons - tens to hundreds of thousands of daltons, particularly when one is talking about large respiratory complexes and other such monsters of biochemistry. Some proteins are notoriously difficult to crystallize - membrane proteins, which reside in a greasy envelope of lipids or are associated to one via a greasy anchor at one end of the protein, are one of the continuing challenges for protein crystallographers. Instead of trying to line up in three dimensions small, cute, cuddly atoms and molecules, one is attempting to line up large, bulky, recalcitrant proteins into crystals on the scale of a few hundred micrometers in each direction ideally.

As to how to do this....there is a curious blend of physical chemistry, one's intuition, and black magic. (OK, I'm kidding about the black magic. But if you happen to have any goats available for moonlight sacrifices.....) The idea is that one needs to precipitate the protein out of solution slowly and in an orderly fashion. Salts are often added to the buffer so as to take up water molecules in a solvation shell which would otherwise be surrounding the protein, as well as to affect the surface residues of the protein. Organic molecules such as polyethylene glycol are also added, which draw away water from the proteins in a similar fashion. Other chemicals may be added as needed - for example, dithiothreitol or 2-mercaptoethanol to prevent formation of disulfide bonds, or perhaps if one is working with an enzyme, the enzyme's preferred substrate so as to see where the substrate binds relative to the active site. One can also play with temperature, the amount of protein, and the geometry of the crystallization process (do you have your protein hanging above a reservoir of these compounds, sitting down surrounded by these compounds, or being injected into a microfluidics device?).

Even after all of this, you may still not end up with a protein crystal. Or, you may not end up with a large enough protein crystal - one might have a batch of so-called "microcrystals," so-called since they're best measured not in hundreds of micrometers but far fewer micrometers. What might look like messy precipitate might be nanocrystals, but it could just be unordered protein precipitate after all. My lab's specialty - solid state NMR - is able to deal with such cases, where crystals don't form large enough crystals for x-ray diffraction studies or fails to form crystals at all.

However, to my astonishment, I managed to grow a very few crystals back in the spring of x-ray diffraction size and so I've been following up on that as well as more expected pursuits. You see, there are some interesting questions that arise from thinking about the currently available crystal structures for the system I work with when considering temperature effects and under what conditions one finds my protein (to wit, crystals grown in a refrigerator don't necessarily correlate to an organism which does not grow at such temperatures). So, I figured, why not? However, as was once related to me by a protein crystallographer, "Sometimes it's just a matter of setting enough trays and wells before you start getting crystals." So, as to what I actually do when attempting to grow crystals? Let me show you.

First, I take one (or two, or three, or however many I need) crystallization tray(s) (we use the well known standard from Hampton Research, their VDX Plate), and apply a small bit of vacuum grease to each and every circular well ring. This makes sure that when I apply the cover slide (which will come later), I have a reliable seal and will not have contamination from the outside. This is done by gently heating some vacuum grease until it melts and dipping a small glass flask upside down into the grease, and then carefully applying the greased rim of the flask to the well ring. When I first started doing this, it would take almost an hour to do this for an entire tray (24 wells) - I was very concerned about dripping grease into the wells and, in general, making a mess of the entire process. Now it takes ten minutes on a good day. I'll usually make sure that my precipitation/crystallization solutions are prepared and and ready to be added, either directly or after a small bit of mixing if I'm using a high concentration stock solution of polyethylene glycol. The protein solution is in its appropriate buffer and at a high enough concentration, and has been mixed with the substrate if I'm doing such a trial. Now, I use pretty high concentrations of protein at times (over 200 milligrams per milliliter on occasion), so I often have to use centrifugal concentration methods - like these from Fisher - to get my protein to the desired protein concentration. If I'm cocrystallizing my protein with its substrate, I'll typically do this before concentrating - this way I can monitor the binding with UV/Vis spectra and then be sure that my substrate is in there before spending all that time concentrating my protein to a thin layer of protein sludge (well, not really sludge per se, just rather viscous solution). I then take some small plastic microscope cover slips, add a small bit of protein sludge, add a small bit of precipitation/crystallization solution, and then place the slide over a filled well. This is one of those places beginners can botch things up - if you take too long in flipping the slide over, it can run and you get something smeared out over more area than a neat small drop's worth. You then gently press down and end up with a sealed well, protein hanging above your solution. You do this 12, 24, 48, 72, ..... times and you wait. You need to check daily under a microscope (just a regular light microscope, nothing fancy is really needed for just monitoring growth or lack thereof of your protein crystals, and we have a digital camera to take pictures of anything interesting looking) and wait. Did I mention the waiting? This is the babysitting, and what usually takes the longest. You can make up your solutions, prepare your tray with the grease seals, concentrate your protein, and mix everything up in a day - the waiting is what really takes the time. One day of work - a week or longer (far, far longer in some cases) of watching and waiting.

I've had middling to fair success thus far - one of the proteins I worked with grew crystals pretty easily (then again, the conditions were already known) and the other one, my main interest, is coming along reasonably well. I seem to be able to get microcrystalline material without too much difficulty, and am playing around with conditions to see if I can get larger crystals. I'm also going to try such interesting techniques as microseeding - taking a small microcrystal and dropping it into a freshly set well to act as a growth initiator for a larger crystal - and magnetic field crystallization - where one basically sets up a magnetic field surrounding your trial in order to grow larger crystals. (As for an actual explanation of how magnetic fields affect protein crystallization, your guess is as good as mine - there are lots of ideas, but nothing firm or even reliable. One can wave one's hands about, but in the end.....remember the comment about black magic before?) Something that I need to be careful about is how conditions for crystal growth may not be optimal for doing NMR - too much salt in microcrystals or precipitate can cause unwanted heated during an NMR experiment, considering that we're pumping in high power radiofrequency waves; too much precipitant (polyethylene glycol) might cause unwanted signals in the resulting spectra of the protein.

Ultimately, though, it's well worth the time - if I can devise better ways to precipitate out protein (forming large, x-ray grade crystals or not), the easier my adventures in preparing future NMR samples should be, as well as the tantalizing idea of having comparative crystallographic data to work with when trying to synthesize what is already known with my original research.

Sites To Look At:

Crystallography 101 - An introduction to x-ray crystallography at Lawrence Livermore.

Crystals for Fun and Education - Hampton Research has some fun and cool crystallization projects for kids (and those who are still kids at heart).

2004-07-04T16:42:00.000-04:00

I like bacteria.

This may not be totally surprising - after all, bacteria are exceptionally useful production factories for chemists and biophysicists interested in macromolecular structure and function and enzymology. They are a cheap, effective method of overexpressing proteins, nucleic acids, and other molecules of current interest. They are also of course targets in and of themselves, being the reason for a number of diseases and medical maladies.

But no, you see, I really like bacteria. I think they're fascinating in and of themselves, without appeals to medical relevance or to biotechnological advantages. I used to work with cyanobacteria and green sulfur bacteria, both of which are photosynthetic organisms. I could make up a batch of solution containing not much more than inorganic salts and a few organic acids, inoculate with a colony or frozen culture, shine some light on them while in a warm water bath, and I'd end up with a dark green broth of dense photosynthetic bacteria. Out of a sulfur-laden bottle of smelly media I would grow some of the most fascinating bacteria out there. Did you know green sulfur bacteria are able to grow meters - up to ~70 meters - under the surface of water, where the transmitted light is only a slight percentage of that available at the surface? Their light harvesting apparatus are contained in what are known as chlorosomes - a monolayer of lipids and proteins - where long rods of aggregated chlorophyll are able to efficiently harvest light. The vast diversity of metabolism amongst bacteria is also endlessly fascinating and could keep a person busy for a lifetime. Modified glycolytic pathways where adenosine diphosphate (ADP) - not adenosine triphosphate (ATP) - serves as a phosphate donor, metalloproteins that utilize such "unconventional" metals like tungsten and vanadium....the list goes on. I cannot describe what a thrill it was to finally start learning these sorts of things when I reached university - while my experiences in high school biology were overwhelmingly positive, I left with the impression that bacteria weren't all that interesting. Then when you find out that there is spatial organization in bacteria - after having been inundated with "eukaryotes have internal organization and organelles, prokaryotes have neither of them" - you suddenly feel like the wool has been lifted from your eyes. When I first began to read about Carl Woese and his work in classifying microorganisms - and his proposal of the Archaea - it was wonderful to see how the main division that I had always been taught (prokaryotes and eukaryotes) was being rethought.

I may yet end up an honest-to-Darwin biologist after all.

2004-06-28T00:15:00.000-04:00

I don't normally speak about politics here, mainly since I really don't think anyone comes here for my politics (admit it, you come here for the occasional bit of science, dark grad student humor, and waiting for me to make some bad science jokes), but I figure I have to say this much, at least.

I went to see Fahrenheit 9/11 Friday evening with a few of my friends. Overall, I thought the movie could have been a bit more tightly edited and Moore could have strengthened the connections between the points he makes in the movie. However, if it takes a film to get people talking about public policy and the absolutely cracked out excesses of the Bush Administration and the direction of the U.S., then it is well worth the controversy and ensuing drama that has arisen. As I mentioned to a few people elsewhere, I don't think this movie is best served by praising it or attacking it, because everyone else is doing that. If it starts to inspire more honest and forthright discussion and action about what direction this country has been going and how to change it for the better, that's perhaps the best response ever.

I also saw Michael Moore heading out of the theater complex after one showing as I was waiting in line waiting to go into another theater for my showing. I would have ran after him and asked him something marginally intelligent, but I figured I should at least see his movie. Besides, the theater was packed, a good seat in NYC is hard to find at times. Heh.

2004-06-27T23:45:00.000-04:00

In the June 30th issue of the Journal of the American Chemical Society, there's a paper by a MIT research group on an application of two-dimensional (2-D) infrared (IR) spectroscopy to protein structure and dynamics, namely antiparallel (AP) beta-sheets (see here for a convenient visualization of beta sheets). The potential usefulness of this work may or may not be immediately clear, if it's not, I urge you to keep reading I think it's quite fascinating.

For those who never had the pleasure of taking organic chemistry or certain other classes (or for those who have, you've since pushed some of them into your subconscious), infrared spectroscopy measures vibrational transitions in molecules. In analytical chemistry, particularly the type that is done in organic chemistry courses, this yields information regarding the presence or absence of various functional groups (e.g. a carbonyl or amide moiety), which typically relates to the bond strength and the masses of the atoms involved. IR data is normally presented in units of wavenumbers (which equal 1/wavelength of radiation in cm) and in terms of transmittance (so absorption of light by a certain vibrational transition shows up as a low amount of transmittance at that specific wavenumber). This sort of analysis is not particularly quantitative in nature IR spectroscopy's ability to serve as a general probe for functional groups means there are tradeoffs with being able to extract useful and local structural and dynamic information. There is also the aspect that repetitive motifs in a molecule's structure can add up to give a broadened, featureless resonance. For an example, the fundamental backbone structure of a protein repeats regularly (see this Birkbeck College page for instance), so any resonances that originate from the backbone would likely overlap. One transition in proteins, the so-called amide I transition, is quite overlapped in IR spectra, being primarily the stretching of the C=O bond coupled with some peptide bond (CN) stretching and CCN bending.

This amide I transition is in fact the window through which the group investigated secondary structure motifs in their 2-D IR experiments. If you look at the previously linked structure of antiparallel beta sheets, you'll notice that the short range interactions (hydrogen bonding) that give the beta sheet the appearance of being a sheet and not just a collection of protein strands are from the N-H of one strand to the C=O of another strand just the components which contribute to the amide I transition. Hydrogen bonding is a short-range interaction, sensitive to conformation and dynamics of the hydrogen donor and acceptor this makes it quite useful for probing protein dynamics (such as folding or conformational changes), as well as interactions of the protein with other proteins, nucleic acids, and small molecules. When one considers that infrared spectroscopy has an instrinsic timescale in the picosecond range, measuring the fast kinetic processes of protein folding becomes possible. The experimental setup is based upon a four wave mixing experiment three pulses of light are directed into the sample, and based upon the optical properties of the sample (as well as the parameters of the light pulses), a fourth signal is generated and then emitted from the sample which contains the information of interest. The samples used included a model polypeptide (poly-L-lysine, with a molecular weight range from 70,000 daltons to 150,000 daltons) and the following proteins concanavalin A (Con A), ribonuclease A, lysozyme, and myoglobin. Con A is exclusively composed of antiparallel beta sheets excluding any unstructured loops and strands, and the amount of AP beta-sheets decreases until you reach the exclusively helical myoglobin, with the same qualifer. The two-dimensional descriptive is seen in how the data from these experiments is presented and analyzed with the initially excited transition plotted against the resultant signal. This information coupled with the the model derived in the paper for 2-D IR spectroscopy of beta-sheets can provide insight into conformational dynamics, given frequencies, lineshapes, splitting patterns and crosspeaks in the two-dimensional plots. I was thinking of explaining more about the physical basis of this work, but I didn't want to overshadow the biological relevance in this post. Perhaps I will keep it in mind for future posts.

To wrap it up, the MIT group was able to identify patterns and data with 2D spectroscopy not extracted if at all observed in one dimensional IR spectroscopy with a fairly reasonable model underlying it all. Obviously, much more work must be done before its full biological utility is realized, especially in extending this idea to more complicated (e.g., mixed secondary structure) proteins, as well as to proteins interacting with other biopolymers and relevant molecules.

(For those interested in looking at visualizing biological macromolecules for themselves after reading this, I highly recommend Swiss PDB Viewer available at the ExPASy Proteomics Server - it's free and available for Mac, Windows, Linux, and SGI. I personally have used this for a while now, including for my fabled second year defense earlier this year as well as for giving talks and for other sundry tasks. You can make very nice pictures using POV-Ray, a ray-tracing program which is very easy to use with Swiss PDB Viewer. While the PDB is available on my side bar as a useful link, I'll pass along the PDB IDs for the proteins used in this study just so you can see for yourself - Concanavalin A :1NLS; ribonuclease A: 1FS3; lysozyme: 132L; and myoglobin: 1WLA.)

2004-06-16T12:41:00.000-04:00

When reading a story or article about some aspect of modern biology, my interest always piques when there is mention of how recent structural studies of the constituent parts has enabled new insights into the function of said system. The main reason for this interest is twofold - one, I rather like hearing that structural biology does have something to contribute to interesting problems in biology and medicine; and two, I always wonder just how reliable the structural work actually is, and not in the sense of the resolution of the structure.

Kleywegt and Jones (1995;Structure, 3: 535-540) describe some of their always interesting work in trying to improve validation of macromolecular structures, namely, intentionally tracing a protein backwards and attempting to fit it to the obtained electron density to see what differences arise. The intentionally traced backwards model of the protein had an R-factor (to slightly oversimplify, a statistical measure to compare the experimentally observed diffraction pattern to the expected diffraction pattern given the determined structure) of 0.214 (21.4%), while the model that had been obtained with a more conservative protocol (without being traced backwards) had an R-factor of 0.251 (25.1%). As the authors mention in this paper, the threshold tossed about in the crystallography community for an R-factor whereupon structures could be problematic floated around 25% and then upwards. Thankfully, the authors showed that other measures (particularly an updated R-factor, the so-called R(free)-factor where one randomly excludes some of the reflections from the refinement, introduced by Axel Brunger) clarified which was the incorrect structure and protein trace. The rise in the usage of NMR spectroscopy in structural biology sets off an interesting train of thought - structures derived by NMR are typically presented as an ensemble of individual structures, with an RMSD value determined for ensemble of structures. The RMSD is typically for the protein backbone at the very least. The idea is that a low RMSD suggests a higher quality structure - which really can't be justified in my opinion. The wonderful thing about NMR is that you can probe protein dynamics (as well as the dynamics of whatever the protein is interacting with). What follows next is perhaps obvious to the astute visitor - if the RMSD is not within a few angstroms (or is glaringly enormous along certain stretches of the protein), there is likely something interesting going on there with protein motion, solvation, protein-protein interactions, protein-ligand interactions, or some other biologically relevant process. And that is what should really get you excited, not that you can say you have an RMSD of 0.87 angstroms.

One of the other things is that you needn't abandon your basic chemical/biological intuition. If there look to be ridiculous conformations in the determined structure, they may very well be mistakes or artifacts. If the Ramachandran plot (where one plots the backbone torsion angles phi and psi against one another, see here at Birkbeck College for a more detailed explanation) has points where you really shouldn't expect to see them, there may be something wrong. There is an enormous database (the Cambridge Structural Database) where an enormous number of high resolution small molecule structures can be found - comparing the statistical trends in structures from there to what you see in proteins can be an enlightening exercise. Given that a number of the small molecule structures were determined by neutron diffraction methods, this can also be a way to critically examine hydrogen bonding as well. And in an even more basic fashion, if it simply doesn't make sense, then nothing compels you to take that structure as a gold standard. If the positioning of the electron transfer cofactors seems odd given what you know about the physiological rates, then it may very well be an artifact of crystallization. (Go read a couple of structure papers, with an emphasis on the materials and methods section. See how many crystallize proteins at non-physiological - even non-ambient laboratory - temperatures.) Does it look like there may be some electrostatic repulsion between the two surfaces of the interacting macromolecules? It may be that there needs to be a bound water to form some hydrogen bonds and minimize that repulsion.

The main reason I find this topic important is because researchers outside the structural biology community utilize this sort of information and expect that they are looking at something which reflects what the macromolecule really looks like, for lack of a better phrase. There needs to be care taken that if you're working with a protein or other macromolecule that does something interesting, you should be able to assay for that activity so you're not spending all that effort on examining an inactive assembly of amino acids (or what have you). It's crucial that we not lose sight of the fact that these beautiful structures should be able to be connected to the system's physical properties and biological structure.

2004-05-26T22:56:00.000-04:00

So, it's been a while since I last bothered to write anything in this blog of mine.

Things That Have Happened Since I Last Posted: Passed sixth cumulative exam (fulfilling that departmental requirement), endured my (significantly less agonizing than feared) second year oral defense, finally packed a solid state NMR sample more or less on my own, acquired and processed solid state NMR data for an actual biological sample with a good helping of assistance, synthesized some marginally novel substrates for my project, downed six rum and cokes in three hours during the departmental awards dinner....well, that last one is really only notable since the beverages were free. It also looks like I will pick up another side project or two to go along with my primary research project and current side project. The putative side project looks to be a bit more physically oriented than my current projects, which is of course perfectly fine, I was kind of hoping I'd get to do some of it. (Yes, I am being deliberately cagey about what it is I'm working on - but once papers come out in the indefinite and not necessarily near future, I'll share.)

So, looking at a couple of referrals to this page, I see Google searches for things related to magic angle spinning (MAS) NMR, which is wholly not surprising. I also saw "vodka nmr spectrum" which is kind of interesting. If the person who was looking for that still reads my blog, I will be more than happy to run your vodka samples - if any - as long as you are willing to send me an entire bottle of each to study. Please contact me at my email address and I will let you know where you can ship your samples. There was also "photosynthesis for idiots" - which, for the record, is carbon dioxide + water + sunlight --> carbohydrates + dioxygen. That is about as user friendly as photosynthesis gets. There have also been a few searches for the famed protein synthesis video at Stanford, narrated by Paul Berg. A classic bit of cinema, I tell you, and I hope you enjoyed it as much as I did.

I think that will be all for now, and if things go well, my next post may actually have something of scientific interest.

2004-04-14T21:05:00.000-04:00

I went to a really interesting seminar today by Jonathan Wilker today on the chemistry and biochemistry of marine adhesives found in mussels and barnacles. The "glue" that mussels use to attach themselves to surfaces and other mussels is apparently a heavily cross-linked protein matrix that is initiated by the addition of iron that generates not only cross linking but also an organic radical that may be how the "glue" attaches to a surface, including such "slippery" surfaces as Teflon (polytetraflouroethylene). The amino acid that is involved in the cross-linking is 3,4-dihydroxyphenylalanine - aka DOPA, the amino acid derivative that has been used for treating Parkinson's disease. The sidechain of DOPA is rather similar to the catechol-like binding site that is seen in enterobactin, a molecule used in iron transport and regulation in bacteria. Now, while this work has lots of obvious applications in medicine (e.g. surgical adhesives that can work while wet, are rather strong, and are far less toxic than harsher adhesives) as well as industry (waterproof adhesives would be useful, not to mention ways to better protect ships and maritime structures against barnacles without leaching heavy metals into the water via the paint), what really strikes me as fascinating is the same basic chemistry - metal binding to catechol - being used for both metal regulation and transport (in the case of enterobactin) and for synthesis of a biomaterial (the mussel protein-based adhesive). I've heard some vaguely similar things for Parkinson's - DOPA being used to replace the endogenous pool of DOPA due to the pool being chewed up by the presence of metals and therefore not able to carry out its function - but that's about all I can remember. While I am sure I could find other cases of similar chemistry being carried out in different contexts in different organisms, metal binding to catechol moieties being used across bacteria to invertebrates to vertebrates for such a diversity of purposes is really astonishing when you think about it. An experiment which any one of us could run - titrate iron into a solution of catechol or vice versa and measure the binding - is being used every moment around us in completely different contexts and different species. This is part of the reason why I decided to become a scientist - you can see not only how things are connected, but how by using the same basic process widely varying ends are achieved, and in both cases, it amazes the mind.

Anyway, I'm going to think about this in a sense of youthful wonder while studying for the cumulative exam tomorrow on Prof. Yuste's work (mentioned in a previous post).

2004-04-10T09:13:00.000-04:00

Enzymatic function dependent on membrane structure. I thought this was really fascinating, not least because lipids tended to be overlooked in my biochemistry class and I always thought they were far more interesting molecules than something to only learn about in relation to metabolism. We of course covered the standard canard about lipids and sundry chemical derivatives and relations, but most of the class focused on things like protein structure and function, as well as lots of metabolism in the second semester. (Of course, my real complaint about second semester biochem - it was a smallish honors course with ~ 20 people - was that the professor didn't teach a single bit of photosynthesis even though the regular sections covered it. It must have been the fact that most people in the class were premeds and he decided against it. Given that I worked in a photosynthesis lab for over two years, you can imagine where my interests lie.) What will be most interesting is to see how these scientists follow it up with in vivo studies. Lipids are known to play structural roles - for a photosynthesis example, the cyanobacterial photosystem I structure has a few lipids in there which apparently play a role in keeping the structure together. Given that there is so much interesting biology that takes place at the membrane or associated with it - witness the great deal of interest in lipid rafts - it will be extremely fascinating to keep an eye on this in the near and not-so-near future.

I am apparently a REBWAGSOH as well. Heed the warning at the linked page before watching the movie. If you are not also a REBWAGSOH, you will likely be very perturbed. And that's an understatement.

Must be off to help my friend move. There is free beer in it for me, so....gosh, I'm such a typical graduate student.

2004-04-08T20:43:00.000-04:00

In other news, I attended a seminar today by Rafael Yuste on two-photon imaging of dendritic spines and cortical microcircuits. The first part of his talk was about dendritic spines (small protrusions from the dendrites - the part of the neuron that accepts inputs from other cells - that are the actual site of the synapse). He explained how optical imaging coupled with some genetic engineering (introducing green fluorescent protein into the neuron) allows for studying neurons that are still living and functional. Previously, these spines were considered to be too small and too challenging for study, including too small for even a microelectrode. One of the more interesting things that was mentioned that is being done is measuring membrane potentials via spectroscopic methods. While it seems that the dendritic spine (which looks like an almost pinched off vesicle) has some biochemistry that occurs when stimulated, this chemistry doesn't reach its way to the dendrite due to the thin bottleneck (diffusion limited) that connects the synaptic head to the dendrite. However, does the electrical signal propagate? The other aspect was tracing cortical microcircults in tissue. Basically, you stimulate a neuron, and see what other neurons are stimulated. And so on and so forth. From what was presented, there are basic underlying circuits in the mammalian tissues studied thus far (and as my memory lets me recall). Hmm, why would there be this similarity? I wonder. It was really quite fascinating, and it's very interesting to see biology, chemistry, physics, and engineering come together in a single problem.

Time for me to leave the lab at last, it seems. Today was only a half day. Mind you, the grad student definition of a "half day" is 12 hours when it comes to work.

2004-04-08T18:42:00.000-04:00

While the IDiocy in World magazine has already been ably tackled by the fine folks over at The Panda's Thumb, I think we should take a far more serious perspective if the rather unimaginative predictions come to pass....

********************

2009

From World Magazine: By 2009, however, when Darwin's followers had hoped to stage a triumphal celebration of their leader's 200th birthday, millions of people were laughing at the emperor with no clothes.

In other news: Scientists worldwide wonder what is meant by "leader." William Dembski is refuted by people who can tell mathematics from utter crap for the 87th time that year. A slight increase of recently minted American-educated Ph.Ds take postdoctoral positions in Europe and Asia compared to the past five years.

2010

From World Magazine: Although a few biologists had been arguing against DNA reductionism for decades, biologists guided by intelligent-design theory in 2010 discovered the true nature of the information that guides embryo development.

In other news: Their manuscript is rejected by every respectable journal in the world for being a load of warm cat vomit. Best attempts to educate IDiots to how science actually works fails for 42nd time that week. Philosophers of science would rather suck said warm cat vomit through a straw than bash their head against a wall (that is, try to explain how science works to the ID advocates) given the utter ignorance of IDiots regarding what reductionism is and is not.

2010-2015

From World Magazine: All three of these developments-teaching the controversy, educating people about the lack of evidence for evolutionary theory, and using intelligent-design theory to make progress in biomedical research-were bitterly resisted by Darwinists in the first decade of this century. Defenders of the Darwinian faith engaged in a vicious campaign of character assassination against their critics in the scientific community. Meanwhile, their allies in the news media conducted a massive disinformation campaign, aimed primarily at convincing the public that all critics of Darwinism were religious zealots.

In other news: While the IDiots and allies managed to con the American people and government into thinking they were making actual progress via ID "research", not to mention making up false conspiracies regarding the people who dared expose them for the frauds they were, scientific progress in Europe, Asia, and other parts of the world progresses nicely give that scientists don't have to spend disturbingly high amounts of time fighting this nonsense.

2015

From World Magazine: By 2015 it was well on its way to joining its intellectual cousins, Marxism and Freudianism, in the dustbin of discarded ideologies.

In other news: Nearly all American-educated Ph.Ds. take postdoctoral positions overseas due to the atrocious environment in the U.S. for conducting research. A "reverse brain drain" is in full effect, reinvigorating the global economies due to emerging discoveries and technologies that are almost a daily fact of life. Pharmaceutical companies move a vast amount of R & D overseas due to the fact that modern biomedical research does in fact have roots in evolutionary biology. The U.S. economy suffers.

2020

From World Magazine: By 2020, Darwinism was effectively dead.

In other news: In the U.S. only. The U.S. government finally had in place restrictions for funding graduate students and strict laws intended to keep U.S.-incorporated pharmaceutical firms from moving so much overseas. Firms reincorporated overseas, wherein over the past decade and change had shifted so much economic clout that the U.S. market was not utterly necessary. While the rest of the world benefited from the advances that had been made, the IDiots finally saw the error of their ways. The thinly shielded wallet overtook, at last, the thick-walled brain. They begged and pleaded. Prof. Emeritus P.Z. Myers of the Univ. of Minnesota took the surrender terms on Dec. 1, 2020. The IDiots were exiled to the Galapagos Islands, where they'd have to see Darwin's finches for the remainder of their natural lives.

Also, Dr. Michael Harris, Professor of Biochemistry & Biophysics at Oxford, is awarded the 2020 Nobel Prizes for Physics, Chemistry, and Physiology or Medicine. He attributes his enormous success to the positive and welcoming environment he found overseas from his native U.S. and comments that "I didn't have to worry about saying that I based my work on evolution."

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The above was parody, for those of you who may be humor-impaired. And an excuse to call myself a Nobel laureate. :)

ETA: The occasional stream of consciousness made its way in there, I hopefully cleared that up.

2004-04-04T13:59:00.000-04:00

So, in a postscript to my post from March 24th, my letter to the editor was in fact published. If you are so inclined you can take a look here. As a friend of mine mentioned to me the other week (considering there was a terrible op/ed earlier this term by an Intelligent Design advocate), "What are they putting in the water up there?"

My second year report has been written and passed along for editing and feedback. Writing it was very interesting, if for no other reason that I had to condense such a quantity of information into such a cramped format (five pages, double spaced, reasonable font size, including all figures/tables/graphs/equations). This post by Paul Myers came back to my mind while I was writing up my report. There's another hallmark of technical writing that I ended up employing regularly - why bother blathering on about what you did when you can save a paragraph or two and just cite a reference? I remember when I was a bit less experienced in my career trajectory where I'd feel compelled to explain at agonizing length in a lab report or such. Now, I feel little guilt to put in the superscript and the citation in the references. Because, rest assured, someone probably has already tried that trick to do X, Y, or Z in some obscure journal and all you need to do is find it. (Earliest reference for my second year report is 1967, four page article on how to synthesize some amino acid derivative. Explaining how to do the synthesis is probably a paragraph or two, which I did not have space for by any means.)

My blogging should be relatively more frequent from here on out. While I do have much planned for the upcoming week, it is not nearly as full as last week. The only remaining obstacles for the month include my group meeting presentation in a week and my second year defense for later this month, so it should be relatively manageable.

2004-03-28T12:11:00.000-05:00

I have my second year report due next Monday, so while it is more or less already written - distributed among sundry progress reports, PowerPoint presentations from group meetings, emails as well as other things - it needs to be put together and cleaned up. As such, any blogging until then will be sporadic and not very verbose. Well, more sporadic and even less verbose than my recent posts.

A paper on prion biochemistry recently struck my interest. The paper is freely available here, being from the open-access journal Public Library of Science-Biology. I would comment further, but must get to lab for a bit this afternoon. If I have time, I'll try to comment later on this today or tomorrow.

2004-03-24T21:19:00.000-05:00

For those of legal age in their jurisdiction, you might find the DNA cocktail to be a suitable experiment to carry out. (In the spirit of this post by Paul Myers.)

While I suspect that anyone who reads this blog has already visited there, I just want to offer my support and a plug for The Panda's Thumb. It's off to a great start and I will be watching it with great interest.

In less entertaining evolution-related news, I read this editorial in the local student newsrag the other day. I was going to mention it yesterday, but blogging was not in the cards. Read that second paragraph carefully. Darwin's work is derided as either 1.) a manifestly failed ideology, 2.) a specious belief system, or 3.) a trendy theory. Your choice. Yes, I'm writing a letter to the editor, this will be the second I've written this semester with regard to evolution. Disclaimer: I am in favor of including the U.S. Constitution and the Federalist Papers in the Core Curriculum here, so it's not the basic argument which has me riled up.

Time to start writing that letter among other things. (Second year report for one.)

2004-03-22T17:07:00.000-05:00

I meant to post a response to this post over at Pharyngula the other day here, but I slacked off on mostly everything yesterday. It was a day long overdue.

Back in the mid-1990s, I was studying the martial arts in my hometown, and one of the aspects that was brought into the curriculum after I earned my black belt was traditional Asian medicine. It was primarily shiatsu/acupressure, although the underlying principles (according to the Chinese tradition such as ch'i/qi, meridians, etc.) were introduced to us. It was related to what is known about anatomy (nervous system, circulatory system) so I never got the entire "I'm being inculcated into a cult" sensation. The inclusion of it in a martial arts curriculum is actually rather admirable, that one learns to heal (such as it may be) as well as to harm. Because, quite frankly, getting a massage after a hard hour or two of working out is a darn good thing. When I left for university, when I mentioned I had learned some of the methods, I got a lot more requests to give massages. Especially from the female half of the species. I wasn't complaining.

A bit later that decade when I was in university, I ended up working retail at a vitamin/health food store. The hours were good, the pay decent and the work was more than manageable. The most amusing thing was that my fellow co-workers were quite realistic and forthright (when asked) about the entire deal - our manager was a former restaurant chef and our asst. manager was an active competitive bicyclist who had done his undergrad work in biology/nutrition. I saw and spoke with everyone from competitive athletes (who usually have a pretty good idea of what they need to do to prepare themselves for their respective events) to people with various medical conditions who had spoken with their physician and worked out a reasonable, rational plan for taking care of themselves to people who didn't know anything but had heard something on the radio. The worst were those who had latched onto the first "wellness guru" that had come across their purview.

Given that I check the American Chemical Society Publications page on a more than regular basis, I keep seeing that The Journal of Natural Products has not yet gone out of print, so the idea that nature - through its profligate plants, bacteria, invertebrates, and the rest - produces interesting and useful chemicals that may be of medicinal benefit is certainly a reasonable one. As for chiropractic, well, there are chiropractors who are attempting to bring their field into respectability and not spend all their time into conning people with their chiropractic theory. Acupuncture always struck me as "we're going to do something very minimal and non-intrusive, and if you get better, we'll say that it works on a subtle level. If it doesn't work, well...."

In short, something works or it doesn't work, at least when looking at a large enough sample size. If it works, there is a very good rationale for it somewhere based on what we know about anatomy, physiology, biochemistry, neuroscience, etc. Sure, there are always the anecodotal tales of some treatment working - but in any adequately sized population you'll find some people who make progress regardless of treatment.

Back to the lab bench I go...

2004-03-20T14:18:00.000-05:00

So, upon the highly useful suggestion of Smijer, I have switched over to HaloScan for comments and trackbacks. It does look nicer than the last bit of code I was using, so it should probably stand like this for a while.

In slightly more interesting science oriented news, I came across this interesting looking paper from the most recent issue of Accounts of Chemical Research. The authors Eric Wise and Ivan Rayment (Univ. of Wisconsin, Department of Biochemistry) discuss the area of how new enzyme functions evolve from current enzyme functions through divergent evolution. They specify three basic routes for divergent enzyme evolution:

1.) the substrate specificity is conserved in the duplicated gene product,
2.) the reaction mechanism is conserved, or
3.) the active site architecture is conserved.

In the first case, the new protein from the duplicated gene still binds the original substrate but catalyzes a different reaction by way of a new mechanism. In the second, the reaction mechanism is conserved (more specifically, a particular mechanistic feature of the original enzyme) but the substrates which are involved may be quite different. With the third case, the arrangement of the enzyme's active site remains as a template for catalysis of a different reaction with a different substrate. While I am still in the process of reading the paper, Wise and Rayment work within the TIM barrel family (so-called because triose phosphate isomerase - aka TIM - was one of the earliest characterized enzymes both in terms of biochemistry and structure, which happens to have a barrel-like structure composed of 8 alpha helix/beta strand motifs) and pick three case studies for each route: the tryptophan biosynthetic pathway for route 1, the enolase superfamily for route 2, and OMPDC (orotidine monophosphate decarboxylase) superfamily for route 3.

Something to think about for the rest of the day while I'm in lab working, if nothing else. I may have more to say about this topic later on.

ETA - Silly ACS link is buggered up, the citation is Eric L. Wise and Ivan Rayment. (2004) "Understanding the Importance of Protein Structure to Nature's Routes for Divergent Evolution in TIM Barrel Enzymes." Acc. Chem. Res. 37 (3): 149-158.

2004-03-19T23:33:00.000-05:00

So, being an overworked and underpaid grad student I've added one of those quick and dirty comment scripts that are floating about for Blogspot, at least for the time being. So if anyone wants to talk at me, you can do it here. (I also figure this will avoid me accidentally deleting emails that someone may send to me since I don't have a comment feature.)

Given my recent success with SDS-PAGE, I am going to have to retract my previously muttered complaint that gel electrophoresis is black magic. On the other hand, in order to get it to work, I had to completely switch my buffer/gel system, not to mention significant alteration of my sample preparation. So it's gray magic....

For those wondering what my blog title means ("The Magic Angle"), a brief explanation. For those of you who have suffered through organic chemistry, you have likely been exposed to the delightful technique of nuclear magnetic resonance (NMR). Now, when most people think of NMR, it's a common analytical method done to elucidate the structure of small molecules in solution. For those with a slightly more masochistic bent, you can do NMR of macromolecules - including proteins, nucleic acids, and carbohydrates - in solution. For those with a particularly masochistic bent, you can do NMR of materials and macromolecules in the solid state. That happens to be one of the things I will be doing shortly (once I get some more wet biochemistry wrapped up). The thing is, in solution, you have your molecule/macromolecule of interest tumbling around and going through essentially all possible orientations to the main magnetic field. These fluctuations help to cancel out various interactions between atoms and give you the nice, sharp peaks that one sees in a solution state NMR spectrum. In the solid state, you do not have these sorts of fluctuations due to the rigidity (except in very rare cases) and these interactions make your spectral lines broad and overlapping. In order to help cancel out some of these interactions, one must spin the sample about the axis of the magnetic field at a so-called "magic angle" which is determined by the term which modulates the strength of these interactions. For those who may be interested, the term of interest is 3*cos^2(theta) - 1, where solving for theta gives you 54.74 degrees. I should probably see if I can get TeX support for this blog as well...hmm. In any case, expect far more verbose posts about spectroscopy and NMR in the future, it is one of my favorite topics.

For those who didn't take organic chemistry (or other suitable science class) as an undergraduate.....I promise I'll do a nice long post about NMR from start to finish one of these days. It really is an incredible field of research, from the fundamentals to the applications.

2004-03-18T21:41:00.000-05:00

While not nearly as numerous as the anti-evolution kooks out there, the anti-relativity kooks on Usenet and elsewhere are almost as bothersome. One of their most common lines of argument is the work of physicist Dayton Miller, who summarized his work in a review called "The Ether-Drift Experiment and the Determination of the Absolute Motion of the Earth" that was published in Reviews of Modern Physics, Volume 5 (July 1933) pp. 203-242. If they are feeling generous, they might grudingly admit that the reanalysis of Miller's data (published by R.S. Shankland, S.W. McCuskey, F.C. Leone, and G. Kuerti in Reviews of Modern Physics, Vol. 27, No. 2 (April 1955), pp. 167-178.) provides the "relativist conspiracy" a way out. They might claim that no one has bothered to do any more experiments so as to avoid being confronted with the stark reality of an ether. I would point them to a delightful paper which came out last year that can be found for free here. So yes, there are modern Michelson-Morley experiments being conducted with the very latest in technology confirming the special theory of relativity and no ether. Not only that, but these experiments are being used to provide experimental boundary conditions for the latest in particle physics and cosmology.

If you read the arXiv paper linked to above, you'll see the data was collected over the course of a year and change, with adjustments in data analysis made for cryogenic liquid fills and such. If you're so inclined, ask the etherites how Miller collected his data. There's a bit of difference in the experimental setup as you'd find out.

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As I was informed by my dear friend Kir, the legendary protein synthesis video narrated by Paul Berg (Nobel Laureate at Stanford University) can be found at ftp://biochem.stanford.edu/pub/pberg for your viewing pleasure. I should warn you, the files are VERY large so you may want to find a fast, robust connection before you download it. For those of you who have never seen it, it truly is a mixture of the 1960s and protein biochemistry. Heh. I first saw it in my introductory genetics class and have never forgotten it.

2004-03-17T23:04:00.000-05:00

I should warn anyone who reads this blog that it is a work in progress and that the format will slowly evolve over the next few days (weeks, months...). I am currently trying to figure out if I can host this blog on some webspace I already have or go find a different host. Along with this goes my eventual promise to install some sort of comment feature one of these days.

The topic this evening is something that I ended up discussing with some folks elsewhere when it first came out (August of last year, or thereabouts). It has to do with this paper. In this paper, the researchers decided to determine the structure and work out some of the biochemistry involved for a presumed methyltransferase (an enzyme which sticks a -CH3 group onto a molecule from a cobalt-containing cofactor) from Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. Now, the reason for choosing this enzyme to study is rather straightforward when you're able to decode the jargon - it allegedly catalyzes the last step in vitamin K (menaquinone) biosynthesis, which this bacterium is able to synthesize and we humans are not. Given that multi-drug resistant strains of M. tuberculosis are a serious public health concern, it's a pretty worthwhile endeavor to determine the structure so as to provide material for the structure-based drug design community. So, the brave biochemists figured out how to express the protein, purify it, do some biochemistry, and ultimately determined the structure to a pretty good level of resolution. However, when the researchers actually looked at the structure and examined the biochemical data, it didn't quite match up with its proposed function as a methyltransferase.

In the end, though, the interesting thing that comes out of this paper is the blunt reminder that genomics is a starting point for biological research, not the glorious end accomplishment that it may occasionally be made out to be in the media. Now, while I don't necessarily advocate doing structural biology on every single open reading frame from every organismal genome sequence (I'll leave that for the NIH structural genomics consortiums to tout), it looks like there is no substitute for doing honest biochemistry in trying to determine function.

This warms my dark little heart, given that I have unfond recollections of molecular genetics classes where a wanton disregard of enzymology and actual biochemistry always grated on me. I also have concerns about the high-throughput genome types insisting that high throughput methods are the way to go when it comes to other areas of biological research, but I think I'll hold off on them for another day.

2004-03-16T00:55:00.000-05:00

This is my first post. Hmmm. I would make it memorable, but let me bore you with some biographical detail.

Name: Michael Harris (FYI - Mike or Michael is perfectly acceptable, I have no vested interest in either. Given that three of my friends in high school were also named Michael, I have gotten used to not being called by my first name.)

Birth: I was born on a rainy Tuesday (16 May 1978) in Abington, Pennsylvania. You can do the math to figure out my age.

I currently live in New York City, where I am a second year graduate student in the department of chemistry at Columbia University. My research interests are roughly in the interface between physics, chemistry, and biology. My current research involves conformational dynamics of proteins, with the intent of doing spectroscopic and crystallographic studies as well as theoretical/computational studies. I am jointly advised by Prof. Ann McDermott and Prof. Richard Friesner, both of the department. My undergraduate degree is from Pennsylvania State University (University Park, PA), where I worked in the biochemistry department for Prof. John Golbeck in the area of photosynthesis.

Recent favorite books include the City Watch novels from Terry Pratchett's Discworld, Moral Politics by George Lakoff, At Swim, Two Boys by Jamie O'Neill, and Principles of Magnetic Resonance by C.P. Slichter. (It *is* good, I tell you!). Recent favorite albums.....*haven't bought an actual album in a while*....probably Liz Phair's eponymous release from last year. My favorite musical acts - besides the dirty mouthed goddess Liz - include U2, Phish, The Who, Sarah McLachlan, Dwight Yoakam, as well as others.

I really love New York City. Sure, being cloistered up here in Morningside Heights does have its downsides (oh why couldn't we be like NYU and right in the middle of the city?) but there are some hidden treasures away from the tourist-afflicted areas. I have what seems like a permanent affinity for vodka and rum. I am marginally obsessed with both Tolkien and the Harry Potter books.

There will be more, I swear. Presuming I keep up with this thing.