chemical biology

Not simple analogues, but ligands for biological switches

A while ago I blogged about a paper where a set of structures analogous to estrogen were made. Now a follow-up paper has appeared in Protein Engineering, Design and Selection. The aim was actually not to make simple analogues of estrogen, but to use the compounds to create specific receptor proteins.

Starting from the human estrogen receptor α, the authors employed directed evolution: they changed the residues in proximity of the ligand by mutagenesis, screened the resulting mutants, and selected the best receptor mutants for the next round. After the third round of directed evolution, they came up with an optimized mutant that bound to CV3320 with an EC50 of 55 nM, while the affinity to 17β-estradiol was reduced by a factor of 10 (4 nM).

CV3320 and estradiol

While the authors admit that the selectivity over 17β-estradiol could still be improved, it still is a nice piece of work that demonstrates the power of directed evolution. This way, a protein receptor for a substrate that does not occur in nature can be made. Such a receptor can be used to make biological switches.

By March 3, 2009 0 comments chemical biology

Death by Chocolate

For those of you who don’t know, Dr. Joe Vinson is iconic to the chemical community (believe it or not, even more so than Soderquist).  The American Chemical Society frequently hosts his seminars on some of life’s guilty little pleasures, coffee and chocolate.  I recently had the chance to sit in on his “Science of Chocolate” seminar.  And after and hour of lecturing about the history and chemical make up of chocolate, he took questions from the audience.  When I used to housesit for my aunt, I remember her telling me to be careful not to feed the dog chocolate because it could kill them.  I also recall coming across a warning by the ASPCA about the dangers of cocoa bean fertilizer. 

With my curiosity, I decided to ask the expert.  “Why is chocolate toxic to dogs?”  There was a bit of laughter behind me after I posed the question.  Vinson claimed that the theobromine was responsible.  “You would think that for a 100 pound dog it would be okay to feed them chocolate safely.  But you can’t.”  He then took the next question while I sat there completely unsatisfied with the response. 

So (like my daschund and miniature pinscher) I went digging.  Despite the name, theobromine has nothing to do with halogens.  Theobromine (or more IUPAC-y, 3,7-dimethylxanthine) is a structural derivative of caffeine.  In fact, several species of plants synthesize caffeine by converting xanthosine into theobromine.  The biosynthesis is concluded by N-methylation of theobromine by caffeine synthase (using S-adenosyl-L-methionine or SAM).  Recently, Crozier and co-workers mentioned that several groups have reported identical biosynthetic routes to caffeine (Coffea Arabica – coffee; Camellia sinensis – tea; Theobroma cacao – cacao; see Phytochemistry 2008, 69, 841-856).  At any rate, both theobromine and caffeine are stimulants (caffeine much more so). 

It appears that theobromine metabolism has only been moderately studied in the scientific community; most research has revolved around human metabolism.  Arnaud and Welsch (two research chemists at Nestlé in Switzerland) used 14C-labeled theobromine to determine the metabolic breakdown of the alkaloid in rats (J. Agric. Food Chem., 1979, 27, 524-527).  They determined that theobromine and methyl uracil were the major radioactive components in the urine (accounting for 85% of total radioactivity).  Other side products included 7-methylxanthine, 7-methyluric acid, 3-methyluric acid and several others.  Interestingly, they noted large similarities in the chemical composition of urine samples in both humans and rats that had been given theobromine.  However, there were quantitative differences between the two species.  Along with their paper, they actually printed pictures of 2D-TLC plates of urine samples of humans and rats.

By comparison, it appears that the canid (or canine) biochemistry for metabolizing theobromine is strangely unique relative to humans (and rats for that matter).  The consensus opinion appears to be that dogs are unable to metabolize and then excrete theobromine efficiently.  Upon ingestion of a theobromine-containing substance, dogs have been reported to excrete “small quantities of an unidentified but apparently unique metabolite” (Drug Metab. Disposition 1984, 12, 154-160).  It also appears that the toxicity associated with the inability to metabolize theobromine causes an increased concentration of intercellular free calcium, which is consistent with significant CNS stimulation and tachycardia (J. Agric. Food Chem., 2005, 53, 4069-4075).  Physiologically, theobromine ingestion in dogs is linked to epileptic seizures, heart attacks and death. 

Bottom line: stick to the peanut butter.  It’s much safer.

By November 24, 2008 12 comments chemical biology

Biology in 4D

Hello everyone. Mitch has asked me to contribute to this blog. This may be somewhat difficult as I am a biophysicist which leaves the topic of this blog as the one branch of science left out of the name of my field. Perhaps it would be better if I refer to myself as a biophysical chemist (or would that be physical biochemist? chemical biophysicist?)

Anyway, as the token biologist, I wanted to bring your attention to a commentary in Cell (doi:10.1016/j.cell.2008.06.013) describing the role of Pixar-style computer animations in the future of biology education. Although the article is an interesting read, what I really wanted to show you all is the author’s website (www.molecularmovies.org) which houses a collection of these animations. (Warning: visiting this site can be hazardous to your research productivity)

There are tons of other really great computer animations on the site (though some are not so great in terms of explaining things. Alas, the one video related to organic synthesis falls into the not-so-great category). My personal favorite is the movie on apoptosis (programmed cell death), which features one of my favorite protein complexes, the apoptosome (or as I like to call it, the seven-membered ring of death).

Now, as a biophysicist, I think that these videos are great because they illustrate some very important concepts in biology. The apoptosis video shows how many processes in biology resemble overly complex Rube-Goldberg Machines. Other videos on the site, especially those by Drew Barry, offer a glimpse into an important field of research: protein dynamics. Chemists are used to thinking of catalysts as fairly static entities. Sometimes a catalyst can be as simple as a surface that acts binds a reactant and primes it for subsequent reaction. In contrast, the catalysts in biology, enzymes and ribozymes, are rarely static. The video on DNA replication (video available at the WEHI website) shows the dynamic nature of these biological catalysts. The animation shows the E. coli replisome, a large multienzyme complex, as it copies DNA. The components of this complex have a number of different enzymatic activities that all need to be synchronized and coordinated in order for replication to proceed. Despite all these complicated interactions, the E. coli replisome proceeds at a rate of about 1000 nucleotides per second and with an error rate of about 1 per 109 nucleotides.

Of course, one has to remember that these videos are animations, not realistic simulations. While they are based on empirical results (e.g. crystal structures, biochemical assays, single molecule experiments), the animators do take some creative liberties with the videos. For example, I doubt anyone has observed buzzing and clicking sounds that accompany Brownian motion and enzyme catalysis in many of these videos. Indeed, the animators (with good reason) don’t show two concepts that are becoming increasingly important in understanding biological dynamics: the stochasticity of events in the cell (e.g. polymerases don’t move along at a constant rate) and the very crowded environment of the cell.

Most significant, however, is that while many of these videos depict the dynamics of various enzymes, not much is known about the actual motions of these enzymes and enzyme complexes. When biologists discuss conformational changes, these protein movements are often identified by looking at static “snap-shots” of an enzyme in two different stages of a reaction. Rarely are the kinetics of the transition measured directly, and the techniques that can directly observe conformational changes (e.g. Förster resonance energy transfer) give limited spatial information. Furthermore, the single molecule experiments that give arguably the best kinetic information about enzyme catalysis and protein motion often have limited temporal resolution (it’s hard to go below the millisecond time scale). Computational methods (e.g. molecular dynamics) can give detailed videos of molecular motion with both high spatial and temporal resolution, but modern computers can simulate only tens of nanoseconds, orders of magnitude below the timescale of most large protein motions. NMR spectroscopy has the advantage of being able to access a large range of time scales, but NMR measurements are limited to small systems and can access only dynamics of an enzyme in equilibrium. Being able to somehow synthesize and connect the information from timescales ranging from bond rotations and vibrations to conformational change and allostery is a tough task, but doing so may offer huge insights into the fundamental chemical and physical principles governing enzyme catalysis. Recent attempts to do so (Henzler-Wildman et al. 2007, doi:10.1038/nature06407, doi: 10.1038/nature06410) have been very promising, though there is still much work left for us biophysicists.

By July 23, 2008 3 comments chemical biology

Metabolite Identification – The TV Show

This was mentioned awhile ago by Bethany Halford in the Comments Section of the ever rancorous TheChemBlog. The video is produced as a chemists’ CSI TV episode. I realize most of you don’t have a background in metabolite identification[1], but it’s still a rather acute chemistry(analytical chemistry) spoof. Click screen shot of video below.

The video is an advertisement gimmick for Thermo Fischer Scientific, obviously enough Wink. A link to the original video can be viewed from this web page: http://www.biocompare.com/video/thermo/smi/

[1]: Not saying, I have a background in metabolite identification, but I do have more experience with it than you might expect for a nuclear chemist.

Note 1: Also covered by Closeted Chemistry
Note 2: Made an entertaining game room for the graduate student procrastinators in the audience: http://www.chemicalforums.com/index.php?board=62.0

Mitch

By May 13, 2007 0 comments chemical biology