Oct 232008
ResearchBlogging.orgThe practical aim of the investigation of allostery is the manipulation of this property as a means to aid human health and industry. We already have in hand the sequences of numerous enzymes that carry out unique and useful chemical reactions, and recent advances suggest that we will in the near future be able to design man-made enzymes that efficiently carry out completely novel reactions. Making the fullest use of these abilities demands that we be able to regulate the enzymatic activity of interest. Fortunately, just as nature possesses a rich array of enzymatic activities, it also holds a number of binding proteins, so we don’t have to start from scratch. The trick is that there must be some way to communicate the binding event from one domain into the active site of the other domain. In last week’s edition of Science researchers from the University of Pennsylvania and the University of Texas Southwestern Medical Center claim to have achieved just that.

The core idea of their approach is disarmingly simple. Identify a protein that has a long-range conformational response to a binding event, and locate the distal region on its surface where this response gets read out. Then, on an enzyme of interest, find a region of the surface that has an energetic connection to the active site. Join the proteins at these surfaces and voila! Now you have a regulatory switch for your enzyme!

The reality, of course, is likely to be trickier. In order for efficient communication between sites to occur via these pathways, the structural dynamics of allostery at the points of attachment must be compatible. For nearly all proteins, the precise nature of the conformational reactions that drive communication are essentially unknown. Thermodynamic mutant cycle analysis cannot provide detailed mechanistic information, and structural dynamics experiments from NMR and other techniques can provide only general information about what occurs on these pathways. Only molecular dynamics simulations are likely to give us the information we need to tune the allosteric control precisely. Absent that, all you can do is just stick things together and hope for the best, which is essentially what Lee et al. did.

Of course, they didn’t go in totally blind. Lee et al. used the statistical coupling analysis (SCA) technique pioneered by Dr. Ranganathan to identify distal surface sites linked to the light sensitivity of a PAS domain and the enzymatic activity of a bacterial dihydrofolate reductase (DHFR). I’ve mentioned this technique before in connection with Ranganathan’s research on the PDZ domain. The SCA results indicated that a surface loop of DHFR was energetically linked to its active site. The analysis also indicated that a region encompassing the N- and C- termini of the LOV2 PAS domain was likely to be a readout for its detection of light. These results accorded with existing knowledge about these proteins. The result with the PAS domain was particularly convenient. Because the N- and C- termini are adjacent, it meant that the PAS domain could simply be inserted at a loop site. Also conveniently, the surface identified for DHFR was a loop.

Thus, Lee et al. inserted the PAS domain at two sites in DHFR. One was the loop identified by SCA, and the other was a control site equally distant from the active site but not predicted to be linked. Figure 3 of the paper shows the key result: insertion of the PAS domain at the SCA-identified site (A site), but not the control location (B site), resulted in a modest light-dependence of the hydride transfer rate for DHFR. All of the A site chimeras had substantially reduced DHFR activity, similar to the effect of a G121V mutation. Interestingly, shifting the insertion site by even a single residue completely abolished the light-dependence of the activity.

Granted, the light dependence is less than twofold at room temperature; this approach did not generate a genuine light-dependent on/off switch for DHFR. However, for the reasons I mentioned before, a perfect switch is hardly something that could have been expected. What this experiment does do is prove that this approach is workable. Conceivably, with further tuning the hybrid PAS-DHFR can be made to carry out its catalytic function exclusively in the presence (or absence) of light. Since PAS domains bind a wide array of ligands, the approach can probably be adapted for various chemical triggers.

On a more fundamental level, the authors claim that this result supports the view that specific surface locations in many domains may be evolutionarily-conserved loci for allosteric control. This does not mean that every PAS domain (or PDZ domain, or DHFR) actually possess allosteric properties, but it does imply that all of them have the potential to exert or receive allosteric influences. If this is true, then it may be possible to adapt a wide array of binding modules as allosteric regulators for natural and designed enzymes. As our understanding of intradomain signaling improves, our ability to make use of these approaches will only increase.

J. Lee, M. Natarajan, V. C. Nashine, M. Socolich, T. Vo, W. P. Russ, S. J. Benkovic, R. Ranganathan (2008). Surface Sites for Engineering Allosteric Control in Proteins Science, 322 (5900), 438-442 DOI: 10.1126/science.1159052

Aug 232008
ResearchBlogging.orgThe ability to sense and respond to magnetic fields is a fundamental aspect of behavior in many animals. While migratory birds famously use the earth’s magnetic field to navigate during, magnetic field responses occur in all manner of animals, from eels to invertebrates. Even the lowly fruit fly, best known as a reminder that you really should have taken the garbage out two days ago, can react to magnetism. While various explanations have been put forward in different species, magnetosensitivity remains fairly mysterious. In this week’s Nature, researchers from the University of Massachusetts Medical School show that the blue-light photoreceptor cryptochrome plays an essential role in allowing fruit flies to detect magnetic fields.

Cryptochrome (or Cry) inherited the ability to receive blue light along with its photolyase domain, which is homologous to a prokaryotic, light-dependent DNA repair protein. Cry proteins, which are present in all animals, do not perform any DNA repair work, but instead play a role in regulating the circadian rhythm. While it is not clear in all cases whether Cry’s ability to absorb blue light is biologically significant in clock regulation, it is known that fruit flies (Drosophila melanogaster) use Cry to synchronize their circadian clocks. Previous experiments had suggested that the ability of fruit flies to detect magnetic fields was somehow related to photoreception, and that short wavelengths (like those sensed by Cry) had different effects from longer ones.

Gegear et al. devised a relatively simple experiment to test the importance of Cry in Drosophila magnetosensing. They placed a T-junction in a box, with a magnetic coil on one side and a non-magnetic coil on the other. They released flies into the junction, with (trained) or without (naive) performing an earlier run where the magnetic field was associated with a sucrose reward. They shined a light into the box and used filters to investigate the role of specific wavelengths.

They discovered that several strains of Drosophila could be trained to go to the magnetic field, although the degree of preference and the nature of the naive response differed substantially between strains. Gegear et al. chose the strain that showed the greatest response in full-spectrum light (and displayed a tendency to avoid the magnetic field in the naive state) to perform the filter experiment. Cutting off all wavelengths of light shorter than 500 nm abolished both the naive and trained responses to the magnetic field in these flies, as did filtering out all wavelengths shorter than 420 nm. If only wavelengths shorter than 400 nm were cut off, some of the trained and naive response returned. Simply dimming the light was not enough to replicate the effect of filtering. These experiments indicate that magnetic sensitivity in these flies requires light in the blue to ultraviolet range.

In order to prove that cryptochrome specifically is necessary for this magnetic sensitivity, Gegear et al. took advantage of our tremendous knowledge of fly genetic manipulation to create mutant flies that did not have a functional Cry gene. No matter what wavelengths of light were used in the T-junction experiment, these flies did not respond to the magnetic field. Crossing these Cry-null mutants with normal flies restored magnetosensitivity. The authors also performed experiments to show that the circadian rhythm was not itself essential to magnetic response in the flies.

Because this is a genetic experiment, it cannot address the question of whether Cry is both the blue-light photoreceptor and the magnetosensor. Going just on what we have in this paper, it is also possible that Cry acts upstream of another magnetosensor protein or is part of its downstream signaling pathway. However, in light of research that shows the flavin photoreception in other cryptochromes induces the formation of magnetically-sensitive radicals, some of which I discussed last year, it certainly seems possible that Drosophila cryptochrome does the whole job itself. As I mentioned in the case of the previous article, though, there is not yet any understanding of a mechanism by which information about magnetic field could be transduced from Cry radicals into the nervous system.

Dorosophila Cry differs from other plant and animal Cry proteins in significant ways, so it’s unclear whether these results have any relevance for other organisms. However, the finding that Cry is essential to Drosophila magnetosensitivity suggests at least the possibility of parallel systems in migratory birds and other species that use magnetic fields.

Robert J. Gegear, Amy Casselman, Scott Waddell, Steven M. Reppert (2008). Cryptochrome mediates light-dependent magnetosensitivity in Drosophila Nature, 454 (7207), 1014-1018 DOI: 10.1038/nature07183

Oct 312007

Blogging on Peer-Reviewed Research

The annual movements of birds are frequently cited as one of the great wonders of nature, and rightly so. This widespread behavior’s initial evolution and subsequent refinement into the amazingly specific systems we observe in the modern world are testaments to the enormous power of natural selection to shape not only the physiological systems that enable long-range directional flight, but also the social mechanisms that specify destination and mode of travel. In general terms, we understand what biological components enable migratory behaviors, but the specifics of the system are not yet completely understood. We know that birds have a magnetic compass, and that their choice of direction is also light-dependent to some extent. Today in PLoS ONE, Miriam Liedvogel and colleagues attempt to eplain this linkage with biophysical experiments.

The target of their studies is cryptochrome, a eukaryotic protein related to the bacterial DNA repair protein photolyase. Cryptochrome is a blue-light photoreceptor, known to play a role in light-detection in plants, and important for circadian rhythm in mammals. Eukaryotic cryptochromes have a photolyase-like domain that contains a flavin chromophore, and a large, intrinsically unstructured C-terminal domain that is important for signaling. The lovely and talented Carrie Partch showed that photoreception in the photolyase homology region disrupts stable structure in the C-terminal domain of arabidopsis cryptochrome, and that this is important for signaling (shameless promotion). Liedvogel et al. show in their paper that the flavin nucleotide reacts to the incidence of blue light by forming pairs of radicals. This is important because the electronic behavior of these radicals can be affected by external static or oscillating magnetic fields. Moreover, cryptochromes may be active in the eye during magnetic orientation, as Mouritsen’s lab showed previously.

Liedvogel et al. demonstrate the existence of radical pairs using a transient absorbance experiment following irradiation of a 20 µM cryptochrome solution with a blue laser. Their Figure 1 (to see it, follow the link above—PLoS is free) shows absorbance maxima characteristic of flavin radicals that persist for a few milliseconds following excitation. These peaks are not present in absorbance spectra averaged over a longer period of time (Figure 3). Liedvogel et al. propose that these radicals, though persisting for only a few milliseconds, nonetheless last long enough to plausibly interact with the earth’s magnetic field.

So… problem solved? Not exactly—Liedvogel et al. do a pretty good job explaining the limitations of this research and the attendant models. Although this work opens up some intriguing possibilities it certainly leaves us with more questions than answers. Specifically, it would be nice to know whether this radical-pair formation actually causes some kind of signal; given the relatively short lifetime of these species this is not at all certain. Also it would be important to determine whether the presence of magnetic fields in any way modulates cryptochrome signaling. This is also a case where the nature of the experiment may be misleading us—a dilute solution excited by a laser may not really resemble a cellular milieu stimulated by ambient light. To be fair, this cuts both ways; cofactors present in the cell could inhibit or enhance radical formation.

In this regard, it will be important to perform in vivo experiments, at least at the level of cultured cells. Experiments of that nature are likely to be very difficult, however, given the highly specific context in which the signaling occurs. Given that the experiment entailed the creation of a cDNA library from bird retinal cells under the appropriate signaling conditions, a yeast two-hybrid screen under conditions of blue-light irradiation may be a possibility.

The subsequent challenge will be to test whether and how a magnetic field alters signaling. The problem there is one of controls—the earth’s magnetic field is pretty difficult to escape. However, once signaling partners are isolated, one could set up experiments involving NMR spectrometers… not as direct devices for investigation, but rather as part of the experimental setup. The magnetic field inside (and thus, with an unshielded instrument, outside) an NMR spectrometer has a directionality, and not all of them are oriented the same way. Some point away from the core of the earth, others towards it. The magnetic field of a typical MRI machine is oriented parallel to the ground. In addition, the field of almost any superconducting magnet is vastly greater than that of the earth. Growth experiments performed in the leaking field outside of these spectrometers could be compared to experiments performed in the ambient magnetic field in order to gain insight into the effects of external polarization on cryptochrome signaling. Dilute-solution experiments performed within an NMR spectrometer may also be of value.

Liedvogel et al. argue that the cryptochrome would have to be tethered or immobilized in some way in order for its interactions with the magnetic field to encode information. This is certainly the simplest possibility, but may not go far enough. Simply tethering the molecule is unlikely to prevent isotropic or near-isotropic tumbling, especially considering the unstructured nature of the c-terminal domain that seems to govern signaling. However, tethering the protein at a single point might work if the actively signaling species is short-lived, experiences primarily local interactions, and is dispersed along a line or plane of tissue. In this case, the averaging due to tumbling might be countered by a gradient imposed by the arrangement. This is especially attractive because this form of activation appears to be low-yield: increased average activity of a tethered ensemble rather than a rare specific signal incidence might be the mechanism.

This is an interesting paper that amounts to a feasibility study. Liedvogel et al. demonstrate that blue-light stimulation can induce the formation of radical pairs, and it is known that the behavior of these species are affected by magnetic fields. This shows that it is possible for cryptochromes to play a direct role in migratory magnetic navigation. Additional evidence suggests that they play some role, but this may be coincidence; for instance, magnetic compass calibration may be a system controlled by the circadian rhythm function of cryptochromes. Actually proving the existence of a direct role for cryptochromes in orientation control will require substantially more work.

Liedvogel M, Maeda K, Henbest K, Schleicher E, Simon T, et al. (2007) Chemical Magnetoreception: Bird Cryptochrome 1a Is Excited by Blue Light and Forms Long-Lived Radical-Pairs. PLoS ONE 2(10): e1106. doi:10.1371/journal.pone.0001106