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GEN News Highlights : Jun 20, 2011
Scientists Use MIPs to Crystallize Proteins for Research and Screening
Studies show that MIPs act as efficient nucleants for hard to crystallize molecules.!--h2>
Scientists claim molecularly imprinted polymers (MIPs) can be used as effective nucleants for the formation of large single crystals of otherwise hard to crystallize proteins. A team led by Subrayal M. Reddy, Ph.D., at the U.K.’s University of Surrey, and Naomi E. Chayen, Ph.D., at the U.K.’s University College, London, and colleagues, found that using MIPs in screening experiments also led to successful hits that would have been missed using other types of nucleant.
Reported in PNAS, the work showed that MIPs sped the formation of protein crystals even in cases where crystals would eventually have been generated, and in some cases improved diffraction. The team’s work is described in a paper titled “Protein crystallization facilitated by molecularly imprinted polymers.”
MIPs are polymers formed in the presence of a molecule that, once extracted, leaves complementary cavities behind as an imprint that allows highly selective rebinding of the original molecule. In the field of protein research, MIPs have been used for applications including protein purification/isolation, replacement of biological antibodies in immunoassays, catalysis, and biosensors for medicine, the researchers note. However, MIPs haven’t before been used to facilitate protein crystallization, and the difficulties in obtaining useful crystals still remains a bottleneck in proteomics and structural biology.
The ultimate way to obtain high-quality protein crystals is to control nucleation, which is the first step that determines the entire crystallization process, the authors continue. A variety of substances have been evaluated as nucleants, but their desirable properties are offset by the fact they have no specificity for individual proteins. The U.K.-based team hypothesized that MIPs could represent ideal nucleants because they are designed to specifically attract a template protein.
The researchers evaluated the ability of hydrogel-based MIPs (hydroMIPs) imprinted with seven proteins to induce nucleation of their own cognate proteins, as well as other proteins. The seven proteins used for imprinting were lysozyme, trypsin, catalase, haemoglobin, intracellular xylanase IXT6-R217W, alpha crustacyanin, and human macrophage migration inhibitory factor (MIF). With the exception of the catalase MIP, the resulting polymers were able to induce the formation of crystals of nine tested proteins, including in some cases non-cognate proteins with similar molecular weights.
These successes included generating crystals of HIV protein complexes that diffracted at 4.2Å: other techniques used to crystallize this complex have failed to produce crystals with diffraction beyond 9Å, the authors note. In the case of human MIF, the use of both its cognate and one other MIP induced the formation of crystals with a diffraction resolution of 1.2Å using a rotating anode x-ray source, whereas previous crystallization approaches have needed synchrotron sources to achieve the same resolution, the authors report. A number of the MIPs were capable of inducing the crystallization of noncognate proteins with a molecular weight of the same order of magnitude.
When the experiments were repeated at the same conditions using other known nucleants including human hair, horse hair, zeolites, and bioglass powder, only lysozyme and trypsin generated crystals, and these were small multiple crystals compared with the large single crystals produced using the MIPs.
The researchers then moved on to test whether MIPs could also improve the hit rate in index screens for alpha crustacyanin, MIF, intracellular xylanase IXT6-R217W, and trypsin (which is relatively easily crystallized) as a control. The three test proteins were chosen because alpha crystacyanin and intracellular xylanase IXT6-R217W have not produced useful crystals to date, while MIF requires higher resolution crystals, they note.
Under suitable conditions the index screen resulted in four to five hits for each of the three target proteins when their cognate MIPs were present, whereas no hits were obtained in the absence of MIPs. The hits appeared between 24 hours and four days after setting up the trials. When the experiments were repeated using other known nucleants, hits were only generated for the control substance trypsin. “The results demonstrate that in the presence of MIPs, 8–10% of the screening trials of the target proteins produced hits that would have been missed even when other nucleants were applied,” the authors note. Interestingly, when they then set up studies to see if noncognate MIPs would also give rise to hits, only the experiment for alpha crystacyanin failed to generate any hits. This supported the earlier findings that MIPs could generate crystals of noncognate proteins.
The team says its results support previous theoretical and experimental evidence suggesting that crystal nucleation proceeds in two steps: aggregation of molecules into a dense fluid droplet, and then ordering. The newly reported data combined with atomic force microscopy data indicate that MIPs promote aggregation of protein molecules to form a protein-rich phase, which at a later stage becomes crystalline, they state. “It therefore appears that in these cases, MIPs may function by facilitating the nucleation and stabilization of droplets of the protein-rich liquid phase, at conditions that would be quite far from the liquid–liquid phase separation conditions in the absence of nucleant.”
The researchers admit that when they started their research the expectation was that each MIP would work exclusively on its congnate protein. The observation that some MIPs, such as those imprinted with lysozyme or trypsin, also induced the crystallization of other proteins with similar molecular weights could be particularly useful, they comment, for example when a related MIP is available for a difficult to crystallize target protein that is too scarce to allow imprinting in its own right.
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