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Structure of Integral Membrane Proteins Readily Solved Using an Alternative In Meso In Situ Serial Crystallography Technique

By LabMedica International staff writers
Posted on 16 Jun 2015
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Image: In meso in situ serial crystallography (IMISX) equipment that was used to quickly and accurately provide detailed blueprints of protein structure (Photo courtesy of Dr. Martin Caffrey, Trinity College Dublin).
Image: In meso in situ serial crystallography (IMISX) equipment that was used to quickly and accurately provide detailed blueprints of protein structure (Photo courtesy of Dr. Martin Caffrey, Trinity College Dublin).
A recent paper discussed the benefits of using synthetic cyclic olefin copolymer (COC) plates to replace glass as the base for generation and growth of crystals of membrane and soluble proteins for high-resolution X-ray crystallographic structure determination.

Membrane proteins perform critical functions in living cells related to signal transduction, transport, and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, the structural and functional understanding of membrane proteins lags behind that of soluble proteins, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique, which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels, and G protein-coupled receptors.

A mesophase is a phase of matter intermediate between a true liquid and a true solid that exists in a liquid crystal. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials.

Investigators at Trinity College (Dublin, Ireland) described an alternative approach for the in meso in situ serial crystallography (IMISX) method, which showed that the use of COC plates provided many advantages over glass plates and was compatible with high-throughput in situ measurements. The novel IMISX technique was demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein.

The synthetic COC was chosen for several reasons. To begin with, it is commercially available in sheets of varying thicknesses and is inexpensive. Further, it is relatively watertight, optically transparent, UV-transmitting and non-birefringent. As a plastic, COC is chemically inert and is a weak absorber and scatterer of X-rays.

A paper published in the June 2015 online edition of the journal Acta Crystallographica D described the IMISX protocol, which required less than 10 micrograms of protein and generated structures with resolutions ranging from 0.18 to 0.28 nanometers.

Senior author Dr. Martin Caffrey, professor of membrane structural and functional biology at Trinity College, said, "This is a truly exciting development. We have demonstrated the method on a variety of cell membrane proteins, some of which act as transporters. It will work with existing equipment at a host of facilities worldwide, and it is very simple to implement. The best part of this is that these proteins are as close to being "live" and yet packaged in the crystals we need to determine their structure as they could ever be. As a result, this breakthrough is likely to supplant existing protocols and will make the early stages of drug development considerably more efficient."

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