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Scientists in Illinois and Pennsylvania have developed a method of chemically synthesising mirror image forms of a natural antifreeze protein originally isolated from the Canadian snow flea. Then, they have used X-ray crystallography to capture a snapshot of the protein in its natural and enantiomeric (mirror image) form. The study represents a first in crystallography, the researchers say, and could have implications for transplant medicine. In 2005, researchers in Canada reported the discovery of a new "antifreeze" protein in the tiny snow flea, Hypogastrura harveyi. The protein protects the flea from the depths of the frozen Arctic winter by preventing ice crystals from growing within its body. Snow fleas, a species of springtail, live in soil, in mosses or in leaf litter, are dark blue, about 1-2 mm in length, and eat decaying matter. This discovery, reported in Science, revealed snow flea antifreeze protein, sfAFP, to be an 81-amino acid that is glycine-rich, about 45% of its amino acids are glycine, which makes it very distinct from other antifreeze proteins. Moreover, the protein has no sequence homology with any known protein, the team reported. At the time, the research team of Laurie Graham and Peter Davies, from the Department of Biochemistry at Queen's University in Kingston, Ontario, suggested that the compound might be adapted for use in extending the shelf life of human organs for transplantation. The same material might also be used to improve ice cream, again by preventing the formation of ice crystals that spoil the product's smooth texture. Now, Stephen Kent, Brad Pentelute, and Zachary Gates of the Department of Chemistry and Institute for Biophysical Dynamics at the University of Chicago, and colleagues, Jennifer Dashnau and Jane Vanderkooi of the Department of Biochemistry and Biophysics, at the University of Pennsylvania, have at last experimentally deciphered the complete molecular structure of the snow flea antifreeze protein. Obtaining the complete structure is critical for making larger amounts of the protein, which exists naturally in only minute quantities in the insects themselves. The larger synthetic quantities could be used for further research and potential medical and commercial applications, the researchers suggest. Using total chemical protein synthesis, pioneered by the Kent laboratory, the US team made a synthetic version of the protein, snow flea antifreeze protein, sfAFP, and demonstrated that it has the same activity in inhibiting ice crystal formation as the natural protein. They also synthesised the mirror image, enantiomeric form, of the natural sfAFP using peptide segments built from synthetic amino acids in the D, as opposed to the natural L form. The circular dichroism (CD) spectrum of the D form revealed it to be identical but for having the opposite sign to the natural and synthetic sfAFP. More importantly, the researchers showed that the enantiomeric form also had the same antifreeze properties. Given that there are no proteins, antibodies, enzymes, or receptors, composed of D amino acids in the human body, the enantiomeric sfAFP will, the researchers hope, be less likely to trigger a potentially harmful antibody reaction from use in preservation of a transplant organ. The D form of sfAFP would also be more resistant to destruction as a substrate of natural enzymes, making it potentially more effective than the native form for use in organ and tissue preservation, the researchers explain. "Our most significant advance was to use co-crystallization of the two mirror image forms of the protein to determine the previously unknown crystal structure of this unique protein," says Kent. "That is a first in the history of protein X-ray crystallography." Understanding the mechanism of the inhibitory effect of sfAFP on ice crystal formation could improve the outlook for the practical medical and consumer applications. The inhibition is more complicated than the kind of antifreeze effect associated with the colligative properties of a solution of sodium chloride in water, for instance. Researchers assume that as the temperature drops, the proteins adhere to the surface of sub-microscopic seed crystals of ice that begin to form through their backbone of functional groups. The presence of the protein on the surface of the ice presumably stops further water molecules from accruing on the growing face of the seed crystal. "It will be useful to have a reliable source of high-purity sfAFP in amounts (multiple tens of milligrams) useful for study by advanced physical techniques," the researchers say. Their total chemical synthesis coupled with the experimental structure of the folded sfAFP molecule will now enable researchers to systematically alter the covalent structure of sfAFP and potentially strip the protein down to the bare essentials that will have the same desired inhibitory effect as the natural sfAFP. Related links:
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Frozen mirror image proteins
Kent and colleagues unravelling snow flea antifreeze |
