Biofoams
‘Once we have collected the nests, we then have to spend many hours tediously hand-separating the eggs (hundreds of them) from the foam, being careful not to damage the eggs to avoid contamination of the foam. In Trinidad, we had the luxury of lab facilities at the local university for basic sample processing and storage. In Malaysia, this was done in Rosalind Tan’s grandmother’s kitchen or on the porch, in a typical Malaysian family house, surrounded by normal domestic activities,’ Cooper reminisces.
The blue protein Studying the foam nests back in Glasgow, the researchers found that the colour of those of Polypedates leucomystax come from a remarkable protein – ranasmurfin – that is bright blue when purified. In 2008, they collaborated with crystallographer Jim Naismith at St. Andrews University, UK, to solve the crystal structure of this protein. Ranasmurfin turned out to be a
crystallographer’s dream – it forms crystals of such outstanding quality that the researchers didn’t even need the sequence of the polypeptide chain. They could identify most of the amino acids directly from the electron density map and cleared up the last ambiguities using mass spectrometry.1 They discovered a new protein fold
– the large scale pattern according to which the local structural elements, such as α-helices and β-sheets, arrange themselves to form the whole, independently folding structure unit, which may be a complete small protein or a domain of a larger protein. New folds only emerge rarely nowadays, in spite of the exponentially growing number of new protein structures being solved. The researchers also found a novel kind of crosslink between amino acid side chains. Within each of the two subunits of the protein, two lysine residues react with a tyrosine to form an orthoquinone. The orthoquinone groups from both subunits combine to form an indophenol unit covalently holding the subunits together.
‘The unusual protein crosslinking associated with ranasmurfin may be another way of helping long-term
stability in such nests.’ Alan Cooper University of Glasgow, UK
26 Chemistry&Industry • November 2013 A comparable
protein-based biofoam is latherin, the surfactant protein of horse sweat and saliva
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The indophenol binds a zinc ion, and they are together responsible for the blue colour, as the researchers demonstrated via synthesis of analogous model complexes. This type of modification has never been observed before, and it would have been impossible to predict it on the basis of the gene sequence. As in the case of the widely used Green Fluorescent Protein (GFP), whose chromophore is also the product of an autocatalytic merger of amino acids within the protein structure, this highlights the importance of directly studying biological materials of interest, rather than deriving information only from genomes. The unusual modification may also help to stabilise the protein foam. Normally, biochemists fear the appearance of foam on their protein solutions, as it is usually the sign of denaturation – loss of the folded structure – and the loss of biological function. Only in rare cases, such as ranasmurfin and a small group of other frog proteins known as ranaspumins, is foaming a positive activity related to the natural function of a protein. ‘We still do not know the true biological function of ranasmurfin,’ Cooper admits. ‘The foaming in these nests seems to rely more on entrapment of bubbles in the sticky, viscous mixture, as opposed to the surfactant behaviour seen in the ranaspumins. The unusual protein crosslinking associated with ranasmurfin
may be another way of helping long-term stability in such nests.’ Kennedy adds that other ideas such as that ranasmurfin may act as a sunscreen, camouflage, or act against microbial or insect attack on the nests and their precious contents, still need to be examined. Some other species of tropical frogs
also produce foam nests. Vania Melo at the Federal University of Ceará at Fortaleza, Brazil, has studied foam nests of tropical frogs found in northeast Brazil. She has identified a surfactant protein from Leptodactylus vastus, Lv- ranaspumin, which seems to be unrelated to the ranaspumins analysed in Glasgow.2 In fact, the amphiphilic properties required for a surfactant protein can be realised with many different amino acid sequences. Therefore, it is not surprising that evolution came up with very different versions of surfactant proteins, possibly based on modifying genes that initially served an entirely unrelated function. Marine invertebrates such as the intertidal tunicate Pyura praeputialis also use foam to protect their spawn, but their foam is based on carbohydrate surfactants, not proteins, as Juan Carlos Castilla and his team at Santiago de Chile have shown.3
Sweating it out Apart from the frogs, other examples of comparable protein-based biofoams are few and far-between. The nearest example of biofoam production among
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