The biophysical chemistry of frog foam nest proteins

The tropical frog, Physalaemus pustulosus, which is widespread throughout South and Central America, protects its offspring during development by encapsulating them in a foam nest. The nest material protects the tadpoles from environmental and predatory dangers for several days. Previous work within the Cooper group has identified the constituents of the nest to be based almost entirely on proteins and carbohydrates, and have sequenced six proteins termed Ranaspumins. Of these six proteins, RSN-2 (Ranaspumin 2) is of particular interest due to its lack of similarity to any other proteins published in the protein data bank. Proceeding on the hypothesis that RSN-2 has an important role in the foam structure, the recombinant protein has been cloned and expressed in bacteria and subsequently the solution structure has been derived experimentally by NMR. Using surface techniques including Langmuir-Blodgett depositions, surface pressure analysis and atomic force microscopy, the surface structure has been studied. A proteomic and chromatographic separation and identification strategy was used to identify a large number of peptide and polypeptide fragments present in various abundances. This dramatically adds to the six proteins already discovered and the initial carbohydrate analysis performed by collaborators. The large number of peptides is typical of amphibian secretions and they are almost certainly functional to some degree. The solution structure of RSN-2 was calculated as a five-tum helix, running perpendicular over a five-strand sheet. The elongated amino-terminus was very flexible and relatively unstructured in solution whereas the carboxy-terminus was anchored to the sheet. Analysis of nitrogen relaxation and chemical shifts suggested that there are turn regions in rapid motion; one region with slow dynamics (possibly cis-trans proline isomerisation) and a cleft undergoing slow conformational exchange. The region between the helix and first strand is flexible and is postulated as a hinge for surface unfolding. Recombinant RSN-2 was established to be a powerful surfactant and provides surface pressures of 20-30 mNm-1 at approximately monolayer coverage. Hydrophobic patches were observed to partition at the air interface, and it is proposed that this is a result of reversible denaturing at the surface. RSN-1 and other proteins were examined briefly and are good candidates for further investigation due to their interesting postulated functions