Crystallization and preliminary X-ray diffraction of the surfactant proteinLv-ranaspumin from the frogLeptodactylus vastus

Abstract

Lv-ranaspumin is a natural surfactant protein with a molecular mass of 23.5 kDa which was isolated from the foam nest of the frog Leptodactylus vastus. Only a partial amino-acid sequence is available for this protein and it shows it to be distinct from any protein sequence reported to date. The protein was purified from the natural source by ion-exchange and size-exclusion chromatography and was crystallized by sitting-drop vapour diffusion using the PEG/Ion screen at 293 K. A complete data set was collected to 3.5 Å resolution. The crystal belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 51.96, b = 89.99, c = 106.00 Å . Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54%.

Frog Foam Nest Protein Diversity and Synthesis

“…Regarding to the MS spectra obtained from the three analyzed spots, it was noticed that the three proteins exhibited very similar spectra suggesting that they have the same or a very similar amino acid sequence with microheterogeneities. The different isoelectric points can be due to few modifications in the amino acid sequence as confirmed by Hissa et al (2014) and Hissa et al (2012) showing the presence of at least four isoforms in the protein amino acid sequence of Lv-RSN-1. It is also important to mention that Lv-RSN-1 has no glycosylation or phosphorylation.…”

“…The different isoelectric points can be due to few modifications in the amino acid sequence as confirmed by Hissa et al. () and Hissa et al () showing the presence of at least four isoforms in the protein amino acid sequence of Lv‐RSN‐1. It is also important to mention that Lv‐RSN‐1 has no glycosylation or phosphorylation.…”

“…). Lv‐RSN‐1 is a monomeric protein with molecular mass of 23.5 kDa, presenting surfactant activity and novel tridimensional conformation ( Hissa et al., , Hissa et al., ).…”

“…We have described the protein Lv-ranaspumin (Lv-RSN-1) (Hissa et al, 2008), the major protein in the foam nest of the pepper frog L. vastus A. Lutz 1930 (Heyer, 2005), endemic from northeastern Brazil (De Sá et al 2014). Lv-RSN-1 is a monomeric protein with molecular mass of 23.5 kDa, presenting surfactant activity and novel tridimensional conformation ( Hissa et al, 2012, Hissa et al, 2014.…”

Unique Crystal Structure of a Novel Surfactant Protein from the Foam Nest of the Frog Leptodactylus vastus

“…The lack of a complete amino acid sequence hampered the search for a reliable molecular-replacement model, and obtaining phases was problematic initially. [10] We solved the monoclinic structure by using ab initio phasing with the program ARCIMBOLDO, [11] which was facilitated by the predicted (and later confirmed) high a-helical content. The resolution of the electron density was sufficient to identify most amino acids in the polypeptide chain; this helped in assembling the complete sequence, by grouping the peptides obtained by manual de novo sequencing.…”