H-1-NMR ASSIGNMENT AND GLOBAL FOLD OF NAPIN BNIB, A REPRESENTATIVE 2SALBUMIN SEED PROTEIN

Napin BnIb is a representative member of the 2S albumin seed proteins, which consists of two polypeptide chains of 3.8 and 8.4 kDa linked by two disulfide bridges. In this work, a complete assignment of the H-1 spectra of napin BnIb has been carried out by two-dimensional NMR seq uence specific methods and its secondary structure determined on the b asis of spectral data, A calculation of the tertiary structure has bee n performed using similar to 500 distance constraints derived from una mbiguously assigned NOE cross-correlations and distance geometry metho ds. The resulting global fold consists of five helices and a C-termina l loop arranged in a right-handed spiral. The folded protein is stabil ized by two interchain disulfide bridges and two additional ones betwe en cysteine residues in the large chain, The structure of napin BnIb r epresents a third example of a new and distinctive folding pattern fir st described for the hydrophobic protein from soybean and nonspecific lipid transfer proteins from wheat and maize. The presence of an inter nal cavity is not at all evident, which rules out in principle the nap in BnIb as a carrier of lipids. The determined structure is compatible with activities attributed to these proteins such as phospholipid ves icle interaction, allergenicity, and calmodulin antagonism. Given the sequence homology of BnIb with other napins and napin-type 2S albumin seed proteins from different species, it is likely that all these prot eins share a common architecture. The determined structure will be cru cial to establish structure-function relationships and to explore the mechanisms of folding, processing, and deposition of these proteins. I t will also provide a firm basis for a rational use of genetic enginee ring in order to develop improved transgenic plants.