Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro

A heat-soluble protein present in substantial quantities in Typha latifolia pollen was purified to homogeneity. The protein was subjected to cyanogen bromide cleavage, and the peptides produced were separated by HPLC chromato graphy and sequenced. The two sequences determined were found to be related to the putative D76 LEA protein from Brassica napus seeds and one of them to the D-7 LEA protein from upland cotton. This suggests the pollen protein to be a member of the LEA group III family of proteins. The secondary stru cture of the protein in solution and in the dry state was investigated usin g Fourier transform IR spectroscopy. Whereas the protein in solution was hi ghly unordered, being largely in a random coil conformation, the conformati on was largely alpha -helical after fast drying. Slow drying reversibly led to both alpha -helical and intermolecular extended beta -sheet structures. When dried in the presence of sucrose, the protein adopted alpha -helical conformation, irrespective of drying rate. The effect of the protein on the stability of sucrose glasses was also investigated. The dehydrated mixture of sucrose and the LEA protein had higher glass transition temperatures an d average strength of hydrogen bonding than dehydrated sucrose alone. We su ggest that LEA proteins may Flay a role together with sugars in the formati on of a tight hydrogen bonding network in the dehydrating cytoplasm, thus c onferring long-term stability. (C) 2001 Elsevier Science B.V. All rights re served.