Abstract
Nonspecific lipid transfer proteins (ns-LTPs) are small, basic proteins and are ubiquitous in plant kingdom. Ns-LTPs are subdivided into two subfamilies that differ in molecular mass, nsLTP1 (~9 kDa) and nsLTP2 (~7 kDa). They were thought to be involved in the formation of cutin and suberin because of the existing in extracellular membranes and containing lipid transfer ability. The in vivo biological function is not very clear, but it has been suggested that they are involved in responses towards stresses and plant defense. In order to study the lipid binding mechanism of rice nsLTP1, we crystallized the crystals of native and complexed nsLTP1s from rice with saturated fatty acids in different carbon length from 10 to 18. The crystals of rice nsLTP1 have been grown at room temperature from 45~50% PEG 600, 0.2 M (NH4)2SO4 in 0.1 M Tris buffer, pH 5.4, within one week to the dimensions of 0.3mm*0.3mm*0.1 mm. They diffract to at least 2.5Å upon exposure Cu Kα radiation X-rays. After preliminary data analysis, the space group of nsLTP1-myristate and palmitate complexes belong to C2221 in unit cell parameters of a = 48.04, b = 49.75, c = 72.70, a = b = g = 90.00. The space group of nsLTP1-stearate complex was determined to be C2 in unit cell parameters of a = 49.07, b = 53.80, c = 75.92, a = b = 90.00, g = 96.31. The data sets of myristate-, palmitate-, and stearate-nsLTP1 complexes were processed to 2.0~2.4 Å. Furthermore, in order to investigate the differences of nsLTP1s in functionality and characteristics between different species. A novel nsLTP1 has been isolated from lotus seeds. 81 residues (about 90%) amino acid sequence of lotus seed nsLTP1 was determined by N-terminal Edman degradation of the intact protein as well as the peptide fragments resulted from trypsin and thermolysin digestions. The secondary structure of lotus seed nsLTP1 is predominately α-helix as determined by circular dichroism spectroscopy. We have analyzed the thermodynamic of the stability by using denaturant- and heat-induced unfolding experiment. It was found that the nsLTP1 is a highly stable protein, which apparently does not denature at temperatures up to 96℃ and it is most stable in pH4 than other pH conditions. This high stability may be important for the biological function of LTP1.