Abstract
Plant non-specific lipid transfer proteins (ns-LTPs) are isolated from various plants, including rice, barley and maize. Ns-LTPs can bind to variety of lipids and transfer phospholipids between membranes. Three dimensional structures of ns-LTP1s are composed of four helical segments and a C-terminal tail with four disulfide bonds connecting these secondary components together. The four helix segments forms a hydrophobic cavity inside the protein which is believed involved in the lipid binding activity. The property that ns-LTPs possess a hydrophobic cavity makes itself as an interesting model for protein folding study. In this study, we investigated the folding pathway of rice ns-LTPs in terms of disulfide formation and breakage. We conducted oxidative folding, reductive unfolding and disulfide bond scrambling experiments to elucidate the folding pathway of rice ns-LTPs. We used HPLC to collect folding and unfolding intermediates and analyzed by the disulfide bond pattern determination and circular dichroism spectrometry. The disulfide bond patterns were determined by trypsin digestion, mass spectrometry, Edman sequencing, CN-induced cleavage and tandem mass. In our studies, folding pathway of rice LTP1 showed a higher complexity than rice ns-LTP2. We identified four denatured structures of scramble rice ns-LTP1 in scramble unfolding experiments and two reductive unfolding intermediates along the rice ns-LTP1 unfolding. Rice ns-LTP2 showed a simple two-state mechanism in reductive unfolding, scramble folding and unfolding.