Spectroscopic Characterisation of the Lipid-binding Properties of Wheat Puroindolines

Abstract

The lipid-binding properties of wheat puroindolines (PINs) make these proteins likely candidates to play an important role in the stability of lipid films in the gas cells of bread dough, an important aspect of bread making. Therefore, the interaction between PINs and water-soluble model lipids was investigated by fluorescence emission and circular dichroism (CD) spectroscopy. The secondary structure of PIN-a and PIN-b in solution, as determined by far-UV CD measurements, resembled that of plant non-specific lipid transfer protein (LTP). However, PINs contain a tryptophan-rich loop located at the exterior of the protein. It was shown by fluorescence emission spectroscopy and near-UV CD spectroscopy that this domain is involved in the lipid binding. Fluorescence titration experiments of PIN and its synthetic tryptophan-rich domains with the zwitterionic lipidn-hexadecylphosphocholine (C16PN) revealed that the binding was cooperative. For the binding of the charged lipids,n-hexadecacylphosphoglycol (C16PG) and hexadecyl-trimethylammoniumbromide (CTAB), a clear effect of ionic strength of the solution was obtained. Organisation of lipids into micelles was not a prerequisite for binding to PINs. Most likely, wheat PIN binds to monomeric lipid molecules via its tryptophan-rich domain, rendering the protein more hydrophobic. The overall secondary structure of wheat PINs did not change significantly upon binding to lipids.