Three-dimensional crystallization of the light-harvesting complex from Mantoniella squamata (Prasinophyceae) requires an adequate purification procedure

Abstract

We present a new purification procedure for the light-harvesting complex of Mantoniella squamata whereupon three-dimensional crystallization succeeded. Previous purification methods were based on density centrifugations as the only separating principle. We have extended this preparation procedure by applying anion-exchange and molecular-sieve chromatography techniques. Purity and stability of the complex were proved by denaturing and non-denaturing polyacrylamide-gel electrophoresis, and spectroscopic measurements. With respect to contaminating lipids the purified pigment-protein complex was examined by thin-layer chromatography and the aggregation and/or oligomeric states were investigated by gel filtration, fluorescence emission, and electron microscopy. Molecular-mass determination by gel filtration gave evidence for a trimeric organization of the purified native complex. Our purification procedure resulted in mixed protein-detergent micelles, small and homogeneous, the ideal material to start crystallization trials. We found involuntary aggregation to be easily detected by additional fluorescence emission at red-shifted wavelength (700 nm) as compared to the main emission at 680 nm. Electron-microscopic images proved this emission at 700 nm to be really due to aggregation, which could be prevented if sufficient detergent were present in the samples during the whole purification procedure. The ultimate proof that our efforts have been successful was the appearance of hexagonal crystals.