Two-dimensional crystallization, transmission electron microscopy and image processing of keyhole Limpet Haemocyanin (KLH)

Abstract

KLH, the respiratory protein haemocyanin of the keyhole limpet Megathura crenulata, has been obtained as a high g pellet from the cell-free haemolymph and purified by gel permeation chromatography on a Biogel A15m column. The leading major protein peak eluted has been found to contain haemocyanin multi-decamers, followed by a second major peak containing single di-decamers, with small amounts of decamer and partly dissociated material following in the later fractions. The purified KLH di-decamer has been for two- dimensional crystallization studies with the negative staining-carbon film technique, in the presence of polyethylene glycol. In the side-on orientation, KLH has been found to produce two-dimensional crystals with a half-molecule linear displacement in consecutive rows. This is shown to be due to a specific association and two-dimensional crystal nucleation of the molecules in this arrangement. When oriented end-on, KLH has been found to form close- packed hexagonal monomolecular arrays which are not truly crystalline. This is because the five-fold rotational symmetry of the cylindrical macromolecule is not readily compatible with the hexagonal molecular packing. Computer-processed averaged images have been produced from the side-on KLH two-dimensional crystals and the end-on arrays, which reveal the principal molecular features of this homo-oligomeric protein complex to a resolution of ca 2.7 nm.