Hydrophobic interaction chromatography of Micrococcus lysodeikticus F 1-ATPase. A method to detect conformational flexibility of the molecule

Abstract

Hydrophobic interaction chromatography of purified ATPase from Micrococcus lysodeikticus (E.C. 3.6.1.3.), a complex oligomeric protein, induces extensive conformational changes in it. In this report, we describe some physicochemical properties of the enzyme forms obtained. They can be summarized as follows. (1) The subunit stoichiometry of the enzyme is altered by the adsoption and desorption process since most of the forms obtanied are defective in γ and δ subunits. An important reduction in the molar proportion of α subunit is also observed; (2) the fluorescence spectra of the different forms show progressive tyrosine residues which roughly correspond to the extent and stregth of the interaction existing before elution of the enzyme; (3) circular dichroism measurements reveal changes of the secondary structure of the F 1-ATPase undergoing an increase in α-helical content; (4) the ordered, active forms eluted from the hydrophobic chromatography columns are less stable than the native protein, as shown by dialysis experiments. These results while supporting the use of hydrophobic chromatography as a simplified model of membrane-membrane protein interaction, also indicate the need for caution in its application to the purification of complex membrane proteins.