Interfacial Enzymes: Membrane Binding, Orientation, Membrane Insertion, and Activity

Most interfacial enzymes undergo activation upon membrane binding. Interfacial activation is determined not only by the binding strength but also by the specific mode of protein-membrane interactions, including the angular orientation and membrane insertion of the enzymes. This chapter describes biophysical techniques to quantitatively evaluate membrane binding, orientation, membrane insertion, and activity of secreted phospholipase A₂ (PLA₂) and lipoxygenase (LO) enzymes. Procedures for recombinant production and purification of human pancreatic PLA₂ and human 5-lipoxygenase (5-LO) are also presented. Several methods for measurements of membrane binding of peripheral proteins are described, i.e., fluorescence resonance energy transfer (FRET) from tryptophan or tyrosine residues of the protein to a fluorescent lipid in vesicles, changes in fluorescence of an environment-sensitive fluorescent lipid upon binding of proteins to membranes, and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. These methods produce the apparent binding constant, the protein-to-lipid binding stoichiometry, and the Hill cooperativity coefficient. Experimental procedures for segmental isotope labeling of proteins and determination of the orientation of membrane-bound proteins by polarized ATR-FTIR spectroscopy are described. Furthermore, evaluation of membrane insertion of peripheral proteins by a fluorescence quenching technique is outlined. Combination of the orientation and membrane insertion provides a unique configuration of the protein-membrane complex and hence elucidates certain details of the enzyme function, such as the modes of acquisition of a membrane-residing substrate and product release. Finally, assays for determination of the activities of secreted PLA₂, soybean LO, and human 5-LO are described.