Domain Structure and Molecular Conformation in Annexin V/1,2-Dimyristoyl- sn-Glycero-3-Phosphate/Ca 2+ Aqueous Monolayers: A Brewster Angle Microscopy/Infrared Reflection-Absorption Spectroscopy Study

Abstract

Annexins comprise a family of proteins that exhibit a Ca 2+-dependent binding to phospholipid membranes that is possibly relevant to their in vivo function. Although substantial structural information about the ternary (protein/lipid/Ca 2+) interaction in bulk phases has been derived from a variety of techniques, little is known about the temporal and spatial organization of ternary monolayer films. The effect of Ca 2+ on the interactions between annexin V (AxV) and anionic DMPA monolayers was therefore investigated using three complementary approaches: surface pressure measurements, infrared reflection-absorption spectroscopy (IRRAS), and Brewster angle microscopy (BAM). In the absence of Ca 2+, the injection of AxV into an aqueous subphase beneath a DMPA monolayer initially in a liquid expanded phase produced BAM images revealing domains of protein presumably surrounded by liquid-expanded lipid. The protein-rich areas expanded with time, resulting in reduction of the area available to the DMPA and, eventually, in the formation of condensed lipid domains in spatial regions separate from the protein film. There was thus no evidence for a specific binary AxV/lipid interaction. In contrast, injection of AxV/Ca 2+ at a total Ca 2+ concentration of 10 μM beneath a DMPA monolayer revealed no pure protein domains, but rather the slow formation of pinhead structures. This was followed by slow (>2 h) rigidification of the whole film accompanied by an increase in surface pressure, and connection of solid domains to form a structure resembling strings of pearls. These changes were characteristic of this specific ternary interaction. Acyl chain conformational order of the DMPA, as measured by ν symCH 2 near 2850 cm −1, was increased in both the AxV/DMPA and AxV/DMPA/Ca 2+ monolayers compared to either DMPA monolayers alone or in the presence of Ca 2+. The utility of the combined structural and temporal information derived from these three complementary techniques for the study of monolayers in situ at the air/water interface is evident from this work.