Structure and orientation of a glycosylphosphatidyl inositol anchored protein at the air/water interface

The monolayer organization of intestinal alkaline phosphatase (AP), a glycosylphosphatidyl inositol (GPI) anchored dimeric protein (AP-GPI), was monitored using polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and Brewster angle microscopy (BAM). The behavior of the monolayer was reversible during the compression-expansion cycles as shown by BAM. At high surface pressure, we observed formation of condensed protein domains. Upon decompression, the protein clusters remained stable over a period of several tenths of a minute, and then the condensed domain disappeared. These results suggest strong interactions between proteins forming clusters, which could be involved in the formation of membrane microdomains. The protein did not unfold as evidenced by PM-IRRAS, whatever the initial spreading surface pressure used. The PM-IRRAS spectra obtained during compression of a native AP-GPI monolayer induced slight reversible modifications on PM-IRRAS spectra. From the amide I/amide H ratio and the a-helix band position, it can be deduced that the protein was anchored at the interface with the GPI standing up and the great axis of the ellipsoid model of AP-GPI remained parallel to the interface. The PM-IRRAS spectra obtained during compression and their simulated spectra were consistent with the existence of small movements of intramolecular protein domains. Such movements may be related to the allosteric properties of mammalian alkaline phosphatases.