Polarization-Modulated Infrared Spectroscopy and X-Ray Reflectivity of Photosystem II Core Complex at the Gas-Water Interface

Abstract

The state of photosystem II core complex (PS II CC) in monolayer at the gas-water interface was investigated using in situ polarization-modulated infrared reflection absorption spectroscopy and x-ray reflectivity techniques. Two approaches for preparing and manipulating the monolayers were examined and compared. In the first, PS II CC was compressed immediately after spreading at an initial surface pressure of 5.7 mN/m, whereas in the second, the monolayer was incubated for 30 min at an initial surface pressure of 0.6 mN/m before compression. In the first approach, the protein complex maintained its native alpha-helical conformation upon compression, and the secondary structure of PS II CC was found to be stable for 2 h. The second approach resulted in films showing stable surface pressure below 30 mN/m and the presence of large amounts of beta-sheets, which indicated denaturation of PS II CC. Above 30 mN/m, those films suffered surface pressure instability, which had to be compensated by continuous compression. This instability was correlated with the formation of new alpha-helices in the film. Measurements at 4 degreesC strongly reduced denaturation of PS II CC. The x-ray reflectivity studies indicated that the spread film consists of a single protein layer at the gas-water interface. Altogether, this study provides direct structural and molecular information on membrane proteins when spread in monolayers at the gas-water interface.

Insertion of Lipidated Ras Proteins into Lipid Monolayers Studied by Infrared Reflection Absorption Spectroscopy (IRRAS)

“…In the following years, a series of investigations was performed to determine the orientation of peptides and proteins at the air-water interface and also incorporated into lipid monolayers (44). In most cases the PM-IRRAS measurements were performed only at a constant angle of incidence of ;75°due to technical reasons and the value of the amide-I/amide-II ratio was taken to determine the orientation of the secondary structure units (45)(46)(47)(48).…”

Determination of the Contribution of the Myristoyl Group and Hydrophobic Amino Acids of Recoverin on its Dynamics of Binding to Lipid Monolayers

“…This method overcomes the water absorption limitation using rapid polarization modulation of the incident beam which allows the observation of infrared absorption bands of phospholipids and proteins at the monomolecular level (42,43). Furthermore, it is possible to extract information from the infrared spectra on conformation and orientation of molecules such as peptides and phospholipids present at the air/water interface (6,(44)(45)(46)(47)(48)(49)(50)(51)(52) as well as the kinetics of hydrolysis of monolayers by enzymes (53)(54)(55)(56).…”

Anisotropic Optical Constants of Bacteriorhodopsin in the Mid-Infrared: Consequence on the Determination of α-Helix Orientation

“…In purple membranes, bR exists as a two-dimensional cr ystal. 7±10 Bacteriorhodopsin was thus chosen because it is structurally very stable when spread in monolayers at the air/water interface compared to other proteins, 11,12 and it is very easy to handle. Moreover, it has been previously well characterized by ATR 13 and by electron 8,14 and Xray 9 diffraction, and it shows a de® ned orientation.…”