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.

We have studied surface and spectroscopic properties of Photosystem II core complex (PS II CC) for the first time in monolayers at the nitrogen/water interface. A new instrument was thus specially built to perform absorption and fluorescence spectroscopic measurements directly at the nitrogen/water interface. The effect of initial surface density, incubation time, and compression speed have been studied. When PS II CC was spread at an initial surface pressure of 5.7 mN/m and immediately compressed at a speed of 40 nm 2 /molecule‚min, it retained its native spectroscopic characteristics. Even though a slower speed of compression (10 nm 2 /molecule‚min) produced more homogeneous films, the absorption maxima suffered a blue shift, indicating denaturation of PS II CC. Compression at a speed of 80 nm 2 /molecule‚min produced aggregates of intact PS II CC as indicated by ∆V-A isotherms, absorption spectra, and fluorescence micrographs. We also conclude that spreading of PS II CC at an initial surface pressure of 0.6 mN/m followed by a 30 min incubation time is inadequate to maintain PS II CC surface and spectral properties. Indeed, π-A and ∆V-A isotherms measured in that condition showed transitions which suggested that PS II CC underwent physical changes during compression. Moreover, absorption and fluorescence maxima were blue shifted, indicating that PS II CC is denatured under that condition.

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Photosystem II core complex (PSII CC) absorbs light energy and triggers a series of electron transfer reactions by oxidizing water while producing molecular oxygen. Synthetic lipids with different alkyl chains and spacer lengths bearing functionalized headgroups were specifically designed to bind the Q(B) site and to anchor this large photosynthetic complex (240 kDa) in order to attempt two-dimensional crystallization. Among the series of different compounds that have been tested, oxygen evolution measurements have shown that dichlorophenyl urea (DCPU) binds very efficiently to the Q(B) site of PSII CC, and therefore, that moiety has been linked covalently to the headgroup of synthetic lipids. The analysis of the monolayer behavior of these DCPU-lipids has allowed us to select ones bearing long spacers for the anchoring of PSII CC. Oxygen evolution measurements demonstrated that these long-spacer DCPU-lipids specifically bind to PSII CC and inhibit electron transfer. With the use of atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), it was possible to visualize domains of PSII CC bound to DCPU-lipid monolayers. SNOM imaging has enabled us to confirm that domains observed by AFM were composed of PSII CC. Indeed, the SNOM topography images presented similar domains as those observed by AFM, but in addition, it allowed us to determine that these domains are fluorescent. Electron microscopy of these domains, however, has shown that the bound PSII CC was not crystalline.

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