Structural measurements on membrane psbH protein in different lipid/-detergent environments

Wolfgang Schoefberger¹, Zbyněk Halbhuber¹, Jaroslava Ristvejova¹, Norbert Müller², Rüdiger Ettrich¹ and Dalibor Štys¹

¹Laboratories of High Performance Computing and Biomembranes ,Institute of Physical Biology USB and Institute of Landscape Ecology, ASCR, Zámek 136, 373 33 Nové Hrady, Czech Republic

² Johannes Kepler Universität, Altenbergerstrasse 69, Linz, Austria

One of the key components for the assembly of Photosystem II is the psbH protein [1]. PsbH is one of the proteins expressed in etiolated and illuminated leaves on the same level in higher plants, which indicates that its function may be considered separately from the rest of the multiprotein complex.

The PsbH protein of cyanobacterium Synechocystis sp. PCC 6803 was expressed as a fusion protein with glutathione-S transferase (GST) in E. coli [2]. We isolated the 15N labeled PsbH protein in concentration of 1.1 mg/ml in presence of detergent octyl glucoside (OG). We also isolated non-labeled protein for preliminary lipid titration experiments measured by circular dichroism (CD) spectrometer. Molecular Dynamics experiments on a homolgy model of the PsbH protein were carried out to compare the secondary structure changes with the results from CD.

The liposomes were prepared by reverse-phase evaporation technique from the thylakoid membrane lipids; sulphoquinovosyl diaglyceride (SQDG), digalactosyl diglyceride (DGDG), monogalactosyl diglyceride (MGDG) and phosphatidyl glycerol (PG). The most favourable lipid, which induced complex protein folding, detected as formation of the negative band approx. 222 nm in CD spectra, seemed to be PG. Very similar changes were observed at higher concentration also in SQDG, however folding of clearly different nature was achieved upon titration by DGDG. This indicates that the protein folding may not be directly related to specific binding of lipids, rather we observe two different types of folding in lipid bilayers of two different properties.

The CD measurements revealed folding of the PsbH protein in detergent micelles after addition of sufficiant amount of lipid. We added to each protein sample appropriate amount of the lipid to reach optimal protein/lipid ratio. Unfortunately NMR measurements showed a huge decrease of signal and recording of the remaining ¹⁵N signals into a narrow area. This would indicate very rigid lipid-protein micelles, which relax to fast to be recorded.

Micelle destabilisation using sonication or temperature increase led to only partial improvement, therefore we added into the sample new detergents; CHAPS and digitonin. The simple addition of CHAPS or digitonin did not destabilize micelles sufficiently and we had to remove lipids by dialysis. After dialysis signal recovered, moreover the new peaks indicated the further protein folding. A comparison of the secondary structure content with the molecular dynamics results lead to the conclusion that the combination of digitonin and octyl glucoside is the most effective combination to induce apparent protein folding.

Acknowledgements

This research was supported by AKTION Austria-Czech Republic (No. 40p2), by the Ministry of Education, Youth and Sports of the Czech Republic (MSM6007665808) and by the Academy of Sciences of the Czech Republic (Institutional research concept AVOZ60870520)  

1. Komenda, J., Štys, D. and Lupínková, L. The PsbH protein of photosystem 2. Photosynthetica 41, 1-7, 2003.

2. Halbhuber Z., Petrmichlová Z., Alexciev K., Thulin E. & Štys D., Protein Exp. Purif., 32-1 (2003) 18-27.