Structure differences between protein crystals and solution structures monitored by Raman spectroscopy – case study of PsbP protein

V. Kopecký Jr.¹, K. Hofbauerová^1,3, J. Kohoutová², M. Lapkouski², Ž. Sovová², I. Kutá Smatanová^2,4, R. Ettrich^2,4

¹Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, 12116 Prague 2, Czech Republic

²Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zámek 136, 37333 Nové Hrady, Czech Republic

³Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220 Prague 4, Czech Republic

⁴Institute of Physical Biology, University of South Bohemia, Zámek 136, 37333 Nové Hrady, Czech Republic

kopecky@karlov.mff.cuni.cz

Raman spectroscopy gives a unique opportunity to study protein samples in different phases. It is possible to measure intact protein crystals directly in hanging drops in the crystallization boxes they actually grow and to study the protein structure in crystals as well as chemical reactions in single crystals [1]. Moreover, we apply a new technique of Raman spectroscopy – a drop coating deposition Raman (DCDR) method [2], based on a coffee ring effect, that enables measurements of solutions down to 1 µM concentrations. However, our recent work adverted to subtle differences which correspond to the glass-like phase of the deposited samples [3]. Thus, DCDR protein samples represent a "phase transition" between saturated protein solutions and crystals. This enables to distinguish spectral differences given by the density of molecules in crystals from those caused by protein crystal artifacts.

Here we illustrate the new approach by Raman spectroscopy application on PsbP protein of photosystem II from Spinacia oleracea [4]. The protein is a part of the so-called oxygen-evolving complex, which is involved in photosynthesis. Recombinant PsbP protein was prepared as thrombin-digested recombinant His-tagged PsbP protein overexpressed in Escherichia coli. The crystal structure of PsbP protein from Spinacia oleracea was solved to the resolution of 2.06 Å [5]. Subsequently, Raman spectra of PsbP have been collected from solvent, from the DCDR deposit and from the crystal sample. Mutual comparison of these Raman spectra enabled to point on shortcomings in the crystal structure with respect to the structure in a solution. Moreover, these informations gave the possibility to model unresolved parts of the crystal [4].

References

1. P. R. Carey, J. Dong, Biochemistry, 43, (2004), 8885.

2. D. Zhang, Y. Xie, M. F. Mrozek, C. Ortiz, V. J. Davisson, D. Ben-Amotz, Anal. Chem., 75, (2003), 5703.

3. J. Kapitán J., V. Baumruk, V. Kopecký Jr., R. Pohl, P. Bouř, J. Am. Chem. Soc., 128, (2006), 13451.

4. V. Kopecký Jr., J. Kohoutová, M. Lapkouski, K. Hofbauerová, Ž. Sovová, I. Kutá Smatanová, J. L. Revuelta, J. B. Arellano, R. Ettrich, J. Mol. Biol., (2010), submitted.

5. J. Kohoutová, I. Kutá Smatanová, J. Brynda, M. Lapkouski, J. L. Revuelta, J. B. Arellano, R. Ettrich, Spinacia olerace. Acta Cryst., F65, (2009), 111.

Acknowledgements.

The Grant Agency of the Academy of Sciences of the Czech is gratefully acknowledged for the support (No. KJB101120805)