Protein & Peptide Letters, 2012, 19, 791-794 791 Effect of the Compatible Solute Ectoine on the Stability of the Membrane Proteins Arpita Roychoudhury 1 , Dieter Häussinger 2 and Filipp Oesterhelt 1, * 1 Institute for Physical Biology, Universitätsstr. 1;Geb. 26.12.U1.78, Heinrich Heine University, 40225 Düsseldorf, Germany; 2 Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University Düsseldorf, Moorenstrasse 5, Düsseldorf 40225, Germany Abstract: Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar con- centrations do not interfere with cell metabolism. They are known to protect proteins against external stress like tempera- ture, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by sin- gle molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action. Keywords: Atomic force microscopy, membrane proteins, force spectroscopy, compatible solutes, ectoine. INTRODUCTION Microorganisms produce and accumulate compatible solutes in the cytoplasm aiming for protecting themselves from environmental stress. “Compatible solutes” generally have a low molecular weight, mostly either uncharged or zwitterionic organic molecules including polyols, sugars, amino acids and their derivatives. These types of solutes help maintaining osmotic balance without interfering with the essential cellular processes. Since they have relatively little effect on the cytosolic ionic strength, no special adaptation of the molecular pathways is required even at molar concen- trations [1, 2]. Ectoine is a heterocyclic amino acid (Fig. 1) that serves as a protective substance in many bacterial cells [3, 4, 5]. It allows microorganisms to resist extreme living conditions like drastic temperature variations [2, 6, 7], high salinity [8] and osmotic shocks [9, 10, 11]. Hence it is also known as an osmoprotectant, which is obtained from halophototrophic bacteria. These effects are also based on the direct stabilisa- tion of cytoplasmatic proteins against denaturation [12, 13, 14]. However little is known about the stabilising effects of ectoine on membrane proteins. Figure 1. Chemical structure of Ectoine *Address correspondence to this author at the Institute for Physical Biology, Universitätsstr. 1; Geb. 26.12.U1.78, Heinrich Heine University, 40225 Düsseldorf, Germany; Tel: +49(0)211-81-13805; Fax: +49(0)211-81-15167; E-mail: Filipp.Oesterhelt@uni-duesseldorf.de Transmembrane proteins account for approximately 30% of all proteins and play an important role in almost all cellu- lar processes. They act as sensors, catalysts, receptors, trans- porters and channels and perform essential vital functions. Hence the stabilization of these proteins is very important for maintaining a multitude of molecular pathways and thus cru- cial for the survival of the cell. Here we present experimental results that reveal the stabilising effects of Ectoine against denaturation of a membrane protein. We investigated this effect of ectoine on Bacteriorhodopsin, by performing me- chanical unfolding experiments. Atomic force microscopy [15] is a powerful tool to ob- serve, image and characterize membrane proteins in their physiological environment at the molecular level [16, 17, 18, 19, 20]. Single molecule force spectroscopy offers novel ways to investigate the stabilisation of membrane proteins. It provides detailed insights into inter and intra molecular in- teractions and enables us to go beyond the ensemble average [21, 22, 23]. Force spectroscopy applies a force to single molecules while observing the extraction of the protein from the mem- brane. By contacting the protein with the AFM tip, the pro- tein terminus can be attached to the tip by unspecific or spe- cific (e.g. thiol) interactions [21]. While pulling back the tip we apply an increasing force to the terminus and detect the protein unfolding by measuring length change of its unfolded part, being stretched between the tip and the support reveal- ing (un)folding intermediates. For investigation BR purple membrane patches [24, 25] were adsorbed onto a freshly cleaved mica surface. Unfold- ing experiments were done on single proteins after selecting a patch by imaging and subsequent positioning of the tip above a selected patch (as previously described) [21, 26]. HN N H H 3 C COO - /12 $58.00+.00 © 2012 Bentham Science Publishers