Interaction of Organic Solvents with Protein Structures at Protein-Solvent Interface
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J Mol Model (2013) 19:4701–4711 DOI 10.1007/s00894-012-1507-z ORIGINAL PAPER Interaction of organic solvents with protein structures at protein-solvent interface Morteza Khabiri & Babak Minofar & Jan Brezovský & Jiří Damborský & Rudiger Ettrich Received: 25 December 2011 /Accepted: 13 June 2012 /Published online: 4 July 2012 # Springer-Verlag 2012 Abstract The effect of non-denaturing concentrations of haloalkane dehalogenases and provided an opportunity to three different organic solvents, formamide, acetone and explore effects of organic solvents on the quaternary struc- isopropanol, on the structure of haloalkane dehalogenases ture. Penetration and trapping of organic solvents in the DhaA, LinB, and DbjA at the protein-solvent interface was network of interactions between both monomers depends studied using molecular dynamics simulations. Analysis of on the physico-chemical properties of the organic solvents. B-factors revealed that the presence of a given organic Consequently, both monomers of this enzyme oscillate dif- solvent mainly affects the dynamical behavior of the ferently in different organic solvents. With the exception of specificity-determining cap domain, with the exception of LinB in acetone, the structures of studied enzymes were DbjA in acetone. Orientation of organic solvent molecules stabilized in water-miscible organic solvents. on the protein surface during the simulations was clearly dependent on their interaction with hydrophobic or hydro- Keywords Molecular dynamics . Non-aqueous media . philic surface patches, and the simulations suggest that the Organic solvents . Solvent orientation behavior of studied organic solvents in the vicinity of hyro- phobic patches on the surface is similar to the air/water interface. DbjA was the only dimeric enzyme among studied Introduction : : M. Khabiri B. Minofar R. Ettrich (*) A wide range of chemical agents used in industrial and Institute of Nanobiology and Structural Biology of GCRC, agricultural activities are released into the environment as Academy of Sciences of the Czech Republic, Zamek 136, chemical contaminants. A large number of these compounds 373 33 Nove Hrady, Czech Republic belong to haloalkanes. Several organisms produce various e-mail: [email protected] enzymes which are able to hydrolytically cleave the carbon- : halogen bond in halogenated compounds like halogenated M. Khabiri R. Ettrich – Faculty of Sciences, University of South Bohemia in Ceske alkanes, cycloalkanes and alcohols [1 3]. These enzymes Budejovice, are called haloalkane dehalogenases (HLDs), and they be- Zamek 136, long to the α/β-hydrolase protein superfamily [4]. They use 373 33 Nove Hrady, Czech Republic a hydrolytic mechanism to transform haloalkane into an J. Brezovský : J. Damborský organic halide ion and an alcohol. Structurally these Loschmidt Laboratories, Department of Experimental Biology enzymes are comprised of two different domains [5]. The and Research Centre for Toxic Compounds in the Environment, larger, so-called main domain is conserved in all α/β-hydro- Faculty of Science, Masaryk University, lases and contains a central β-sheet. The smaller domain is Kamenice 5/A13, α β− 625 00 Brno, Czech Republic less conserved within the / fold superfamily, and is : called cap domain. The active site is located between these J. Brezovský J. Damborský two domains in an internal hydrophobic cavity and can be ’ International Clinical Research Center, St. Anne s University reached through a tunnel to the bulk of the solvent [4–6]. Hospital Brno, Pekarska 53, Potential application of HLDs for decontamination of 656 91 Brno, Czech Republic environmental pollutants makes them interesting targets 4702 J Mol Model (2013) 19:4701–4711 for medium engineering to improve their catalytic efficiency amino acids inside the protein structure. In contrast, non- [7]. Three HLDs with known crystal structure and different polar amino acid side chains become more flexible. It has substrate specificity were selected for this study; DbjA from been shown that reorientation of polar amino acid side chains Bradyrhizobium japonicum USDA110 [8], DhaA from Rho- from the surface to the core increases the total number of dococcus rhodochrous NCIMB 13064 [9], and LinB from hydrogen bonds and salt bridges inside the enzyme structure, Sphingomonas paucimobilis UT26 [10]. The substrate spe- making it more rigid in comparison to the enzyme structure in cificities of these HLDs are mainly due to differences in the pure water media [18–20]. Moreover, it has been shown that geometry and the composition of the active site, and the the amount of water molecules on the enzyme surface in non- entrance tunnel connecting the active site to the protein aqueous media is one of the key factors for enzyme stability surface (Fig. 1;[11]). Recently, it has been shown experi- [21]. Taken together, we can say that the effect of organic mentally that the activity of these three enzymes is signifi- solvents on enzyme structure depends on the physicochemical cantly affected by the nature and concentration of different property of amino acids located on the protein surface and the organic solvents [12]. physicochemical property of organic solvent. Water is the essential solvent for life, and so far the water While the general fold of a protein is determined and exclusive properties and its unique capabilities as a solvent preserved by intra-molecular interactions, as are hydrophobic, have drawn broad attention in biochemistry, while inability van der Waals, hydrogen bonding and electrostatic interac- of this unique solvent to solvate many non-polar molecules tions [22], solvent molecules are crucial for the extra- were not considered [13, 14]. It is expected that mixing of molecular hydrophobic interactions and dynamic behavior of water with water miscible solvents such as organic solvents the enzyme. In solution both, enzyme and solvent, have could compensate for some of the shortcomings in water mutual effects. Hereby, not only do general media properties properties [15]. The main advantages of mixtures of water influence the overall enzyme structure, but also particular with organic solvents can be summarized as: (1) speed up solvent molecules close to the protein surface can have spe- general reaction rates, (2) provide an environment for reac- cific interactions with the enzyme. Therefore, the nature of the tions in which hydrophobic interactions are involved in the enzyme surface determines solvent interaction and structure in complex formation between enzyme and substrate and (3) its vicinity. Adding organic solvent molecules that have a dual overcome unwanted water-dependent side reactions [1, 16, nature of both, hydrophobic and hydrophilic properties, to the 17]. Changing the reaction media from physiological to water changes its properties as a physiological solvent [23, non-physiological ones influences the structure and function 24]. These organic solvents are considered as co-solvents, of the enzyme. The solvent accessible surface of the enzyme which enhance hydrophobicity of the medium, i.e., reducing is in the first place influenced by changing the reaction its polarity and hence dielectric constant. media. Many studies show that addition of organic solvents In this work, we investigated the effect of three selected to enzyme water solutions decreases the number of water organic solvents/-water mixtures, formamide 5 %, acetone molecules in the first solvation shell of the protein [18, 19]. 10 %, and isopropanol 20 %, on the tertiary structure of In fact, organic molecules repel water molecules from the DhaA, LinB, and DbjA and the quaternary structure of surface. Consequently, polar amino acid side chains become DbjA, with special focus on the behavior of these organic more rigid, and prefer favorable interactions with other solvents at the protein surface. Fig. 1 3-D models of the crystal structures of dehalogenases DhaA (PDB ID 1CQW), LinB (PDB ID 1 MJ5) and DbjA (PDB ID 3A2M). Structures are represented by ribbons (top) and molecular surfaces (bottom). Tunnels connecting the active site with bulk solvent are shown in green. Hydrophobic surface patches are in orange and hydrophilic surface patches are in blue. The tunnels were calculated by CAVER [46] and structures were visualized in Pymol [39] J Mol Model (2013) 19:4701–4711 4703 Methods water according to the pH memory phenomenon described by Klibanov [15]. Molecular dynamics Initially, all crystal structures were energy minimized in vacuo using a short steepest descent protocol for at least Three-dimensional structures of the organic molecules were 1000 step. The minimized crystal structures were then sol- optimized, and partial charges were calculated in Gaussian 03 vated in pre-equilibrated extended simple point-charge employing the Hartree-Fock method and the 6-31 G* basic (SPCE) water [32], formamide 5 % (v/v), isopropanol set [25]. Geometries were optimized at the MP2/6-31 G* 10 % (v/v) and acetone 20 % (v/v) in a rectangular box with level. Topologies of all organic molecules were generated a minimum distance of 1.5 nm between the protein and the using MKTOP [26] for the all atom optimized potentials for box edges. For each organic solvent solution the pre- liquid simulation (OPLSAA) force field [27–29]. Three dif- equilibration constituted 200 ns of MD simulation to fully ferent mixtures of organic co-solvents with water [formamide equilibrate the water-solvent mixture. After solvation of the 5 % (v/v), acetone 20 % (v/v) and isopropanol 10 % (v/v)]