A systematic method for analysing the protein hydration structure of T4 lysozyme J. Kysilka a,b and J. Vondrášek a * A systematic method for the analysis of the hydration structure of proteins is demonstrated on the case study of lysozyme. The method utilises multiple structural data of the same protein deposited in the protein data bank. Clusters of high water occupancy are localised and characterised in terms of their interaction with protein. It is shown that they constitute a network of interconnected hydrogen bonds anchored to the protein molecule. The high occupancy of the clusters does not directly correlate with water–protein interaction energy as was originally hypothesised. The highly occupied clusters rather correspond to the nodes of the hydration network that have the maximum number of hydrogen bonds including both the protein atoms and the surrounding water clusters. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: protein hydration structure; water; X-ray crystallography; cluster algorithm; interaction enthalpy INTRODUCTION Water evidently affects the majority of biological processes. It assists to proteins during the folding process (Papoian et al., 2004) or stabilises the active conformation of enzymes (Wester et al., 2003; Shaltiel et al., 1998). Water molecules are also abundant at protein–protein interfaces (Papoian et al., 2004; Janin, 1999; Rodier et al., 2005) and mediate the interaction between protein and ligand (Rejto and Verkhivker, 1997; Palomer et al., 2000; Ni et al., 2001). In these cases, water mole- cules form a stabilising hydrogen-bonded network that enables molecular recognition (Rejto and Verkhivker, 1997; Lemieux, 1996). Understanding the structure and dynamics of protein hydration is therefore of great importance for the explanation of biological phenomena. As most of the globular proteins naturally occur in a water-dominated environment, it is crucial to consider their structure, function and dynamics in the context of their interac- tion with ubiquitous water molecules (Levitt and Park, 1993; Rupley and Careri, 1991; Purkiss et al., 2001). Water exhibits its characteristic physico-chemical properties and dynamics on proteins mainly because of the formation of hydrogen bonds (Daniel et al., 2004; Finney, 2004). As the polar groups of the protein have a strong tendency to participate in hydrogen bonding, the surrounding water molecules bind to the protein in the whole range of stability and temporal persistence. As a result of these interactions, water and protein influence each other to a great extent. The water layer covering the protein surface has clearly different properties from the bulk water (Despa et al., 2004; Purkiss et al., 2001). Reversely, the modes of the motion of this hydration shell can trigger large-scale motions of the protein or have a great influence on the protein dynamics. (Ansari et al., 1985; Hayward et al., 1993; Umezawa et al., 2010) Numerous experimental (Billeter, 1995; Ferrand et al ., 1993; Loris et al ., 1994; Nakasako, 2002; Otting, 1997; Otting, 1998; Shou et al ., 2011; Syvitski et al., 2002) and theoretical (Bui et al., 2007; Friedman et al., 2005; Ni et al., 2001; Park and Saven, 2005; Tsui et al., 2000; Umezawa et al., 2007; Virtanen et al., 2010) approaches have been utilised to explore and describe the hydration structure of proteins. The most important fact is that hydration water molecules can be determined by the X-ray crystallography method. However, X-ray studies at ambient temperature reliably display only the most stable bound water molecules—called buried waters (Park and Saven, 2005). These waters form an integral part of the protein structure and contribute significantly to its stabilisation (Ebbinghaus et al., 2007; Park and Saven, 2005; Lu et al., 2007; Takano et al., 1999). It has been shown that they preferably occupy the protein main-chain polar groups, which are not part of any secondary structure and whose hydro- gen bonding is therefore not saturated (Park and Saven, 2005). The positions of these hydration sites tend to be conserved to a great degree in similar structures (Loris et al., 1994; Shaltiel et al., 1998; Sreenivasan and Axelsen, 1992). Cryogenic X-ray crystal structure analysis revealed a large-scale network of hydrogen bonds, (Nakasako, 1999; Yokomizo et al., 2005) which are also observable in molecular dynamics simulation trajectories (Komeiji et al., 1993; Pettitt et al., 1998). These hydration networks can link several secondary structures and greatly influence the dynamic properties of a protein. Water molecule aggregates of various shapes and dimensions cover also hydrophobic residues, and the increase in the number of hydration waters was prominent on flat and electrostatically neutral surface areas (Nakasako, 1999). It is supposed that under normal conditions, the hydrogen bond network undergoes reorganisation, accompanying the motion of * Correspondence to: J. Vondrášek, Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, 166 10 Prague, Czech Republic. E-mail: jiri.vondrasek@uochb.cas.cz a J. Kysilka, J. Vondrášek Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, 166 10 Prague, Czech Republic b J. Kysilka Faculty of Natural Sciences, Charles University in Prague, Albertov 2, 120 00 Prague, Czech Republic Research Article Received: 10 January 2013, Revised: 7 June 2013, Accepted: 8 June 2013, Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jmr.2290 J. Mol. Recognit. 2013; 26: 479–487 Copyright © 2013 John Wiley & Sons, Ltd. 479