The crystal structures of most intracellular lipid binding proteins (LBPs) show between 5 and 20 internally bound water molecules, depending on the presence or the absence of ligand inside the protein cavity. The structural and functional significance of these waters has been discussed for several LBPs based on studies that used various biophysical techniques. The present work focuses on two very different LBPs, heart-type fatty acid binding protein (H-FABP) and ileal lipid binding protein (ILBP). Using high-resolution nuclear magnetic resonance spectroscopy, certain resonances belonging to side-chain protons that are located inside the water-filled lipid binding cavity were observed. In the case of H-FABP, the pH-and temperaturedependent behavior of selected side-chain resonances (Ser82 OgH and the imidazole ring protons of His93) indicated an unusually slow exchange with the solvent, implying that the intricate hydrogen-bonding network of amino-acid side-chains and water molecules in the protein interior is very rigid. In addition, holo H-FABP appeared to display a reversible self-aggregation at physiological pH. For ILBP, on the other hand, a more solvent-accessible protein cavity was deduced based on the pH titration behavior of its histidine residues. Comparison with data from other LBPs implies that the evolutionary specialization of LBPs for certain ligand types was not only because of mutations of residues directly involved in ligand binding but also to a refinement of the internal water scaffold.

Thrombospondin (TSP), a 420-kDa glycoprotein found in human platelet alpha gra-nula, is thought to play an important role in platelet haemostatic function. The aim of this study was to develop a rapid method to isolate TSP for functional and structural studies. For preparation we used two PRPs, each unit containing 2.6 ×1010 platelets. Washed platelets were stimulated with thrombin (1.5 U/ml), protease activity was inhibited by hirudin (5 U/ml) and DIFP (1 mM). Cellular fragments and micro-somes were separated by centrifugation (700g, 5min; 48,000g, 30min). The clear protein solution was further purified by HiTrap Heparin affinity chromatography and a following MonoQ ion exchange chromatography using a NaCl gradient (0-2 M) on an FPLC System. 800 µg/U TSP were isolated within a total separation time of 60min. Purity of the isolated fraction was proved by SDS PAGE showing no additional proteins. TSP-binding studies are now made feasible without long-term planning and set-up. This method may be a helpful tool for further characterisation of platelet TSP receptor.