Crystallinity of Purple Membranes Comprising the Chloride-Pumping Bacteriorhodopsin Variant D85T and Its Modulation by pH and Salinity Daniel Rhinow, † Ivan Chizhik, ‡ Roelf-Peter Baumann, ‡ Frank Noll, ‡ and Norbert Hampp* ,‡,§ Max-Planck-Institute of Biophysics, Department of Structural Biology, Max-Von-Laue-Str. 3, D-60438 Frankfurt, Germany, Philipps-UniVersity of Marburg, Department of Chemistry, Hans-Meerwein-Str. Bldg. H, D-35032 Marburg, Germany, and Material Sciences Center, D-35032 Marburg, Germany ReceiVed: September 6, 2010 Self-assembly of membrane proteins inside the cell membrane critically depends on specific protein-protein and protein-lipid interactions. Purple membranes (PMs) from Halobacterium salinarum comprise wild-type bacteriorhodopsin (BR) and lipids only and form a 2-D crystalline lattice of P3 symmetry in the cell membrane. It is known that removal of the retinylidene residue as well as the exchange of selected amino acids lead to a loss of crystallinity. In PMs comprising the BR variant D85T, we have observed a tunable tendency to form crystalline domains, which depends on pH-value and chloride ion concentration. BR-D85T resembles the function of the chloride pump halorhodopsin. The protonation state of amino acid residues within the binding pocket and chloride binding in the vicinity of the protonated retinal Schiff base affect the overall shape of BR-D85T molecules in the membrane, thereby changing their interactions and subsequently their tendency to form crystalline areas. The combination of small-angle X-ray scattering, atomic force microscopy, and freeze-fracture electron microscopy enables us to analyze the transitions statistically as well as on the single membrane level. PM-D85T is a model system to study membrane protein association upon substrate binding in a native environment. Furthermore, the ability to reversibly modulate the crystallinity of PMs probably will be useful for the preparation of larger artificial crystalline arrays of BR and its variants. Introduction The light-driven proton pump bacteriorhodopsin (BR) is the key protein of halobacterial photosynthesis. 1-3 Within its native host, BR forms 2-D crystalline patches, the so-called purple membranes (PMs), from Halobacterium salinarum, which comprise BR trimers and lipids only. 2,4 BR is the prototype of seven transmembrane helix proteins and shares deep homologies with other microbial retinal proteins. 5-11 High-resolution struc- tural analysis of BR has revealed a detailed molecular picture of light-dependent vectorial proton transport through the cell membrane. 12-17 Furthermore, PM is an excellent model system to study the principles that are responsible for the assembly of integral membrane protein complexes in their native environ- ment. 4 A variety of studies have been performed to elucidate the molecular determinants of PM stability and crystalline assembly. For example, analysis of genetically modified BR variants has revealed several amino acid residues, which are critical for lattice assembly. 4,18 Furthermore, it has been shown that mutations in the core of BR can affect lattice assembly as well. 19 The importance of retinal, covalently bound to lysine 216, for structural integrity of BR and PM lattice assembly has been pointed out in another study. 20 In this work, we demonstrate how a single-point mutation in the core of BR results in PMs, which undergo structural changes from a noncrystalline state to a 2D-crystalline state, depending on the physicochemical conditions. In BR variant D85T, the primary proton acceptor aspartic acid 85 is replaced by threonine, the equivalent residue in halorhodopsin, which converts BR into a chloride pump. 21,22 By means of small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and atomic force microscopy (AFM), we demonstrate that PM-D85T, noncrystalline under most conditions, is con- verted to a highly ordered hexagonal 2-D crystalline state as soon as the physicochemical conditions favor chloride binding of BR-D85T, i.e., pH values below pH 6 and high salinity. A model is presented which explains the crystallization tendency of PM-D85T in terms of conformational changes on the level of single BR-D85T molecules. Materials and Methods Materials. PM wild-type (PM-WT), PM-D85T, PM-D96N, and PM-D96G/F171C/F219L (PM-Tri) were freshly purified according to standard procedures. 23 Small-Angle X-Ray Scattering. Oriented PM multilayers were prepared from PM suspended in distilled water or 0.1 M NaCl. Depending on the conditions desired, the pH was adjusted by phosphate buffer as well as dilute HCl. PM suspensions were deposited on a thin plastic substrate and slowly dried overnight. PM multilayers were analyzed with a SAXS apparatus compris- ing a Phillips PW1830 X-ray generator and a KKK-type Kratky camera (Anton Paar, Graz, Austria) with a sample detector distance of 200 mm. Electron Microscopy. PM-D85T suspensions were placed between two small copper disks and plunged into liquid ethane. Replicas were prepared in a Balzers BAF 400T freeze-fracture unit at 3 × 10 -7 mbar keeping the specimen stage at -130 °C. Shadowing with Pt/C was performed at an angle of 45°, followed by evaporation of pure carbon at 90° reinforcing the heavy metal replica. Replicas were thawed, cleaned from organic * Corresponding author. Phone: +49 6421 2825775. Fax: +49 6421 2825798. E-mail: hampp@staff.uni-marburg.de. † Max-Planck-Institute of Biophysics. ‡ Philipps-University of Marburg. § Material Sciences Center. J. Phys. Chem. B 2010, 114, 15424–15428 15424 10.1021/jp108502p  2010 American Chemical Society Published on Web 10/29/2010