' MATERIALS AND METHODS Materials. 1,2-Dipalmitoyl-sn-glycero-3 phosphoethanolamine (DPPE) and 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (DTPC) were from Avanti Polar Lipids (Alabaster, AL). 6-[9-[2,3-Bis(dodecyloxy)propyl]-3,6,9-trioxanonyl-1-oxycarboxylamino]-2-[di(carboxymethyl)amino]hexanoic acid (NTA-TRIG-DLGE) was custom synthesized by Neuftech Chemicals (Vancouver, BC). HEPES was purchased from Fisher Bioreagents (Fair Lawn, NJ), and all other high-purity salts were from Aldrich (St. Louis, MO). The neoglycolipid Man 9 GlcNAc 2 -DPPE was synthesized according to described procedures. 18 DC-SIGNR Expression and Purification. Restriction fragments from vectors used for bacterial expression of the extracellular domains of three variants of human DC-SIGNR 19 were moved into a vector encoding an N-terminal His 6 tag previously designed for use with DC-SIGN. 18 The vector encodes the sequence Met-Ala-His-His-His-His-His-His-Gly-Glu-Leu, which begins at residue 101 of the full-length DC-SIGNR protein, and corresponds to the middle of the first 23-amino acid repeat in the extracellular domain. The tagged protein was purified by affinity chromatography on mannoseÀSepharose following the protocol used for the untagged extracellular domain. 20 The affinity purification confirms that the CRDs are folded and active. Biochemistry ARTICLE DC-SIGNR Immobilization on Planar Lipid Bilayers. The recombinant proteins with hexahistidine tags at the N-termini of the extracellular segments of DC-SIGNR were immobilized on asymmetric, planar lipid bilayers, which were supported on mica sheets affixed to hemicylindrical silica disks with 1À2 cm radii of curvature. 18 The first layer is a gel phase DPPE monolayer that was transferred at a surface pressure of 38 mN/m by LangmuirÀ Blodgett deposition onto the mica. DPPE was transferred from a pure water subphase at 21°C. At this temperature and pressure, the area per lipid is 43 Å 2 . The outer leaflet is a 100% NTA-TRIG-DLGE monolayer deposited onto the DPPE monolayer at 65 Å 2 per lipid, from a subphase of buffer A (20 mM HEPES, 50 mM NaNO 3 , 3 mM Ca(NO 3 ) 2 , and 50 μM NiSO 4 at pH 7.8) at 21°C. The deposition surface pressure was 35 mN/m, and the NTA-TRIG-DLGE monolayer is in the fluid phase at 21°C. The resulting supported bilayer was kept under buffer A at all times. It was then incubated for 1.5 h with 0.5 μM of either of the hexahistidine-tagged 7-, 6-, or 5-repeat DC-SIGNR variants in buffer A. Nonspecifically adsorbed protein was removed by rinsing the bilayer with buffer A.