Covalent immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembled monolayers for scanning probe microscopy.

We have worked out a procedure for covalent binding of native biomacromolecules on flat gold surfaces for scanning probe microscopy in aqueous buffer solutions and for other nanotechnological applications, such as the direct measurement of interaction forces between immobilized macromolecules, of their elastomechanical properties, etc. It is based on the covalent immobilization of amino group-containing biomolecules (e.g., proteins, phospholipids) onto atomically flat gold surfaces via omega-functionalized self-assembled monolayers. We present the synthesis of the parent compound, dithio-bis(succinimidylundecanoate) (DSU), and a detailed study of the chemical and physical properties of the monolayer it forms spontaneously on Au(111). Scanning tunneling microscopy and atomic force microscopy (AFM) revealed a monolayer arrangement with the well-known depressions that are known to stem from an etch process during the self-assembly. The total density of the omega-N-hydroxysuccinimidyl groups on atomically flat gold was 585 pmol/cm(2), as determined by chemisorption of (14)C-labeled DSU. This corresponded to approximately 75% of the maximum density of the omega-unsubstituted alkanethiol. Measurements of the kinetics of monolayer formation showed a very fast initial phase, with total coverage within 30 S. A subsequent slower rearrangement of the chemisorbed molecules, as indicated by AFM, led to a decrease in the number of monolayer depressions in approximately 60 min. The rate of hydrolysis of the omega-N-hydroxysuccinimide groups at the monolayer/water interface was found to be very slow, even at moderately alkaline pH values. Furthermore, the binding of low-molecular-weight amines and of a model protein was investigated in detail.     

The electrogenic, Na+-dependent I- transport system in plasma membrane vesicles from thyroid glands

Using vesicles from the plasma membrane of hog thyroid, we have characterized its Na+-dependent I- transport system. We have found it to be totally Na+ dependent; K+ cannot substitute and Li+ can partially substitute for Na+; the Na+:I- flux ratio is larger than one; the system is electrogenic, being stimulated by a delta psi negative inside the vesicles. A number of large, lipophilic anions are fully-competitive inhibitors of Na+-dependent I- uptake; the closer their atomic radii are to that of iodine, the smaller their Ki values.     

Membrane topology of light-harvesting protein B870-alpha of Rhodospirillum rubrum G-9+. Amino acid residues in contact with the lipid bilayer as inferred from labeling with photogenerated carbenes

The amino acid residues of the light-harvesting protein B870-alpha of Rhodospirillum rubrum G-9+ that in the chromatophore membranes are in contact with the lipid phase were identified by hydrophobic photolabeling. Three reagents have been used which all contained the trifluoromethyldiazirinylphenyl group as a photo-sensitive precursor of a carbene but which otherwise differed in shape, molecular structure, and in the way they interact with membranes. 3-Trifluoromethyl-3-(m-[125I]iodophenyl)diazirine is a highly lipid-soluble compound, 11-[4-[(trifluoromethyl)diazirinyl]-phenyl]-[10-3H] 9-oxaundecanoic acid is an analogue of a fatty acid, and 1-palmitoyl-2-[11-[(trifluoromethyl)diaziri-nyl] phenyl]-[10-3H]9-oxaundecanoyl]-sn-glycero-3-phosphorylcholine one of a lecithin. Following labeling of chromatophores with these reagents, B870-alpha was isolated and subjected to (solid phase) Edman degradations in order to determine individual amino acid residues labeled. The main features of these results are as follows. 1) Labeling occurred both within the N-terminal segment (residues 1-8) and within the predominantly hydrophobic transmembrane stretch (residues 14-33). 2) Label distribution patterns within segments are indicative of helical structures to which the reagents had access to one face only of the cylindrical envelopes. 3) With regard to the transmembrane segment, the label distribution patterns were similar for all reagents whereas striking differences were noticed within the N-terminal portion. The labeling patterns are consistent with previous models proposing tight association of the transmembrane helix with that of the B870-beta chain. They also suggest that the N-terminal segment forms an amphipathic helix which interacts with the water-lipid interface of the membrane.     

Interaction of the sugar carrier of intestinal brush-border membranes with HgCl2

HgCl2 was used as an inhibitor and potential label for the glucose carrier of intestinal brush-border membranes. Half-maximal inhibition of Na+-dependent D-glucose uptake was reached with micromolar concentrations of HgCl2 when the protein concentration was 1.2 mg/ml. Similar concentrations were found to inhibit the binding of [3H]phlorizin, a reversible competitive inhibitor of sugar transport. Inhibition was reversed by dithioerythritol but only marginally by EDTA. The data support the involvement of a sulfhydryl group in the inhibitory process. Deoxycholate-extracted membranes, which are enriched in specific phlorizin binding activity, were used for labeling studies using 203HgCl2. The polypeptides were separated by gel electrophoresis and analyzed by protein staining and autoradiography. Non-specific 203HgCl2 labeling was minimized by pre-treatment with sulfhydryl reagents which do not inhibit phlorizin binding. Several bands, which are lost from the autoradiographic pattern during a negative purification of the phlorizin binding sites, could be ruled out as essential components of the sugar carrier. The polypeptide profile was also analyzed following proteolysis, which abolished phlorizin binding. Those radioactive bands of which apparent Mr values were alterd by the treatment were considered as possible candidates. Finally, samples in which inhibition was reversed by thiols were also studied. The possible identity of the polypeptide(s) involved in glucose translocation is disussed in the light of these observations.