Self-assembled monolayers from a designed combinatorial library of de novo β-sheet proteins

A variety of naturally occurring biomaterials owe their unusual structural and mechanical properties to layers of β-sheet proteins laminated between layers of inorganic mineral. To explore the possibility of fabricating novel two-dimensional protein layers, we studied the self-assembly properties of de novo proteins from a designed combinatorial library. Each protein in the library has a distinct 63 amino acid sequence, yet they all share an identical binary pattern of polar and nonpolar residues, which was designed to favor the formation of six-stranded amphiphilic β-sheets. Characterization of proteins isolated from the library demonstrates that (i) they self assemble into monolayers at an air/water interface; (ii) the monolayers are dominated by β-sheet secondary structure, as shown by both circular dichroism and infrared spectroscopies; and (iii) the measured areas (500- 600 Å2) of individual protein molecules in the monolayers match those expected for proteins folded into amphiphilic β-sheets. The finding that similar structures are formed by distinctly different protein sequences suggests that assembly into β-sheet monolayers can be encoded by binary patterning of polar and nonpolar amino acids. Moreover, because the designed binary pattern is compatible with a wide variety of different sequences, it may be possible to fabricate β-sheet monolayers by using combinations of side chains that are explicitly designed to favor particular applications of novel biomaterials.

Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.

This paper describes a method based on experimentally simple techniques--microcontact printing and micromolding in capillaries--to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. The technique uses replica molding in poly(dimethylsiloxane) molds having micrometer-scale relief patterns on their surfaces to form a contoured film of polyurethane supported on a glass slide. Evaporation of a thin (< 12 nm) film of gold on this surface-contoured polyurethane provides an optically transparent substrate, on which SAMs of terminally functionalized alkanethiolates can be formed. In one procedure, a flat poly(dimethylsiloxane) stamp was used to form a SAM of hexadecanethiolate on the raised plateaus of the contoured surface by contact printing hexadecanethiol [HS(CH2)15CH3]; a SAM terminated in tri(ethylene glycol) groups was subsequently formed on the bare gold remaining in the grooves by immersing the substrate in a solution of a second alkanethiol [HS(CH2)11(OCH2CH2)3OH]. Then this patterned substrate was immersed in a solution of fibronectin, the protein adsorbed only on the methyl-terminated plateau regions of the substrate [the tri(ethylene glycol)-terminated regions resisted the adsorption of protein]; bovine capillary endothelial cells attached only on the regions that adsorbed fibronectin. A complementary procedure confined protein adsorption and cell attachment to the grooves in this substrate.

Molecular organization of histidine-tagged biomolecules at self-assembled lipid interfaces using a novel class of chelator lipids.

In molecular biology, the expression of fusion proteins is a very useful and well-established technique for the identification and one-step purification of gene products. Even a short fused sequence of five or six histidines enables proteins to bind to an immobilized metal ion chelate complex. By synthesis of a class of chelator lipids, we have transferred this approach to the concept of self-assembly. The specific interaction and lateral organization of a fluorescent fusion molecule containing a C-terminal oligohistidine sequence was studied by film balance techniques in combination with epifluorescence microscopy. Due to the phase behavior of the various lipid mixtures used, the chelator lipids can be laterally structured, generating two-dimensional arrays of histidine-tagged biomolecules. Because of the large variety of fusion proteins already available, this concept represents a powerful technique for orientation and organization of proteins at lipid interfaces with applications in biosensing, biofunctionalization of nanostructured interfaces, two-dimensional crystallization, and studies of lipid-anchored proteins.