Cysteine-Specific Surface Tethering of Genetically Engineered Cytochromes for Fabrication of Metalloprotein Nanostructures

The preparation of oriented metalloprotein nanostructures through introduction of specific and complementary reactive groups on the solid and protein surfaces is critically dependent on the reaction conditions used to prepare the solid surface. Key problems include the hydrolytic stability of the Si─O bond, the low reactivity of simple nucleophilic silane reagents, protein physisorption, and identification of conditions for producing monolayer protein coverages. These problems are largely circumvented by utilizing a two-step linker synthesis, in which the surface is first prepared with a monolayer of (3-aminopropyl)silane (3-APS), and the resulting structure is derivatized with the heterobifunctional reagent N-succinimidyl 6-maleimidocaproate (EMCS). The maleimide functionality is then presented to the protein, into which a single unique cysteine residue has been introduced by genetic engineering techniques. Hydrolytic stability is dramatically enhanced by including a postreaction curing step, in which the solid surface temperature is elevated to drive the alkoxysilane condensation reaction to completion. Finally substituing a gas-phase chemical vapor deposition procedure for the liquid-phase reaction of the 3-APS produces dramatically better control over coverage and quality of the resulting films.