CYSTEINE-SPECIFIC SURFACE TETHERING OF GENETICALLY-ENGINEERED CYTOCHROMES FOR FABRICATION OF METALLOPROTEIN NANOSTRUCTURES

The preparation of oriented metalloprotein nanostructures through intr oduction of specific and complementary reactive groups on the solid an d 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 conditi ons for producing monolayer protein coverages. These problems are larg ely circumvented by utilizing a two-step linker synthesis, in which th e surface is first prepared with a monolayer of (3-aminopropyl)silane (S-APS), and the resulting structure is derivatized with the heterobif unctional reagent N-succinimidyl 6-maleimidocaproate (EMCS). The malei mide functionality is then presented to the protein, into which a sing le 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 completio n. Finally substituing a gas-phase chemical vapor deposition procedure for the liquid-phase reaction of the 3-APS produces dramatically bett er control over coverage and quality of the resulting films.