Nanografting de novo proteins onto gold surfaces

The immobilization of novel proteins onto addressable locations on a flat surface has potential applications in a range of biotechnologies. Here we describe the nanopatterning of a de novo protein onto a gold surface. Patterning was achieved using a technique called nanografting, in which the tip of an atomic force microscope is used to disrupt a preexisting monolayer of alkanethiol molecules on a gold surface, thereby facilitating exchange with alternative thiol-linked molecules from the surrounding solution. The protein used for these studies was chosen from a designed combinatorial library of de novo sequences expressed in E. coli and was engineered to have a glycine-glycine-cysteine tag at its C-terminus, thereby enabling attachment to the gold surface through a single cysteine thiol. The average height of the grafted protein patterns was found to be somewhat higher than expected from the known NMR structure of the protein. Compression of the nanografted patches by an external force (below 10 nN) was reversible but showed some hysteresis. Interestingly, both the energy required to deform the immobilized protein patterns and the energy defined by the hysteresis loop were found to be of the same order as the energy required to unfold the monomeric protein in solution. These studies demonstrate the possibility of preparing nanometer scale protein arrays, lowering significantly the volume requirements of the protein samples necessary to fabricate protein-based biosensor arrays and thereby providing a base for increasing their sensitivity.