Ordered structure formation of bacteriorhodopsin-hDHFR in a plasma membrane

This paper describes a novel surface-processing technique aimed at the in v ivo formation of ordered functionalized structures on surfaces. The essenti al feature of this technique is the utilization of an intrinsic and stable two-dimensional crystal of bacteriorhodopsin (bR) as a template. A simple t echnique to form a functional chimera of the halophilic enzyme dihydrofolat e reductase (hDHFR) with bR is demonstrated. The gene encoding hDHFR from H aloferax volcanii (H. volcanii) was conjugated to that encoding bR (bop) fr om Halobacterium salinarum (H. salinarum). This chimera was expressed in a bop-deficient strain of H. salinarum. The novel bifunctional fusion protein bR-hDHFR was localized in the plasma membrane of H. salinarum and retained the intrinsic characteristics of each component. Microscopic patches compo sed of ordered bR-hDHFR molecules were observed on the plasma membrane by e lectron microscopy. The hDHFR portion of the chimera was detected on the cy toplasmic side of each patch, which confirms that the molecular orientation of the fused proteins was vectorially controlled. The molecular packing of the fusion protein closely resembled the ordered structure of the wild-typ e bR in the "purple membrane". which forms a two-dimensional crystal. This technique for the immobilization of functional proteins on a surface is app licable to a wide range of proteins for organizing ordered supramolecular s urfaces on the nanometer scale.