Film architecture in biomolecular assemblies. Effect of linker on the orientation of genetically engineered surface-bound proteins

This contribution presents strategies for the optimization of supramolecular architecture aimed at controlling the organization of biomolecules at solid surfaces. Myoglobin, modified by site-directed mutagenesis to include a unique cysteine residue, is selectively chemisorbed to self-assembled haloalkylsilylated silica surfaces of varying n-alkyl chain length (n = 2, 3, 8, 11, 15) to yield a series of surface-immobilized recombinant protein assemblies. These supramolecular assemblies are probed using tapping mode atomic force microscopy, wettability measurements, Fourier transform infrared spectroscopy, and linear dichroism spectroscopy to determine how the individual components comprising these structures (substrate, silane coupling layer, and protein) influence macromolecular protein ordering and stability. Surface roughness is found to be a minor contributor in the determination of macromolecular ordering in these assemblies. In contrast, the nature of the underlying silane self-assembled coupling layer is shown to strongly influence both the spatial and functional properties of the chemisorbed protein. Silane coupling layers with short aliphatic chain lengths (n = 2, 3) produce highly trans-conformationally ordered structures upon which differential heme prosthetic group orientation cain be achieved. Long alkyl chain (n greater than or equal to 11) silane-derivatized surfaces also form ordered structures. The stability of myoglobin appended to long chain aliphatic silylated surfaces is poor, however. The apparent protein instability arises due to the increased hydrophobic character of these films. At intermediate alkyl chain length (n = 8), a conformationally disordered coupling layer with a high concentration of gauche defects is produced, regardless of the method of silane deposition or postdeposition processing. Chemisorption of myoglobin to the highly disorganized assembly yields a random orientation of the protein.