Ma. Firestone et al., FILM ARCHITECTURE IN BIOMOLECULAR ASSEMBLIES - EFFECT OF LINKER ON THE ORIENTATION OF GENETICALLY-ENGINEERED SURFACE-BOUND PROTEINS, Journal of the American Chemical Society, 118(38), 1996, pp. 9033-9041
This contribution presents strategies for the optimization of supramol
ecular architecture aimed at controlling the organization of biomolecu
les at solid surfaces. Myoglobin, modified by site-directed mutagenesi
s to include a unique cysteine residue, is selectively chemisorbed to
self-assembled haloalkylsilylated silica surfaces of varying n-alkyl c
hain length (n = 2, 3, 8, 11, 15) to yield a series of surface-immobil
ized recombinant protein assemblies. These supramolecular assemblies a
re probed using tapping mode atomic force microscopy, wettability meas
urements, Fourier transform infrared spectroscopy, and linear dichrois
m spectroscopy to determine how the individual components comprising t
hese structures (substrate, silane coupling layer, and protein) influe
nce macromolecular protein ordering and stability. Surface roughness i
s found to be a minor contributor in the determination of macromolecul
ar ordering in these assemblies. In contrast, the nature of the underl
ying silane self-assembled coupling layer is shown to strongly influen
ce both the spatial and functional properties of the chemisorbed prote
in. 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 surfac
es also form ordered structures. The stability of myoglobin appended t
o long chain aliphatic silylated surfaces is poor, however. The appare
nt protein instability arises due to the increased hydrophobic charact
er of these films. At intermediate alkyl chain length (n = 8), a confo
rmationally disordered coupling layer with a high concentration of gau
che defects is produced, regardless of the method of silane deposition
or postdeposition processing. Chemisorption of myoglobin to the highl
y disorganized assembly yields a random orientation of the protein.