Dynamic Stabilization Of Expressed Proteins In Engineered Diatom Biosilica

Self-assembly of biosilica with biosilica-immobilized recombinant proteins in genetically-engineered diatoms represents a means to construct functional materials in a reproducible and scalable manner, in applications that harness the inherent specificities of proteins to sense and respond to environmental cues. We report that functionalized frustules isolated from the diatom Thalassiosira pseudonana genetically-modified with a biosilica-immobilized single chain fragment variable (scFv) antibody against trinitrotoluene (TNT) or an enhanced green fluorescent protein (EGFP) contained in excess of 200,000 proteins per frustule. To characterize the dynamics of the immobilized proteins, the fluorescence of either a trinitrotoluene (TNT) surrogate compound bound to the scFv or the endogenous fluorescence of EGFP was used to monitor protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Proteins immobilized in isolated frustules exhibited isotropic rotational motions with two-fold reductions in rates of motion indicative of weak macromolecular associations that act to increase the effective viscosity and stabilize proteins. Solvent accessibilities and high-affinity (pM) binding affinities were comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retained binding affinity in the presence of chaotropic agents (i.e., 8 M urea). These results indicated that dramatic increases in protein conformational stability within the biosilica frustule matrices arise through molecular crowding, acting to retain native protein folds and associated functionality. This would permit engineered proteins to function under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.