title: Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices.
abstract: Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.
authors.affiliation: Department of Basic Medical Sciences, Western University of Health Sciences , Lebanon, Oregon 97355, United States.
authors.forename: Yijia
authors.initials: Y
authors.lastname: Xiong
authors.affiliation: Marine Biotechnology Group, Pacific Northwest National Laboratory , Sequim, Washington 98382, United States.
authors.forename: Nicole R
authors.initials: NR
authors.lastname: Ford
authors.affiliation: Marine Biotechnology Group, Pacific Northwest National Laboratory , Sequim, Washington 98382, United States.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Hecht
authors.affiliation: Marine Biotechnology Group, Pacific Northwest National Laboratory , Sequim, Washington 98382, United States.|Department of Microbiology, Oregon State University , Corvallis, Oregon 97331, United States.
authors.forename: Guritno
authors.initials: G
authors.lastname: Roesijadi
authors.affiliation: Department of Basic Medical Sciences, Western University of Health Sciences , Lebanon, Oregon 97355, United States.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Squier
mesh_terms: D017377:Diatoms; D004738:Engineering; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D055550:Protein Stability; D017433:Protein Structure, Secondary; D012822:Silicon Dioxide / Q000737:chemistry*; D057127:Single-Chain Antibodies / Q000737:chemistry*
chemical_list: D057127:Single-Chain Antibodies; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012822:Silicon Dioxide