title: Modulating the activity of protein conjugated to gold nanoparticles by site-directed orientation and surface density of bound protein.
abstract: The key property of protein-nanoparticle conjugates is the bioactivity of the protein. The ability to accurately modulate the activity of protein on the nanoparticles at the interfaces is important in many applications. In the work reported here, modulation of the activity of protein-gold nanoparticle (AuNP) conjugates by specifically orienting the protein and by varying the surface density of the protein was investigated. Different orientations were achieved by introducing cysteine (Cys) residues at specific sites for binding to gold. We chose Escherichia coli inorganic pyrophosphatase (PPase) as a model protein and used site-directed mutagenesis to generate two mutant types (MTs) with a single Cys residue on the surface: MT1 with Cys near the active center and MT2 with Cys far from the active center. The relative activities of AuNP conjugates with wild type (WT), MT1, and MT2 were found to be 44.8%, 68.8%, and 91.2% of native PPase in aqueous solution. Site-directed orientation with the binding site far from the active center thus allowed almost complete preservation of the protein activity. The relative activity of WT and MT2 conjugates did not change with the surface density of the protein, while that of MT1 increased significantly with increasing surface density. These results demonstrate that site-directed orientation and surface density can both modulate the activity of proteins conjugated to AuNP and that orientation has a greater effect than density. Furthermore, increasing the surface density of the specifically oriented protein MT2, while having no significant effect on the specific activity of the protein, still allowed increased protein loading on the AuNP and thus increased the total protein activity. This is of great importance in the study on the interface of protein and nanoparticle and the applications for enzyme immobilization, drug delivery, and biocatalysis.
authors.affiliation: The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, Soochow University , Suzhou 215123, P. R. China.
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Hongwei
authors.initials: H
authors.lastname: Wang
authors.forename: Lin
authors.initials: L
authors.lastname: Yuan
authors.forename: Jingwen
authors.initials: J
authors.lastname: Li
authors.forename: John Law
authors.initials: JL
authors.lastname: Brash
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006046:Gold / Q000737:chemistry*; D043564:Inorganic Pyrophosphatase / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D006046:Gold; D043564:Inorganic Pyrophosphatase
keywords: Au−S bond; gold nanoparticles; protein; site-specific orientation; surface density