DNA Binding to Protein−Gold Nanocrystal Conjugates

• Published in: Bioconjugate chemistry 2006-09, Vol.17 (5), p.1156-1161
• Main Authors: Calabretta, Michelle K, Matthews, Kathleen S, Colvin, Vicki L
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding sites; Deoxyribonucleic acid; DNA; DNA - metabolism; E coli; Electrophoretic Mobility Shift Assay; Escherichia coli; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gold - chemistry; Lac Repressors; Metal Nanoparticles - chemistry; Models, Molecular; Nanocrystals; Point Mutation; Protein Binding; Protein Structure, Tertiary; Proteins; Proteomics; Repressor Proteins - chemistry; Repressor Proteins - genetics; Repressor Proteins - metabolism; Sulfhydryl Compounds
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc0600867

The E. coli DNA binding protein lac repressor (LacI) and a derivative with a designed thiol (T334C) were developed as gold nanocrystal conjugates to assess the effects of conjugation on DNA binding function. The designed derivative was engineered with a solvent-accessible thiol to promote oriented conjugation, avoiding obstruction of the DNA-binding domain by the nanocrystal. Analytical ultracentrifugation (AU) and electrophoretic mobility shift assays (EMSA) were used to evaluate the ability of conjugated repressors to bind the natural operator DNA sequence O1. The results show that LacI does not retain significant DNA binding function when conjugated to gold nanocrystals, presumably because the basic DNA-binding domain is the site for nonspecific conjugation. T334C, with the potential for both directed and nonspecific conjugation, shows enhanced interaction with O1 when conjugated. Interestingly, the order of component addition is a key factor in producing functional lac repressor conjugates.