DNA binding to protein-gold nanocrystal conjugates.
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 O(1). 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 O(1) when conjugated. Interestingly, the order of component addition is a key factor in producing functional lac repressor conjugates.
Calabretta
Michelle K
MK
Department of Chemistry, Rice University, 6100 Main Street, Houston, Texas 77005, USA. mcal@rice.edu
Matthews
Kathleen S
KS
Colvin
Vicki L
VL
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D056968:Lac Repressors; D053768:Metal Nanoparticles / Q000737:chemistry*; D008958:Models, Molecular; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013438:Sulfhydryl Compounds
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D013438:Sulfhydryl Compounds; D006046:Gold; D004247:DNA