Langmuir Monolayer as a Tool toward Visualization of a Specific DNA−Protein Complex

Abstract

Immobilization and imaging of protein molecules and protein-DNA complexes on a Langmuir-Blodgett (LB) substrate have been explored here. We have prepared a nickel-arachidate (NiA) monolayer and characterized it through pressure-area isotherm on a LB trough. Recombinant RNA polymerase from Escherichia coli, where the largest subunit was replaced with the same gene having a series of histidine amino acids at the C-terminus end of the protein, was immobilized over the Ni-arachidate monolayer through a Ni(II)-histidine interaction. A single molecule of RNA polymerase could be seen through intermittent-contact atomic force microscopy (AFM). Under the condition of the formation of the LB monolayer, the enzyme molecules were arrayed and transcriptionally active. Interestingly, they could pick up sequence specific DNA molecules from the subphase in an oriented fashion. On the other hand, preformed RNA polymerase Ni(II)-arachidate monolayers bound DNA haphazardly when no surface pressure was employed.

A Comparative Kinetic and Thermodynamic Perspective of the σ-Competition Model in Escherichia coli

“…cm). At this pH, the carboxyl group of arachidate molecules will remain ionized and interact with the Ni þ2 present in the subphase to form a Ni-arachidate template (19). To form the NiA-HisRNAP monolayer, core HisRNAP was injected just below the preformed NiA monolayer after 30 min to a final concentration of 7.4 nM.…”

Co-evolution of RNA polymerase with RbpA in the phylum Actinobacteria

“…SM07 is a recombinant strain derived from mc 2 155, harboring a chromosomal hexa-histidine tag on the rpoC gene (Mukherjee and Chatterji, 2008). RNA polymerase from E. coli was purified from strain RL916 (gift from Prof. Robert Landick, University of Wisconsin) (Brar et al, 2005). Jmc 2 155 and JRmc 2 155, are the plasmid transformed versions of mc 2 155, carrying the plasmids pJAM2 (Triccas et al, 1998) and pJAM2MsRbpA (Dey et al, 2010), respectively.…”

“…RNA polymerase was purified from M. smegmatis strain SM07 and E. coli strain RL916 using protocols mentioned previously (Mukherjee and Chatterji, 2008, Brar et al, 2005). MsRbpA was purified using Ni-NTA affinity chromatography (Dey et al, 2010).…”

Effect of water and air–water interface on the structural modification of Ni-arachidate Langmuir–Blodgett films

“…Moreover, nickel arachidate (NiA) LB films have been used to immobilize proteins and protein-DNA complexes on some solid substrate [8] through Ni-histidine interaction. This Ni-histidine interaction is commonly used in immobilized metal ion affinity chromatography (IMAC) for the purification of His-tagged proteins and peptides [9].…”