Published: March 11, 2011 r2011 American Chemical Society 3808 dx.doi.org/10.1021/la200225t | Langmuir 2011, 27, 3808–3814 ARTICLE pubs.acs.org/Langmuir Sequential Assembly of an Active RNA Polymerase Molecule at the AirWater Interface Abantika Ganguly and Dipankar Chatterji* Molecular Biophysics Unit, Indian Institute of Science, Bangalore—560012, India b S Supporting Information ’ INTRODUCTION Inside a cell the ability of biomacromolecules to communicate with each other with specificity generates complex assemblies designed to carry out a specific biological function. Traditionally, biochemical and biophysical investigations carried out to study proteinprotein interactions involve responses between isolated biopolymers in homogeneous and dilute solutions. However, within the cell the concentration of these biopolymers is as high as 300400 mg/mL. 1 Recent studies have demonstrated that for biopolymers sharing a common milieu, the effect of macromo- lecular crowding may influence their interactions which cannot be sufficiently evaluated using an ideal condition. 1,2 Thus, our probing techniques need to be adapted to take into consideration nonideality effects in order to accurately describe biological regulatory mechanisms inside a living cell. Several methods have been used to emulate the cellular milieu, which include addition of crowding agents like PEG and Ficoll 2 or the immobilization of one of the interacting partners on a solid support in order to artificially increase the surface density of the molecules. Probing proteinprotein interactions on an immobilized solid support has far-reaching biological as well as technological implications ranging from single-molecule recognition studies to designing biosensor arrays and protein memory chips. 3 Some of the common methods used for immobilization include genera- tion of self-assembled monolayer (SAM) on glass or gold surfaces, 4 covalent coupling of engineered protein molecules on chemically modified gold nanoparticles, and polystyrene beads. 5 The im- mobilization technique becomes infinitely more challenging when one needs to preserve the activity of biological molecules. 6 Direct chemisorptions of thiol-modified proteins on inorganic supports often lead to denaturation problems. In recent years the LangmuirBlodgett technique has emerged as a powerful alter- native to immobilization on solid supports. 7,8 In a Langmuir trough the molar ratios of the various components added to generate a monolayer can be precisely controlled, as opposed to that of a SAM. The use of the LB technique for the study of macromolecules has already been demonstrated. But for biomo- lecules, the use of the LangmuirBlodgett technique has been mostly limited to biopolymers with surface activity that can easily adsorb at the surface thereby modifying the surface properties. 9 We have reported earlier the use of a Ni-arachidate monolayer to immobilize histidinetagged biomolecules at the interface. 1012 In the present study, we have extended this technique to follow proteinprotein interactions in a crowded environment . The Received: January 18, 2011 ABSTRACT: At the heart of understanding cellular processes lies our ability to explore the specific nature of communication between sequential information carrying biopolymers. However, the data extracted from conventional solution phase studies may not reflect the dynamics of communication between recognized partners as they occur in the crowded cellular milieu. We use the principle of immobilization of histidine-tagged biopolymers at a Ni(II)-encoded Langmuir monolayer to study sequence-specific proteinprotein interactions in an artificially crowded environ- ment. The advantage of this technique lies in increasing the surface density of one of the interacting partners that allows us to study macromolecular interactions in a controlled crowded environ- ment, but without compromising the speed of the reactions. We have taken advantage of this technique to follow the sequential assembly process of the multiprotein complex Escherichia coli RNA polymerase at the interface and also deciphered the role of one of the proteins, omega (ω), in the assembly pathway. Our reconstitution studies indicate that in the absence of molecular chaperones or other cofactors, omega (ω) plays a decisive role in refolding the largest protein beta prime (β 0 ) and its recruitment into the multimeric assembly to reconstitute an active RNA polymerase. It was also observed that the monolayer had the ability to distinguish between sequence-specific and -nonspecific interactions despite the immobilization of one of the biomacromolecules. The technique provides a universal two-dimensional template for studying proteinligand interactions while mimicking molecular crowding.