Characterization of molecular interactions between Escherichia coli RNA polymerase and topoisomerase I by molecular simulations.

Escherichia coli topoisomerase I (EctopoI), a type IA DNA topoisomerase, relaxes the negative DNA supercoiling generated by RNA polymerase (RNAP) during transcription elongation. Due to the lack of structural information on the complex, the exact nature of the RNAP-EctopoI interactions remains unresolved. Herein, we report for the first time, the structure-based modeling of the RNAP-EctopoI interactions using computational methods. Our results predict that the salt bridge as well as hydrogen bond interactions are responsible for the formation and stabilization of the RNAP-EctopoI complex. Our investigations provide molecular insights for understanding how EctopoI interacts with RNAP, a critical step for preventing hypernegative DNA supercoiling during transcription.

Investigating direct interaction between Escherichia coli topoisomerase I and RecA.

Protein-protein interactions are of special importance in cellular processes, including replication, transcription, recombination, and repair. Escherichia coli topoisomerase I (EcTOP1) is primarily involved in the relaxation of negative DNA supercoiling. E. coli RecA, the key protein for homologous recombination and SOS DNA-damage response, has been shown to stimulate the relaxation activity of EcTOP1. The evidence for their direct protein-protein interaction has not been previously established. We report here the direct physical interaction between E. coli RecA and topoisomerase I. We demonstrated the RecA-topoisomerase I interaction via pull-down assays, and surface plasmon resonance measurements. Molecular docking supports the observation that the interaction involves the topoisomerase I N-terminal domains that form the active site. Our results from pull-down assays showed that ATP, although not required, enhances the RecA-EcTOP1 interaction. We propose that E. coli RecA physically interacts with topoisomerase I to modulate the chromosomal DNA supercoiling.

Note: Model identification and analysis of bivalent analyte surface plasmon resonance data.

Surface plasmon resonance (SPR) is a widely used, affinity based, label-free biophysical technique to investigate biomolecular interactions. The extraction of rate constants requires accurate identification of the particular binding model. The bivalent analyte model involves coupled non-linear differential equations. No clear procedure to identify the bivalent analyte mechanism has been established. In this report, we propose a unique signature for the bivalent analyte model. This signature can be used to distinguish the bivalent analyte model from other biphasic models. The proposed method is demonstrated using experimentally measured SPR sensorgrams.

Analyzing surface plasmon resonance data: choosing a correct biphasic model for interpretation.

Surface plasmon resonance (SPR) has been widely used as a label-free biophysical technique to quantitatively study biochemical processes. For the SPR data fitting using a single exponential function, the procedure to extract the rate constants is straightforward. However, there is no simple procedure for SPR data fitting with double exponential functions. A highly non-linear fitting procedure is, therefore, used to fit the biphasic SPR data with numerical solutions of the rate equations. This procedure requires some prior knowledge of the underlying interaction mechanism, and the extracted rate constants often have large uncertainties. In this report, we propose a new method of analyzing the biphasic SPR data using the three commonly employed biphasic models. Our method is based on a general analytical solution of the biphasic rate equations, which is much more transparent and straightforward than the highly non-linear numerical integration approach. Our method can be used to determine the underlying biphasic interaction mechanism from the analysis of the SPR data and to extract the rate constants with high confidence levels. We have illustrated the procedures with examples of the data analysis on simulated SPR profiles, and the results are discussed.

Quantitative study of protein-protein interactions by quartz nanopipettes.

In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.

A surface plasmon resonance study of the intermolecular interaction between Escherichia coli topoisomerase I and pBAD/Thio supercoiled plasmid DNA.

To date, the bacterial DNA topoisomerases are one of the major target biomolecules for the discovery of new antibacterial drugs. DNA topoisomerase regulates the topological state of DNA, which is very important for replication, transcription and recombination. The relaxation of negatively supercoiled DNA is catalyzed by bacterial DNA topoisomerase I (topoI) and this reaction requires Mg(2+). In this report, we first quantitatively studied the intermolecular interactions between Escherichia coli topoisomerase I (EctopoI) and pBAD/Thio supercoiled plasmid DNA using surface plasmon resonance (SPR) technique. The equilibrium dissociation constant (Kd) for EctopoI-pBAD/Thio interactions was determined to be about 8 nM. We then studied the effect of Mg(2+) on the catalysis of EctopoI-pBAD/Thio reaction. A slightly higher equilibrium dissociation constant (~15 nM) was obtained for Mg(2+) coordinated EctopoI (Mg(2+)EctopoI)-pBAD/Thio interactions. In addition, we observed a larger dissociation rate constant (kd) for Mg(2+)EctopoI-pBAD/Thio interactions (~0.043 s(-1)), compared to EctopoI-pBAD/Thio interactions (~0.017 s(-1)). These results suggest that enzyme turnover during plasmid DNA relaxation is enhanced due to the presence of Mg(2+) and furthers the understanding of importance of the Mg(2+) ion for bacterial topoisomerase I catalytic activity.

Energetics of the dipole flip-flop motion in a ferroelectric polymer chain.

The authors report on a study of dipole flip-flop "local" transition in ferroelectric polyvinylidene fluoride [P(VDF)] chains, using total energy calculation based on the density functional theory. The calculated results indicate that a simple flipping of a single electric dipole moment is energetically allowed. Furthermore, such a flipping involves no change either in bond length, bond angle, or the orientation of the chain. The calculations also show that on a thin film of ordered chains, strong dipole interactions existing in P(VDF) could cause modulation of the dipole orientation thus forming superlattices on P(VDF) films. These results are in good agreement with recent scanning tunnel microscope experimental measurements. Furthermore, our calculations show that partial flipping may also exist and extend over a length of several monomers during the flip-flop transition.