Purushottam Tiwari

Mass transport through vertically aligned large diameter MWCNTs embedded in parylene

We have fabricated porous membranes using a parylene encapsulated vertically aligned forest of multi-walled carbon nanotubes (MWCNTs, about 7 nm inner diameter). The transport of charged particles in electrolyte through these membranes was studied by applying electric field and pressure. Under an electric field in the range of 4.4 × 10(4) V m(-1), electrophoresis instead of electroomosis is found to be the main mechanism for ion transport. Small molecules and 5 nm gold nanoparticles can be driven through the membranes by an electric field. However, small biomolecules, like DNA oligomers, cannot. Due to the weak electric driving force, the interactions between charged particles and the hydrophobic CNT inner surface play important roles in the transport, leading to enhanced selectivity for small molecules. Simple chemical modification on the CNT ends also induces an obvious effect on the translocation of single strand DNA oligomers and gold nanoparticles under a modest pressure (<294 Pa).

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.

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.

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 cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD) in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP). Here, we first show that surface plasmon resonance (SPR) can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC), which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels.

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.

Investigating molecular interactions between oxidized neuroglobin and cytochrome c

The formation of a complex between neuroglobin (Ngb) and cytochrome c (Cyt c) has an important biological role in preventing apoptosis. Binding of Ngb to Cyt c alone is sufficient to block the caspase 9 activation by ferric Cyt c that is released during ischemic insults. Therefore, a detailed information on the Ngb-Cyt c interactions is important for understanding apoptosis. However, the exact nature of the interactions between oxidized human neuroglobin (hNgb) and Cyt c is not well understood. In this work, we used a combination of computational modeling and surface plasmon resonance experiments to obtain and characterize the complex formation between oxidized hNgb and Cyt c. We identified important residues involved in the complex formation, including K72 in Cyt c, which is otherwise known to interact with the apoptotic protease-activation factor-1. Our computational results, together with an optimized structure of the hNgb-Cyt c complex, provide unique insights into how the hNgb-Cyt c complex can abate the apoptotic cascade without an hNgb-Cyt c redox reaction.

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.