Multimode Analysis of Nanoscale Biomolecular Interactions

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores

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

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

Experimental and Computational Studies Reveal Novel Interaction of Lymphocytes Antigen 6K to TGF-β Receptor Complex

TGF-β signaling promotes migration, invasion, and distant colonization of cancer cells in advanced metastatic cancers. TGF-β signaling suppresses the anti-tumor immune response in a tumor microenvironment, allowing sustained tumor growth. TGF-β plays an important role in normal physiology; thus it is no surprise that the clinical development of effective and safe TGF-β inhibitors has been hampered due to their high toxicity. We discovered that increased expression of LY6K in cancer cells led to increased TGF-β signaling and that inhibition of LY6K could lead to reduced TGF-β signaling and reduced in vivo tumor growth. LY6K is a highly cancer-specific protein, and it is not expressed in normal organs except in the testes. Thus, LY6K is a valid target for developing therapeutic strategies to inhibit TGF-β signaling in cancer cells. We employed in vitro pull-down assays and molecular dynamics simulations to understand the structural determinants of the TGF-β receptor complex with LY6K. This combined approach allowed us to identify the critical residues and dynamics of the LY6K interaction with the TGF-β receptor complex. These data are critical in designing novel drugs for the inhibition of TGF-β in LY6K expressing cancer, induction of anti-tumor immune response, and inhibition of tumor growth and metastatic spread