Docking, molecular dynamics and QM/MM studies to delineate the mode of binding of CucurbitacinE to F-actin

CucurbitacinE (CurE) has been known to bind covalently to F-actin and inhibit depolymerization. However, the mode of binding of CurE to F-actin and the consequent changes in the F-actin dynamics have not been studied. Through quantum mechanical/molecular mechanical (QM/MM) and density function theory (DFT) simulations after the molecular dynamics (MD) simulations of the docked complex of F-actin and CurE, a detailed transition state (TS) model for the Michael reaction is proposed. The TS model shows nucleophilic attack of the sulphur of Cys257 at the beta-carbon of Michael Acceptor of CurE producing an enol intermediate that forms a covalent bond with CurE. The MD results show a clear difference between the structure of the F-actin in free form and F-actin complexed with CurE. CurE affects the conformation of the nucleotide binding pocket increasing the binding affinity between F-actin and ADP, which in turn could affect the nucleotide exchange. CurE binding also limits the correlated displacement of the relatively flexible domain 1 of F-actin causing the protein to retain a flat structure and to transform into a stable ``tense'' state. This structural transition could inhibit depolymerization of F-actin. In conclusion, CurE allosterically modulates ADP and stabilizes F-actin structure, thereby affecting nucleotide exchange and depolymerization of F-actin. (C) 2015 Elsevier Inc. All rights reserved.

Electron transfer amongst flavo- and hemo-proteins: diffusible species effect the relay processes, not protein-protein binding

Hitherto, electron transfer (ET) between redox proteins has been deemed to occur via donor-acceptor binding, and diffusible reactive species are considered as deleterious side-products in such systems. Herein, ET from cytochrome P450 reductase (CPR, an animal membrane flavoprotein) and horseradish peroxidase (HRP, a plant hemoprotein) to cytochrome c (Cyt c, a soluble animal hemoprotein) was probed under diverse conditions, using standard assays. ET in the CPR-Cyt c system was critically inhibited by cyanide and sub-equivalent levels of polar one-electron cyclers like copper ions, vitamin C/Trolox and superoxide dismutase. In the presence of lipids, inhibition was also afforded by amphipathic molecules vitamin E, palmitoyl-vitamin C and the membrane hemoprotein, cytochrome b(5). Such nonspecific inhibition (by diverse agents in both aqueous and lipid phases) indicated that electron transfer/relay was effected by small diffusible agents, whose lifetimes are shortened by the diverse radical scavengers. When CPR was retained in a dialysis membrane and Cyt c presented outside in free solution, ET was still observed. Further, HRP (taken at nM levels) catalyzed oxidation of a phenolic substrate was significantly inhibited upon the incorporation of sub-nM levels of Cyt c. The findings imply that CPR-Cyt c or HRP-Cyt c binding is not crucial for ET. Further, fundamental quantitative arguments (based on diffusion/collision) challenge the erstwhile protein-protein binding-assisted ET hypothesis. It is proven beyond reasonable doubt that mobile and diffusible electron carriers (ions and radicals) serve as ``redox-relay agents'' in the biological ET models/setup studied.

Structural analysis of dihydrofolate reductases enables rationalization of antifolate binding affinities and suggests repurposing possibilities

Antifolates are competitive inhibitors of dihydrofolate reductase ( DHFR), a conserved enzyme that is central to metabolism and widely targeted in pathogenic diseases, cancer and autoimmune disorders. Although most clinically used antifolates are known to be target specific, some display a fair degree of cross-reactivity with DHFRs from other species. A method that enables identification of determinants of affinity and specificity in target DHFRs from different species and provides guidelines for the design of antifolates is currently lacking. To address this, we first captured the potential druggable space of a DHFR in a substructure called the `supersite' and classified supersites of DHFRs from 56 species into 16 `site-types' based on pairwise structural similarity. Analysis of supersites across these site-types revealed that DHFRs exhibit varying extents of dissimilarity at structurally equivalent positions in and around the binding site. We were able to explain the pattern of affinities towards chemically diverse antifolates exhibited by DHFRs of different site-types based on these structural differences. We then generated an antifolate-DHFR network by mapping known high-affinity antifolates to their respective supersites and used this to identify antifolates that can be repurposed based on similarity between supersites or antifolates. Thus, we identified 177 human-specific and 458 pathogen-specific antifolates, a large number of which are supported by available experimental data. Thus, in the light of the clinical importance of DHFR, we present a novel approach to identifying differences in the druggable space of DHFRs that can be utilized for rational design of antifolates.

Mechano-Chemical Coupling at Molecular Level: Forced Dissociation of Selectin/Ligand Binding

Mechano-chemical coupling is a common phenomenon that exists in various biological processes at different physiological levels. Bone tissue remodeling strongly depends on the local mechanical load. Leukocytes are sheared to form the transient aggregates with platelets or other leukocytes in the circulation. Flow pattern affects the signal transduction pathways in endothelial cells. Receptor/ligand interactions are important to cell adhesion since they supply the physical linkages...

Investigation of biotin-streptavidin binding interactions using microcantilever sensors

We report the investigation of biotin-streptavidin binding interactions using microcantilever sensors. A symmetric cantilever construction is employed to minimize the effects of thermal drift and the control of surface chemistry on the backside of the cantilever is demonstrated to reduce the effects of non-specific binding interactions on the cantilever. Three structurally different biotin modified cantilever surfaces are used as a model system to study the binding interaction with streptavidin. The cantilever response to the binding of streptavidin on these biotin sensing monolayers is compared. The lowest detection limit of streptavidin using biotin-HPDP is found to be between 1 and 10 nM limited by the optical measurement setup. Surface characterization using quartz crystal microbalance (QCM) and high-resolution atomic force microscope (AFM) is used to benchmark the cantilever sensor response. In addition, the QCM and AFM studies reveal that the surface density of bound streptavidin on biotin modified surfaces was low, thereby implying that effects other than steric hindrance are responsible for defining cantilever response. (c) 2006 Elsevier B.V. All rights reserved.

Quantifying Cell Binding Kinetics Mediated By Surface-Bound Blood Type B Antigen To Immobilized Antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

Measuring Binding Kinetics Of Surface-Bound Molecules Using The Surface Plasmon Resonance Technique

Surface plasmon resonance (SPR) technology and the Biacore biosensor have been widely used to measure the kinetics of biomolecular interactions in the fluid phase. In the past decade, the assay was further extended to measure reaction kinetics when two counterpart molecules are anchored on apposed surfaces. However, the cell binding kinetics has not been well quantified. Here we report development of a cellular kinetic model, combined with experimental procedures for cell binding kinetic measurements, to predict kinetic rates per cell. Human red blood cells coated with bovine serum albumin and anti-BSA monoclonal antibodies (mAbs) immobilized on the chip were used to conduct the measurements. Sensor-grams for BSA-coated RBC binding onto and debinding from the anti-BSA mAb-immobilized chip were obtained using a commercial Biacore 3000 biosensor, and analyzed with the cellular kinetic model developed. Not only did the model fit the data well, but it also predicted cellular on and off-rates as well as binding affinities from curve fitting. The dependence of flow duration, flow rate, and site density of BSA on binding kinetics was tested systematically, which further validated the feasibility and reliability of the new approach. Crown copyright (c) 2008 Published by Elsevier Inc. All rights reserved.

Changes in the chloride content of ground water in Pinellas County, Florida between 1947 and 1956

In December 1956 the U. S. Geological Survey, in cooperation with the Florida Geological Survey and the Board of County Commissioners of Pinellas County, collected waterlevel and chloride content of water in 94 wells in Pinellas County. First sampled in 1947, resampling and reanalyzing the water from these wells was used to determine the change in the chloride content of the ground water from 1947 to 1956. The chloride content of ground water is generally a reliable indication of the contamination of ground water by sea water, as 90 percent of the dissolved solids of sea water are chloride salts. (PDF contains 15 pages.)

New Oncogenic Drivers in Hepatocellular Carcinoma: Role of the RNA Binding Protein Hu antigen R

156 p. : graf.