Surface plasmon resonance for measuring TBP-promoter interaction

A procedure was developed for real-time measurement of the interaction between an archaeal TATA-binding protein (TBP) with stress-gene promoters from the archaeon Methanosarcina mazeii using surface plasmon resonance (SPR), the BIACORE 3000 equipment, and the SA (streptavidin) Sensor Chip. Measurements were based on the SPR optical phenomenon, which resulted in light extinction when TBP bound a promoter. This process, detected as a change in a particular angle, was recorded in a sensorgram. The BIA-evaluation program allowed the calculation of the equilibrium constant (K(A)) of the interaction of M. mazeii TBP with the promoters of the stress genes grpE, hsp70(dnaK), and hsp40(dnaJ) (0.47, 0.26, and 1.21x10(7)M(-1), respectively) and, for comparison, with the promoter of a non-heat-shock gene, orf16 (0.08x10(7)M(-1)). The association rate (k(a)) of the non-heat-shock gene orf16 was 0.4x10(4)M(-1)s(-1) and those for the stress genes, grpE, hsp70(dnaK), and hsp40(dnaJ) were higher: 2.8, 1.5, and 3.5x10(4)M(-1)s(-1), respectively. The new procedure will allow a comparative analysis of different TPBs and promoters (wild type and mutants) under physiologic and stress conditions, and a correlation of TBP binding parameters with constitutive and stress-induced gene expression.     

Computational Approaches for RNA Structure Ensemble Deconvolution from Structure Probing Data

RNA structure probing experiments have emerged over the last decade as a straightforward way to determine the structure of RNA molecules in a number of different contexts. Although powerful, the ability of RNA to dynamically interconvert between, and to simultaneously populate, alternative structural configurations, poses a nontrivial challenge to the interpretation of data derived from these experiments. Recent efforts aimed at developing computational methods for the reconstruction of coexisting alternative RNA conformations from structure probing data are paving the way to the study of RNA structure ensembles, even in the context of living cells. In this review, we critically discuss these methods, their limitations and possible future improvements.     

Helping Students Reconstruct Conceptions of Thermodynamics: Energy and Heat

Thermodynamics, specifically energy and heat, is a major concept in the foundations of physics and physical science. To determine a strategy to teach thermodynamics meaningfully, the authors conducted classroom action research using interviews to determine secondary physics students' current conceptions of thermodynamics. On the basis of the findings, the authors developed and implemented a science unit to facilitate students' reconstructions of their ideas toward more scientifically appropriate concepts. The lessons, using a learning cycle strategy, and results of the pre- and post-interviews are presented.     

Theoretical investigation of the effect of sugar substitution on the antioxidant properties of flavonoids

Natural flavonoids are secondary phenolic plant metabolites known for their bioactivity as antioxidants. The evaluation of this property is generally done by the estimation of their direct free radical-scavenging activity as hydrogen or electron donating compounds. This paper reviews experimental results available in the literature for a selection of flavonoids and compares them with calculated quantities characteristic of the hydrogen or electron donation. For that purpose, bond dissociation energies, ionization potentials and electron transfer enthalpies are computed by using DFT methods and the ONIOM procedure implemented in the ab initio program Gaussian. This process has been chosen because it can be extended to the study of large molecules. When acid dissociation and interaction with the solvent are taken into account, the results present very good concordance with experimental results, enlightening the complexity of the processes involved in the classical assays which measure the ability of compounds to scavenge the (2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) radical cation (ABTS ⁺) or the 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH^·). This study demonstrates the good accuracy of theoretical calculations in obtaining the relative energies involved in free radical scavenging abilities and its capacity for predictive behaviour. It also highlights the necessity to take into account the pK_a of the compounds and the solvent interaction. The ability of the method to calculate the antioxidant properties of larger molecules are tested on glycosylated flavonoids and the effects of sugar substitution on the antioxidant properties of flavonoids are investigated, pointing out the importance of the charges on the oxygen atoms.     

Molecular dynamics simulations on interaction between bacterial proteins: Implication on pathogenic activities

We perform molecular dynamics simulation studies on interaction between bacterial proteins: an outer‐membrane protein STY3179 and a yfdX protein STY3178 of Salmonella Typhi. STY3179 has been found to be involved in bacterial adhesion and invasion. STY3178 is recently biophysically characterized. It is a soluble protein having antibiotic binding and chaperon activity capabilities. These two proteins co‐occur and are from neighboring gene in Salmonella Typhi‐occurrence of homologs of both STY3178 and STY3179 are identified in many Gram‐negative bacteria. We show using homology modeling, docking followed by molecular dynamics simulation that they can form a stable complex. STY3178 belongs to aqueous phase, while the beta barrel portion of STY3179 remains buried in DPPC bilayer with extra‐cellular loops exposed to water. To understand the molecular basis of interaction between STY3178 and STY3179, we compute the conformational thermodynamics which indicate that these two proteins interact through polar and acidic residues belonging to their interfacial region. Conformational thermodynamics results further reveal instability of certain residues in extra‐cellular loops of STY3179 upon complexation with STY3178 which is an indication for binding with host cell protein laminin.     

Denaturation of MM-creatine kinase by sodium dodecyl sulfate

The denaturation of dimeric cytoplasmic MM-creatine kinase by sodium dodecyl sulfate (SDS) has been investigated using activity measurements, far-ultraviolet circular dichroism, SEC-HPLC, electric birefringence, intrinsic probes (cysteine and tryptophan residues), and an extrinsic fluorescent probe (ANS). Our results show that inactivation is the first detectable event; the inactivation curve midpoint is located around 0.9 mM SDS. The second event is dissociation and it occurs in parallel to tertiary and secondary perturbations, as demonstrated by the coincidence (near 1.3 mM) of the midpoints of the transition curves monitoring dissociation and structural changes. At high total SDS concentration (concentration higher than 2.5 mM), the monomer had bound 170 mol of SDS per mol of protein. In these conditions, electric birefringence experiments suggest that the SDS-CK complex may be described as a prolate ellipsoid with an axial ratio of 1.27 (14 nm×11 nm). These results are compatible with recent models of SDS-protein complexes: the protein decorated micelle structure or the necklace structure.     

Protein-protein interactions: mechanisms and modification by drugs

Protein-protein interactions form the proteinaceous network, which plays a central role in numerous processes in the cell. This review highlights the main structures, properties of contact surfaces, and forces involved in protein-protein interactions. The properties of protein contact surfaces depend on their functions. The characteristics of contact surfaces of short-lived protein complexes share some similarities with the active sites of enzymes. The contact surfaces of permanent complexes resemble domain contacts or the protein core. It is reasonable to consider protein-protein complex formation as a continuation of protein folding. The contact surfaces of the protein complexes have unique structure and properties, so they represent prospective targets for a new generation of drugs. During the last decade, numerous investigations have been undertaken to find or design small molecules that block protein dimerization or protein(peptide)-receptor interaction, or on the other hand, induce protein dimerization.     

Asymmetric Reduction by an NADH Model Compound with l-Prolinamide in the N1-Substituent

An intracellular uricase from Bacillus fastidiosus with high catalytic capacity and strong resistance to xanthine was inactivated in water but could be essentially re-activated in solutions of high ionic strength. By polyacrylamide gel electrophoresis (PAGE), gradient PAGE, sodium-dodecyl-sulfate-PAGE, gel-filtration through Sephadex G200, and activity staining with peroxidase and its chromatogenic substrate, this homotetrameric uricase in water was found to dissociate into inactive homodimers that could form active homotetramers again in solutions of high ionic strength. Sensitivity to low ionic strength of solutions complicates formulation of this uricase as a drug and its elimination requires protein engineering.     

Calculation of nucleation free energy for duplex oligomers in the context of nearest neighbor models

Additive physical properties of DNA double strand polymers have been expanded in terms of 8 irreducible parameters. This provided consistency relations among the corresponding 10 duplex dimer contributions. To allow for oligomer analysis, end parameters were often added, and this would add extra degrees of freedom to the fore mentioned parameters. Statistical mechanics approaches were then connected to the nearest neighbor (NN) approach in the framework of the two‐states model. Ad hoc end effects were thus (wrongly) correlated to nucleation phenomena and this lead to a critique for its role in NN modeling. With this motivation, a new NN model is proposed that accommodates the nucleation free energies. The model relates the nucleation free energy to the mean composition of the chain and permits to obtain a good estimate for the free energy associated only to the Watson–Crick base pairings. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 538–547, 2013.