New insights into the effect of mutations on affibody-Fc interaction, a molecular dynamics simulation approach.

Staphylococcal protein A (SpA) domain B (the basis of affibody) has been widely used in affinity chromatography and found therapeutic applications against inflammatory diseases through targeting the Fc part of immunoglobulin G (IgG). We have performed extensive molecular dynamics simulation of 41 SpA mutants and compared their dynamics and conformations to wild type. The simulations revealed the molecular details of structural and dynamics changes that occurred due to introducing point mutations and helped to explain the SPR results. It was observed in some variants a point mutation caused extensive structural changes far from the mutation site, while an effect of some other mutations was limited to the site of the mutated residue. Also, the pattern of hydrogen bond networks and hydrophobic core arrangements were investigated. We figured out mutations that occurred at positions 128, 136, 150 and 153, affected two hydrophobic cores at the interface as well as mutations introduced at positions 129 and 154 interrupted two hydrogen bond networks of the interface, SPR data showed all of these mutations reduced binding affinity significantly. Overall, by scanning the SpA-Fc interface through the large numbers of introduced mutations, the new insights have been gained which would help to design high- affinity ligands of IgG.

Three-dimensional modeling of streptomycin binding single-stranded DNA for aptamer-based biosensors, a molecular dynamics simulation approach.

Streptomycin (STR) an aminoglycoside antibiotic which is used against bacteria in human and animal infection, have serious side effects on different parts of human body. Therefore, there is a crucial need to detect trace amount of it in serum and food products. Aptamers are oligonucleotides or peptides, which bind their targets with high affinity and specificity. These properties make aptamers as suitable candidates for biosensing applications. A 79-mer ss-DNA aptamer was applied for the detection of small amount of STR in various aptasensors. But there is no structural information on the STR-binding aptamer and molecular details underlying the aptamer-STR binding remain unexplored. In this study we provided a 3D-structural model for 79-mer ss-DNA aptamer from the sequence. Using docking program and molecular dynamics (MD) simulation we predicted the binding pocket of ss-DNA aptamer. Our results show STR streptose ring is buried within the groove of DNA model and capped by non Watson-Crick bases. STR interacts with aptamer through forming stable hydrogen bonds. Our computational findings are in fair agreement with experimental results. With the atomic structural details, we gained new insight into the Apt-STR binding interaction that can help to further optimize aptamer efficiency in biosensing applications.Communicated by Ramaswamy H. Sarma.

Spectroscopy and molecular dynamics simulation study on the interaction of sunset yellow food additive with pepsin.

The interaction of pepsin with sunset yellow food additive (SY) was studied by fluorescence spectroscopy and molecular dynamics simulation. The experimental results indicated that SY can quench the fluorescence of pepsin with static quenching. The apparent binding constant Ka and binding site number n were evaluated at different temperatures. Thermodynamic analysis suggests that SY interact with pepsin spontaneously by van der Waal's forces and hydrogen bond formation. Three-dimensional fluorescence spectra showed that pepsin undergoes a slightly conformation change when it interacts with SY. The molecular dynamics simulation (MD) revealed that the binding site is located mainly on the tyrosine residues at the entrance of the active site of pepsin and the main interactions occurred between SY and pepsin are hydrogen bond and stacking interactions, according to experimental results. Furthermore, the binding between SY and pepsin can inhibit pepsin activity. Our MD results showed that the SY prevents substrate from entering the active site by making a barrier at the entrance of the active site, reducing the pepsin activity.

Insights from engineering the Affibody-Fc interaction with a computational-experimental method.

The interaction between the Staphylococcal Protein A (SpA) domain B (the basis of the Affibody) molecule and the Fc of IgG is key to the use of Affibodies in affinity chromatography and in potential therapies against certain inflammatory diseases. Despite its importance and four-decade history, to our knowledge this interaction has never been affinity matured. We elucidate reasons why single-substitutions in the SpA which improve affinity to Fc may be very rare, and also discover substitutions which potentially serve several engineering purposes. We used a variation of FoldX to predict changes in protein-protein-binding affinity, and produce a list of 41 single-amino acid substitutions on the SpA molecule, of which four are near wild type (wt) and five are at most a factor of four from wt affinity. The nine substitutions include one which removes lysine, and several others which change charge. Subtle modulations in affinity may be useful for modifying column elution conditions. The method is applicable to other protein-protein systems, providing molecular insights with lower workload than existing experimental techniques.

Involvement of mitochondrial/lysosomal toxic cross-talk in ecstasy induced liver toxicity under hyperthermic condition.

The initial objectives of this study were to evaluate the extent of 3, 4-methylenedioxymethamphetamine (MDMA) induced loss of cell viability (cytotoxicity), induction of reactive oxygen species formation and damage to sub-cellular organelles (e.g. mitochondria/lysosomes) in freshly isolated rat hepatocytes under normothermic conditions (37 degrees C) and to compare the results with the effects obtained under hyperthermic conditions (41 degrees C). MDMA induced cytotoxicity, reactive oxygen species formation, mitochondrial membrane potential decline and lysosomal membrane leakiness in isolated rat hepatocytes at 37 degrees C. A rise in incubation temperature from 37 degrees C to 41 degrees C had an additive/synergic effect on the oxidative stress markers. We observed variations in mitochondrial membrane potential and lysosomal membrane stability that are significantly (P<0.05) higher than those under normothermic conditions. Antioxidants, reactive oxygen species scavengers, lysosomal inactivators, mitochondrial permeability transition (MPT) pore sealing agents, NADPH P450 reductase inhibitor, and inhibitors of reduced CYP2E1 and CYP2D6 prevented all MDMA induced hepatocyte oxidative stress cytotoxicity markers. It is therefore suggested that metabolic reductive activation of MDMA by reduced cytochrome P450s and glutathione could lead to generation of some biological reactive intermediates which could activate reactive oxygen species generation and cause mitochondrial and lysosomal oxidative stress membrane damages. We finally concluded that hyperthermia could potentiate MDMA induced liver toxicity probably through a mitochondrial/lysosomal toxic cross-talk in freshly isolated rat hepatocytes.