Synthesis, characterization and biological activity of some Dithiourea Derivatives:

Novel dithiourea derivatives have been designed as HIV-1 protease inhibitors using Autodock 4.2, synthesized and characterized by spectroscopic methods and microanalysis

Structural and functional characterization of peptides derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi

Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant, and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the carboxy-terminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted (Os–C). Here, we report that both peptides were bactericidal (MBC values ranging from 0.94–15mg/ml) to both Gram-positive and Gram-negative bacteria, whereas the parent peptide only exhibited Gram-positive antibacterial activity. The Os peptide was found to be two-fold more active than Os–C against three of the four tested bacteria but equally active against Staphylococcus aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os–C took longer, suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data indicated that in a membrane-mimicking environment, the cysteine-containing peptide has a higher a-helical content. Both peptides were found to be non-toxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity and were 12 times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted.Medical Research Council of South Africa,the National Research Foundation of South Africa, and the University of Pretoriahttp://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1099-1387hb201

Biochemical and thermodynamic characterisation of ligand-binding to class alpha glutathione transferase A1-1

PhD, Faculty of Science (Molecular and Cell Biology), University of the Witwatersrand, 2001.Phenylalanine 51 (F51) in the human class alpha GST forms part of a hydrophobic lockand- key intersubunit motif at the dimer interface. Protein engineering techniques were used to replace the phenylalanine key with serine. The results indicated that the mutant protein is dimeric with a native-like core structure indicating that F51 at the dimer interface is not essential for dimerisation to occur. Replacing F51 with serine impacts on the catalytic and ligandin function suggesting that tertiary structural changes have occurred at/near the active and non-substrate ligand binding sites. The F51S mutant also displays an enhanced exposure of hydrophobic surface as well as ligandin function. The F51S mutant displays a diminished conformational stability when compared to the wildtype protein. The lock-and-key intersubunit motif, therefore, although not essential for dimerisation to occur does stabilise the quaternary structure at the dimer interface. A unique structural feature of the class alpha GSTs is the C-terminal helix (residues 207- 221). In this study, the role of F221 was assessed by deleting it from the C-terminal helix 9 of hGSTA1-1. The results showed that the deletion of F221 does not affect the secondary, tertiary and quaternary structure of the protein as observed using far-UV CD measurements, enzyme activity and conformational stability as probes, respectively. The wild-type protein binds ~ 1.7-fold more ANS than the F221del protein. Binding affinity studies indicated that although both proteins bind ANS with the same affinity, the wildtype protein binds ANS with a higher capacity than the F221del protein. ANS binding to the wild-type and F221del proteins in the presence of urea (0 - 5.5 M urea) indicated that F221 is required for stabilising helix 9 at the C-terminal of hGSTA1-1. Therefore, F221 is not required for catalysis nor does it impact on the conformational stability of the protein. F221 does, however, affect the ligandin function and is required for the stability of helix 9 at the C-terminus of hGSTA1-1. ITC was used to dissect the binding energetics of glutathione (GSH) and glutathione sulfonate (GSO3 -) to the wild-type and Y8F hGSTA1-1 proteins. The contribution of the tyrosyl hydroxyl group to the binding of GSH and GSO3 - indicated that the Y8F mutant binds GSH tighter than the wild-type protein and the wild-type protein, in turn, binds GSO3 - tighter than the Y8F mutant protein. The Y8F mutant displays a larger negative vi DCp than the wild-type protein when complexed with either GSH or GSO3 -. This indicates the burial of a larger solvent-exposed hydrophobic surface area for the Y8F mutant than the wild-type protein. The burial of a large solvent-exposed hydrophobic surface area is related to the immobilisation of helix 9 onto domain I in the presence of active site ligands. The observation that the Y8F mutant displays burial of larger solventexposed hydrophobic surface area suggests that the tyrosyl hydroxyl group controls the dynamics of helix 9 at the C-terminal of hGSTA1-1. The DDG values also suggest that the tyrosyl hydroxyl group stabilises the thiolate anion at the active site in the wild-type protein. The binding energetics of non-substrate ligands (ANS and BSP) to the wild-type human class alpha GSTA1-1 were evaluated. The stoichiometry of the interaction between the wild-type protein and ANS indicated that one molecule of ANS binds per protein monomer. The binding interactions between ANS and the wild-type protein are enthalpically favourable indicating the possibility of hydrogen bond formation. ANS binding to the wild-type protein also resulted in the reduction of non-polar surface area exposed to solvent. It is proposed that the ANS binding site is the region adjacent to domain I that becomes buried when helix 9 is immobilised. The binding of BSP to the wild-type protein involves a high and low affinity set of binding sites. The high affinity binding site binds one molecule of BSP per protein monomer whereas the low affinity site is capable of accommodating a minimum of ~ four BSP molecules. The binding energetics to the high affinity site is both enthalpically and entropically favourable with each term contributing favourably to the favourable Gibbs free energy of binding. Binding to the lower affinity site is not very favourable enthalpically and the major driving force behind the favourable Gibbs free energy of association is the entropic factor. This interaction, therefore, appears to be entropically driven

How the Library can Best Serve the Academic Research Community at Wits

PPT PresentationDr. Yasien Sayed - School of Molecular and Cell Biology - How the Library can Best Serve the Academic Research Community at Wit

Mechanism of drug resistance in HIV-1 protease subtype C in the presence of Atazanavir

AIDS is one of the deadliest diseases in the history of humankind caused by HIV. Despite the technological development, curtailing the viral infection inside human host still remains a challenge. Therapies such as HAART uses a combination of drugs to inhibit the viral activity. One of the important targets includes HIV protease and inhibiting its activity will minimize the production of mature structural proteins. However, the genetic diversity and the occurrence of drug resistant mutations adds complexity to effective drug design. In this study, we aimed at understanding the drug binding mechanism of one such subtype, namely subtype C and its insertion variant L38HL. We performed multiple molecular dynamics simulations along with binding free energy analysis of wild-type and L38HL bound to Atazanavir (ATV). From the analysis, we revealed that the insertion alters the hydrogen bond and hydrophobic interaction networks. The alterations in the interaction networks increase flexibility at the hinge-fulcrum interface. Further, the effects of these changes affect flap tip curling. Moreover, the changes in the hinge-fulcrum-cantilever interface alters the concerted motion of the functional regions leading to change in the direction of flap movement thus causing a subtle change in the active site volume. Additionally, formation of intramolecular hydrogen bonds in the ATV docked to L38HL restricted the movement of R1 and R2 groups thereby altering the interactions. Overall, the changes in the flexibility of flap together with the changes in the active site volume and compactness of the ligand provide insights for increased binding affinity of ATV with L38HL

Tertiary interactions stabilise the C-terminal region of human glutathione transferase A1-1: a crystallographic and calorimetric study

The C-terminal region in class Alpha glutathione transferase A1-1 (GSTA1-1), which forms an amphipathic a-helix (helix 9), is known to contribute to the catalytic and non-substrate ligand-binding functions of the enzyme. The region in the apo protein is proposed to be disordered which, upon ligand binding at the active-site, becomes structured and localised. Because Ile219 plays a pivotal role in the stability and localisation of the region, the role of tertiary interactions mediated by Ile219 in determining the conformation and dynamics of the C-terminal region were studied. Ligand-binding microcalorimetric and X-ray structural data were obtained to characterise ligand binding at the active-site and the associated localisation of the C-terminal region. In the crystal structure of the I219A hGSTA1-1$ S-hexylglutathione complex, the C-terminal region of one chain is mobile and not observed (unresolved electron density), whereas the corresponding region of the other chain is localised and structured as a result of crystal packing interactions. In solution, the mutant C-terminal region of both chains in the complex is mobile and delocalised resulting in a hydrated, less hydrophobic active-site and a reduction in the affinity of the protein for S-hexylglutathione. Complete dehydration of the active-site, important for maintaining the highly reactive thiolate form of glutathione, requires the binding of ligands and the subsequent localisation of the C-terminal region. Thermodynamic data demonstrate that the mobile C-terminal region in apo hGSTA1-1 is structured and does not undergo ligand-induced folding. Its close proximity to the surface of the wild-type protein is indicated by the concurrence between the observed heat capacity change of complex formation and the type and amount of surface area that becomes buried at the ligand–protein interface when the C-terminal region in the apo protein assumes the same localised structure as that observed in the wild-type complex