Protective role of hesperetin against posttranslational oxidation of tryptophan residue of human γD-crystallin: A molecular level study.

Crystallin proteins undergo various posttranslational modifications with aging of eye lens. Oxidation of tryptophan (Trp) residues of a major γ-crystallin namely human γD-crystallin (HGD) was found to be inhibited by a naturally occurring flavonoid hesperetin at relatively low concentration mostly due to its antioxidant activity. Further the molecular interactions between HGD and hesperetin were elucidated on the basis of the quenching of Trp fluorescence of the protein by the flavonoid. Ground state complexation between HGD and hesperetin caused static quenching of the Trp fluorescence of HGD. Binding and quenching constants were in the order of (103- 104 M-1). Energy transfer from protein to hesperetin was suggested by FRET calculations. Thermodynamic parameters reveal significant hydrophobic association between the protein and hesperetin. Synchronous fluorescence and CD spectroscopic results had ruled out conformational changes in the protein due to binding of hesperetin. Docking studies suggested the proximity of hesperetin with Trp 42, which largely corroborates our experimental findings.

2-Aryl benzazole derived new class of anti-tubercular compounds: Endowed to eradicate mycobacterium tuberculosis in replicating and non-replicating forms.

The high mortality rate and the increasing prevalence of Mtb resistance are the major concerns for the Tuberculosis (TB) treatment in this century. To counteract the prevalence of Mtb resistance, we have synthesized 2-aryl benzazole based dual targeted molecules. Compound 9m and 9n were found to be equally active against replicating and non-replicating form of Mtb (MIC(MABA) 1.98 and 1.66 µg/ml; MIC(LORA) 2.06 and 1.59 µg/ml respectively). They arrested the cell division (replicating Mtb) by inhibiting the GTPase activity of FtsZ with IC50 values 45 and 64 µM respectively. They were also capable of kill Mtb in non-replicating form by inhibiting the biosynthesis of menaquinone which was substantiated by the MenG inhibition (IC50 = 11.62 and 7.49 µM respectively) followed by the Vit-K2 rescue study and ATP production assay.

Inhibition of fibrillation of human γd-crystallin by a flavonoid morin.

To inhibit the formation of amyloid fibrils by human γd-crystallin (HGD), a series of four flavonoids (quercertin, rutin, morin and hesperetin) was tested. Only morin had demonstrated significant inhibition of HGD fibrillation. Results from fluorimetric assay techniques (using thioflavin T and ANS), FTIR, circular dichroism and microscopic imaging (fluorescence microscopy and transmission electron microscopy) confirmed HGD fibrillation inhibition by morin. HGD-morin complex formation at ground state resulted tryptophan fluorescence quenching through static mechanism, which was also confirmed by determining the excited-state life time of HGD tryptophan residues. Förster resonance energy transfer occurs from HGD to morin. Synchronous, three-dimensional fluorescence, FTIR and circular dichroism results suggest that major changes in HGD conformation did not occur on binding with morin. The interactions between HGD and morin involve hydrogen bonding and/or van der Waals forces. Docking predictions also support experimental results.Communicated by Ramaswamy H. Sarma.

Elucidation of molecular interactions between human γD-crystallin and quercetin, an inhibitor against tryptophan oxidation.

Different post-translational changes in eye lens crystallin proteins contribute towards the development of cataract. We have studied in vitro oxidative modification of tryptophan (Trp) residues of human γD-crystallin (HGD) towards formation of N-formylkynurenine (NFK) associated with cataractogenesis. This oxidation was found to be inhibited by quercetin at relatively low concentration. Interactions between quercetin and HGD were further studied using fluorescence techniques. Binding and quenching constants were determined as ∼104 M-1. Static quenching of fluorescence due to HGD-quercetin complex formation at ground state was confirmed by finding excited state life time of Trp residues. Energy transfer occurred between the protein and quercetin. Hydrogen bonding and/or van der Waals interactions were involved between HGD and quercetin. Synchronous and three-dimensional fluorescence along with far-UV CD studies suggested no major conformational alterations occurred in HGD due to quercetin binding. Experimental observations were supported by the docking results.Communicated by Ramaswamy H. Sarma.

Inhibition of amyloid fibrillation of human γD-crystallin by gold nanoparticles: Studies at molecular level.

The capability of citrate-stabilized gold nanoparticles (AuNps) has been explored for the inhibition of amyloid fibrillation of human γD-crystallin (HGD), a major protein of eye lens. Citrate-capped AuNps were synthesized, characterized and used further for amyloid inhibition. The results from intrinsic and extrinsic (in the presence of Thioflavin T and ANS) fluorescence based assays and CD spectroscopy clearly suggest that AuNps at nanomolar concentrations can act as an effective inhibitor against fibrillation of HGD. Fluorescence microscopic and transmission electron microscopic images also supported this observation. Considering the inhibitory role of AuNps against HGD fibrillation, interactions between HGD and AuNps were studied to decipher the mechanism of amyloid inhibition. The binding and quenching constants were calculated as ~109 M-1 using the data of tryptophan fluorescence quenching of HGD by AuNps. Ground state complexation between the protein and nanoparticles was predicted. AuNps were not found to cause any major conformational changes in the native protein. Entropy-driven complexation process between the protein and nanoparticles indicates the interactions of AuNps with hydrophobic residues of HGD. Therefore, in the presence of AuNps, the exposure of the hydrophobic patches of HGD during its partial unfolding became restricted, which results inhibition in HGD fibrillation.

Inhibition of copper-induced aggregation of human γD-crystallin by a diimine molecule and investigations on their molecular interactions.

Communicated by Ramaswamy H. Sarma.