Abstract The preferential interactions of bovine serum albumin, lysozyme, chymotrypsinogen, ribonuclease A, and β‐lactoglobulin with polyethylene glycols (PEGs) of molecular weight 200–6,000 have been measured by dialysis equilibrium coupled with high precision densimetry. All the proteins were found to be preferentially hydrated in all the PEGs, and the magnitude of the preferential hydration increased with increasing PEG size for each protein. The change in the chemical potentials of the proteins with the addition of the PEGs had highly positive values, indicating a strong thermodynamic destabilization of the system by the PEGs. A viscosity study of the PEGs showed them to be randomly coiled polymers, as their radii of gyration were related to the molecular weight by R g = a M 0.55 . The thickness of the effective shell impenetrable to PEG around protein molecules, calculated from the preferential hydration, was found to vary with PEG molecular weight in similar fashion as the PEG radius of gyration, supporting the proposal (Arakawa, T. & Timasheff, S.N., 1985a, Biochemistry 24 , 6756–6762) that the preferential exclusion of PEGs from proteins is due principally to the steric exclusion of PEG from the protein domain, although favorable interactions with protein surface residues, in particular nonpolar ones, may compete with the exclusion. These thermodynamically unfavorable preferential exclusion interactions lead to the action of PEGs as precipitants, although they may destabilize protein structure at higher temperatures.
(2020). Two polyphenols with diverse mechanisms towards amyloidosis: differential modulation of the fibrillation pathway of human lysozyme by curcumin and EGCG. Journal of Biomolecular Structure and Dynamics. Ahead of Print.
Abstract The effect of resveratrol, a polyphenol in red wine, on the amyloid fibril formation of human lysozyme (HuL) was investigated, towards elucidating the mechanism of resveratrol action and probing its role as a possible modulator of lysozyme aggregation and toxicity. By using a number of biophysical tools, resveratrol was observed to alter the fibrillization kinetics of HuL and inhibit its fibrillization by binding with weak to moderate affinity to the conformations populated at the early stages of the pathway with concomitant stabilization of these initial conformations. The marginal decrease in the lifetime of HuL in the presence of resveratrol by time‐resolved fluorescence measurements indicated the involvement of a static quenching mechanism in the interaction between HuL and resveratrol. Docking studies predicted the binding of resveratrol to aggregation‐prone regions in HuL, and structure and activity analyses demonstrated the retention of much of the α‐helical structure and activity of HuL in the presence of resveratrol. Resveratrol modulated the fibrillization pathway towards less‐hydrophobic, less‐toxic, off‐pathway aggregates. These results demonstrate that binding of resveratrol to HuL could protect against the formation of pathogenic, cytotoxic aggregates formed in amyloidogenic disorders, such as systemic amyloidosis; thus suggesting its potential as a plausible therapeutic agent against lysozyme amyloidosis.
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Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys(487) resulted in approximately 95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys(172) and Ala(302) to be involved in the binding of ATP and Ser(404) in the binding of CoenzymeA. The influence of mutations of the residues Val(209) and Trp(377) emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C(26)/C(24) fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys(172), Ala(302) and Lys(487) and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the findings of this study will provide valuable inputs in the development of inhibitors against the mymA operon, an important target for the development of antitubercular drugs.
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