Browsing by Author "Kumari, Beeta"

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    Determinants for macromolecular crowding-induced thermodynamic stabilization of acid-denatured cytochrome c to molten globules
    (Elsevier B.V., 2023-07-22T00:00:00) Kumari, Beeta; Shabnam; Yadav, Manisha; Kumar, Manoj; Kushwaha, Pratibha; Prakash Prabhu, N.; Kumar, Rajesh
    The macromolecular crowding effect transforms the acid-denatured ferricytochrome c (cyt cIII) (UA-state) to molten-globule (MGMC-state) at pH 1.85. Crowding-induced stabilization free energy (??G) and preferential hydration ??W were estimated for the UA ? MGMC transition. The magnitudes of ??G and ??W were found to be decreased as dextran 70 (D70) > dextran 40 (D40) > ficoll 70 (F70), which demonstrates that ??G and ??W track the molecular size and shape of the crowder towards refolding and stabilization of UA-state to MGMC-state. Analysis of effects of crowders (D40, D70, F70) on thermal and chemical-denaturations of acid-denatured cyt cIII provided several important information, (i) macromolecular crowding increased the thermodynamic stability of acid-denatured cyt cIII, (ii) concentration, size and shape of crowder control the crowding-induced thermodynamic stabilization of MGMC-state, (iii) crowding effect increased the thermal-denaturation midpoint (Tm) with a slight change in enthalpy (?Hm), suggesting that the steric-excluded volume effect contributes to the crowding-induced increased thermal stability of the acid-denatured protein. Analysis of entropy ? enthalpy plots for D40, D70, and F70 reveals that in addition to the steric-excluded volume effect, the enthalpic contribution is also added to the macromolecular crowding-induced stabilization of acid-denatured cyt cIII. The dilute-medium, compound-crowder, purely entropic-crowder and purely enthalpic-crowder curves were obtained for acid-denatured cyt cIII for D70, D40 and F70. The crossover temperature, Tx was calculated from the dilute and compound-crowder curves. The Tx values measured for D40, D70, and F70 were found to be ? 250.15 K, 272.15 K, and 275.15 K, respectively, which suggests that the Tx value depends on the size and shape of the crowder. Furthermore, the observation of a lower value of Tx and a minor enthalpic component for D40, D70, and F70 is likely due to the formation of weaker soft interactions of acid-denatured cyt cIII with D40, D70, and F70. � 2023 Elsevier B.V.
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    Substitution of carbonate by non-physiological synergistic anion modulates the stability and iron release kinetics of serum transferrin
    (Elsevier B.V., 2022-10-15T00:00:00) Kumar, Rajesh; Sharma, Deepak; Kumar, Navinder; Kumari, Beeta; Shabnam; Kumar, Sanjeev
    Serum transferrin (sTf) is a bi-lobal protein. Each lobe of sTf binds one Fe3+ ion in the presence of a synergistic anion. Physiologically, carbonate is the main synergistic anion but other anions such as oxalate, malonate, glycolate, maleate, glycine, etc. can substitute for carbonate in vitro. The present work provides the possible pathways by which the substitution of carbonate with oxalate affects the structural, kinetic, thermodynamic, and functional properties of blood plasma sTf. Analysis of equilibrium experiments measuring iron release and structural unfolding of carbonate and oxalate bound diferric-sTf (Fe2sTf) as a function of pH, urea concentration, and temperature reveal that the structural and iron-centers stability of Fe2sTf increase by substitution of carbonate with oxalate. Analysis of isothermal titration calorimetry (ITC) scans showed that the affinity of Fe3+ with apo-sTf is enhanced by substituting carbonate with oxalate. Analysis of kinetic and thermodynamic parameters measured for the iron release from the carbonate and oxalate bound monoferric-N-lobe of sTf (FeNsTf) and Fe2sTf at pH 7.4 and pH 5.6 reveals that the substitution of carbonate with oxalate inhibits/retards the iron release via increasing the enthalpic barriers. � 2022 Elsevier B.V.