Department Of Chemistry
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Item Single-molecule analysis of osmolyte-mediated nanomechanical unfolding behavior of a protein domain(Elsevier B.V., 2023-09-16T00:00:00) Bajaj, Manish; Muddassir, Mohd; Choi, Bumjoon; Singh, Priyanka; Park, Jong Bum; Singh, Surjeet; Yadav, Manisha; Kumar, Rajesh; Eom, Kilho; Sharma, DeepakThe small organic molecules, known as osmolytes being ubiquitously present in different cell types, affect protein folding, stability and aggregation. However, it is unknown how the osmolytes affect the nanomechanical unfolding behavior of protein domain. Here, we show the osmolyte-dependent mechanical unfolding properties of protein titin immunoglobulin-27 (I27) domain using an atomic force microscopy (AFM)-based single-molecule force spectroscopy. We found that amines and methylamines improved the mechanical stability of I27 domain, whereas polyols had no effect. Interestingly, glycine betaine (GB) or trimethylamine-N-oxide (TMAO) increased the average unfolding force of the protein domain. The kinetic parameters analyzed at single-molecule level reveal that stabilizing effect of osmolytes is due to a decrease in the unfolding rate constant of I27, which was confirmed by molecular dynamics simulations. Our study reveals different effects that diverse osmolytes have on the mechanical properties of the protein, and suggests the potential use of osmolytes in modulating the mechanical stability of proteins required for various nano-biotechnological applications. � 2023 Elsevier B.V.Item 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, RajeshThe 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.Item 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, SanjeevSerum 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.Item Analysis of the effect of 1-Allyl-3-Methylimidazolium chloride on thermodynamic stability, folding kinetics, and motional dynamics of horse cytochrome c(Elsevier B.V., 2022-09-08T00:00:00) Garg, Mansi; Sharma, Deepak; Kumar, Rajesh1-allyl-3-methylimidazolium chloride (AMIMCl) acts as a potential green solvent for proteins. The present work provides a possible pathway by which the structural, kinetic, thermodynamic, and folding properties of horse cytochrome c (cyt c) are affected in green aqueous-AMIMCl systems. Analysis of the effect of AMIMCl on thermodynamic stability, refolding/unfolding kinetics, and motional dynamics of cyt c provided important information, (i) AMIMCl decreases the thermodynamic stability of reduced cyt c and also strengthens the guanidinium chloride (GdmCl)-mediated decrease in thermodynamic stability of protein, (ii) AMIMCl reduces the thermal-fluctuation of Met80-containing omega-loop of natively-folded compact state of carbonmonoxycytochrome c (MCO-state) due to polyfunctional interactions between the AMIM+ and different groups of protein, (iii) AMIMCl shifts the kinetic chevron plot, ln kobs[sbnd][GdmCl] to the lower concentration of GdmCl, (iv) AMIMCl shifts the refolding and unfolding limps to vertically downwards and upwards, respectively, and (v) AMIMCl reducing the unfolding free energy estimated by both thermodynamic and kinetic analysis. � 2022Item Effect of imidazolium based ionic liquids on CO-association dynamics and thermodynamic stability of Ferrocytochrome c(Elsevier B.V., 2020-11-05T00:00:00) Garg, Mansi; Kumar, Sandeep; Kaur, Anupamjeet; Goyal, Bhupesh; Kumar, RajeshAnalysis of kinetic and thermodynamic parameters measured for CO-association reaction of Ferrocytochrome c (Ferrocyt c) under variable concentrations of 1-butyl-3-methylimidazolium with varying anion ([Bmim]X) (X = Cl?, I?, Br?, HSO4?) at pH 7 revealed that the low concentration of [Bmim]X (?0.5 M) constrains the CO-association dynamics of Ferrocyt c and typically follows the order: [Bmim]HSO4 > [Bmim]Cl > [Bmim]Br > [Bmim]I. At relatively higher concentrations (>0.5), the chaotropic action of [Bmim]+ dominates which consequently increases the thermal-fluctuations responsible to denature the protein and thus accelerates the speed of CO-association reaction. Analysis of thermal denaturation curves of Ferrocyt c measured at different concentrations of [Bmim]X revealed that the [Bmim]X decreases the thermodynamic stability of protein and typically follows the order: [Bmim]I > [Bmim]Br > [Bmim]Cl > [Bmim]CH3COO > [Bmim]HSO4, demonstrating that the effect of [Bmim]X on thermodynamic stability of protein is not in accordance to Hofmeister series effect of anions because instead of increasing the kosmotropic anion carrying [Bmim]X ([Bmim]CH3COO and [Bmim]HSO4) also decreases the thermodynamic stability of protein. � 2020 Elsevier B.V.Item Structural, kinetic and thermodynamic characterizations of SDS-induced molten globule state of a highly negatively charged cytochrome c(Oxford University Press, 2019) Jain, R; Sharma, D; Kumar, Rakesh; Kumar, RajeshThis study presents the structural, kinetic and thermodynamic characterizations of previously unknown submicellar concentrations of SDS-induced molten globule (MGSDS) state of a highly negatively charged basedenatured ferricytochrome c (U B -state) at pH ∼12.8 (±0.2). The far-UV CD, near-UV CD, ANS-fluorescence data of UB-state in the presence of different concentrations of SDS indicate that the submicellar concentrations of SDS (≤0.4mM) transform the UBstate to MG SDS -state. The MG SDS -state has nativelike α-helical secondary structure but lacks tertiary structure. The free energy change (ΔG° D) for U B → MG SDS transition determined by far-UV CD (∼2.7 kcal mol -1 ) is slightly higher than those determined by fluorescence (∼2.0 kcal mol -1 ) at 25°C. At very low SDS and NaCl concentrations, the MG SDS -state undergoes cold denaturation. As SDS concentration is increased, the thermal denaturation temperature increases and the cold denaturation temperature decrease. Kinetic experiments involving the measurement of the CO-association rate to the base-denatured ferrocytochrome c at pH ≈12.8 (±0.2), 25°C indicate that the submicellar concentrations of SDS restrict the internal dynamics of base-denatured protein. © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.Item Analysis of the pH-dependent thermodynamic stability, local motions, and microsecond folding kinetics of carbonmonoxycytochrome c(Academic Press Inc., 2016) Kumar, RajeshThis paper analyzes the effect of pH on thermodynamic stability, low-frequency local motions and microsecond folding kinetics of carbonmonoxycytochrome c (Cyt-CO) all across the alkaline pH-unfolding transition of protein. Thermodynamic analysis of urea-induced unfolding transitions of Cyt-CO measured between pH 6 and pH 11.9 reveals that Cyt-CO is maximally stable at pH?9.5. Dilution of unfolded Cyt-CO into refolding medium forms a native-like compact state (NCO-state), where Fe2+?CO interaction persists. Kinetic and thermodynamic parameters measured for slow thermally-driven CO dissociation (NCO?N+CO) and association (N+CO?NCO) reactions between pH 6.5 and pH 13 reveal that the thermal-motions of M80-containing ?-loop are decreased in subdenaturing limit of alkaline pH. Laser photolysis of Fe2+-CO bond in NCO-state triggers the microsecond folding (NCO?N). The microsecond kinetics measured all across the alkaline pH-unfolding transition of Cyt-CO produce rate rollover in the refolding limb of chevron plot, which suggests a glass transition of NCO en route to N. Between pH 7 and pH 11.9, the natural logarithm of the microsecond folding rate varies by?<?1.5 units while the natural logarithm of apparent equilibrium constant varies by 11.8 units. This finding indicates that the pH-dependent ionic-interactions greatly affect the global stability of protein but have very small effect on folding kinetics. ? 2016 Elsevier Inc.Item Role of Macromolecular Crowding on Stability and Iron Release Kinetics of Serum Transferrin(American Chemical Society, 2017) Kumar, Sandeep; Sharma, Deepak; Kumar, RajeshThe macromolecular crowding influences the structural stability and functional properties of transferrin (Tf). The equilibrium as well as kinetic studies of Tf at different concentrations of crowding agents (dextran 40, dextran 70, and ficoll 70) and at a fixed concentration of dextran 40 under different concentrations of NaCl at pH 7.4 and 5.6 (?1) revealed that (i) the crowder environment increases the diferric-Tf (Fe2Tf) stability against iron loss and overall denaturation of the protein, (ii) both in the absence and presence of crowder, the presence of salt promotes the loss of iron and overall denaturation of Fe2Tf which is due to ionic screening of electrostatic interactions, (iii) the crowder environment retards iron release from monoferric N-lobe of Tf (FeNTf) by increasing enthalpic barrier, (iv) the retardation of iron release by crowding is enthalpically dominated than the entropic one, (v) both in the absence and presence of crowder, the presence of salt accelerates the iron release from FeNTf due to ionic screening of electrostatic interactions and anion binding to KISAB sites, and (vi) the crowders environment is unable to diminish (a) the salt-induced destabilization of Fe2Tf against the loss of iron and overall denaturation and (b) the anion effect and ionic screening of diffusive counterions responsible to promote iron release from FeNTf. ? 2017 American Chemical Society.