Determinants for macromolecular crowding-induced thermodynamic stabilization of acid-denatured cytochrome c to molten globules

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 (& UDelta;& UDelta;G) and preferential hydration & UDelta;& UGamma;W were estimated for the UA & RARR; MGMC transition. The magnitudes of & UDelta;& UDelta;G and & UDelta;& UGamma;W were found to be decreased as dextran 70 (D70) > dextran 40 (D40) > ficoll 70 (F70), which demonstrates that & UDelta;& UDelta;G and & UDelta;& UGamma;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 (& UDelta;Hm), suggesting that the steric-excluded volume effect contributes to the crowdinginduced 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 aciddenatured 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.

Automated summary

The macromolecular crowding effect transforms acid-denatured ferricytochrome c (cyt cIII) (UA-state) to molten-globule (MGMC-state) at pH 1.85. The magnitudes of crowding-induced stabilization free energy (∆G) and preferential hydration (∆ΓW) were found to be decreased as the size and shape of the crowder towards refolding and stabilization of UA-state to MGMC-state. Macromolecular crowding increased the thermodynamic stability of acid-denatured cyt cIII and the size and shape of crowder control the crowding-induced thermodynamic stabilization of MGMC-state.