Thermodynamic and pH stability of recombinant human L- and H-ferritins were probed by differential scanning calorimetry and 8-anilino-1-naphthalenesulfonate (ANS) binding in the pH range 2-7. At pH 2.0-2.8 they were dissociated into subunit monomers and in this pH interval the H-subunit displayed a single calorimetrically-revealed domain with properties of a molten globule-like state: low enthalpy (6.3-8.0 J/g or 169-172 kJ/mol) and Tm of thermal unfolding (approximately 50 degrees C), a wide transition range (approximately 20 degrees C) and high ANS binding. In contrast, at pH 2 the L-ferritin subunit showed two calorimetric domains with Tm of 35 and 40 degrees C with similar unfolding enthalpies and with moderate extent of interactions, as indicated by the ratio of calorimetric enthalpy (293.9 kJ/mol) and van't Hoff enthalpy (174.2 kJ/mol) for the thermal transition. A pH increase from 2.0 to 2.8 determined the coupling of the two domains into a single cooperative folding unit and drastic increase of the transition temperature (from 37 to 80 degrees C). The contacts between the two domains in the L-subunit appeared to contribute to about 30% of the total stabilization free energy. The unfolding enthalpies, heat capacity changes and pronounced ANS binding of the L-subunit at pH 2.0-2.8 indicated that part of the structure lacked 'meltable' tertiary interactions. The results indicate that H- and L-subunits are stabilized by largely different intra-chain interactions with a critical contribution to L-subunit stability of embedded salt bridge(s) absent in the H-subunit. Study holds ProTherm entries: 3456, 3457, 3458, 3459, 3460, 3461, 3462 Extra Details: differential scanning calorimetry; ferritin subunits;,protein folding; protein stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:21 p.m.
|Number of data points||21|
|Proteins||Ferritin light chain ; Ferritin heavy chain|
|Assays/Quantities/Protocols||Experimental Assay: dHcal pH:2.8 ; Experimental Assay: Tm pH:2.8 ; Experimental Assay: dHvH pH:2.8 ; Experimental Assay: dHcal pH:2.4 ; Experimental Assay: Tm pH:2.4 ; Experimental Assay: dHvH pH:2.4 ; Experimental Assay: dHcal pH:2.2 ; Experimental Assay: Tm pH:2.2 ; Experimental Assay: dHvH pH:2.2 ; Experimental Assay: dHcal pH:2.0 ; Experimental Assay: Tm pH:2.0 ; Experimental Assay: dHvH pH:2.0|
|Libraries||Mutations for sequence MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIQKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES|