Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [Delta(DeltaG)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change (DeltaC(P)), resulting in higher DeltaG values over a wide temperature range (0-100 degrees C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization. Study holds ProTherm entries: 15177, 15178, 15179, 15180, 15181, 15182, 15183, 15184, 15185, 15186, 15187, 15188, 15189, 15190, 15191, 15192, 15193, 15194 Extra Details: combined mutant; cytochrome c; differential scanning calorimetry (DSC); protein stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:45 p.m.
|Number of data points||44|
|Proteins||Cytochrome c-552 ; Cytochrome c-551 ; Cytochrome c-552 ; Cytochrome c-551|
|Assays/Quantities/Protocols||Experimental Assay: dCp ; Experimental Assay: dHcal ; Experimental Assay: ddG ; Experimental Assay: Tm ; Derived Quantity: dTm|
|Libraries||Mutations for sequence EDPEVLFKNKGCVACHAIDTKMVGPAYKDVAAKFAGQAGAEAELAQRIKNGSQGVWGPIPMPPNAVSDDEAQTLAKWVLSQK ; Mutations for sequence NEQLAKQKGCMACHDLKAKKVGPAYADVAKKYAGRKDAVDYLAGKIKKGGSGVWGSVPMPPQNVTDAEAKQLAQWILSIK|