Thermophile Hydrogenobacter thermophilus cytochrome c(552) (HT) is a stable protein with denaturation temperatures (T(m)) of 109.8 and 129.7 degrees C for the oxidized and reduced forms, respectively [Uchiyama, S., Ohshima, A., Nakamura, S., Hasegawa, J., Terui, N., Takayama, S. J., Yamamoto, Y., Sambongi, Y., and Kobayashi, Y. (2004) J. Am. Chem. Soc. 126, 14684-14685]. The removal of a single hydroxyl group from the hydrophobic core of HT, through the replacement of a Tyr by Phe, resulted in further elevation of the T(m) value of the oxidized form by approximately 6 degrees C, the T(m) value of the reduced one remaining essentially unaltered. As a result, the redox potential of the mutant with higher stability in the oxidized form exhibited a negative shift of approximately 20 mV relative to that of wild-type HT in an enthalpic manner. These findings indicated that the redox function of a protein can be enthalpically regulated through the stability of the oxidized form by altering the contextual stereochemical packing of hydrophobic residues in the protein interior using protein engineering. Study holds ProTherm entries: 20154, 20155, 20156, 20157 Extra Details: Oxidized form. hydroxyl group, hydrophobic core, higher stability, negative shift.
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:52 p.m.
|Number of data points||8|
|Proteins||Cytochrome c-552 ; Cytochrome c-551 ; Cytochrome c-552 ; Cytochrome c-551|
|Assays/Quantities/Protocols||Experimental Assay: Tm details:Additives DTT (0.5 mM), ; Experimental Assay: Tm details:Additives ; Derived Quantity: dTm details:Additives DTT (0.5 mM), ; Derived Quantity: dTm details:Additives|
|Libraries||Mutations for sequence EDPEVLFKNKGCVACHAIDTKMVGPAYKDVAAKFAGQAGAEAELAQRIKNGSQGVWGPIPMPPNAVSDDEAQTLAKWVLSQK ; Mutations for sequence NEQLAKQKGCMACHDLKAKKVGPAYADVAKKYAGRKDAVDYLAGKIKKGGSGVWGSVPMPPQNVTDAEAKQLAQWILSIK|