The irreversible thermal denaturation of the F1 portion of the chloroplast ATP synthase (CF1) was examined by differential scanning calorimetry, ATPase activity loss, and release of bound nucleotides. In nearly all cases, the loss of ATPase activity closely paralleled the temperature dependence of the excess heat capacity. Although the irreversible nature of the denaturation precluded thermodynamic interpretation, a kinetic analysis was feasible. A two-state kinetic model was found to fit the calorimetric data very well. The activation energies of thermal denaturation calculated from calorimetric data were very close to those determined from Arrhenius plots of the apparent first-order rate constants of loss of ATPase activity versus reciprocal of the temperature. The nucleotide binding site occupancy profoundly influenced the temperature at which thermal denaturation occurred. In particular, the temperature at which the maximum in excess heat capacity occurs (Tm) was increased about 8 degrees C by occupancy of tight, noncatalytic ATP binding sites and by an additional 3-4 degrees C by the presence of nucleotides in the medium during heating. The thermal denaturation of CF1, an enzyme composed of nine polypeptide chains, is highly cooperative in that it obeys the simple two-step kinetic model. Since the removal of the epsilon and delta subunits has little effect on thermal denaturation, the major forces that stabilize CF1 must, thus, be between the alpha, beta, and gamma subunits. Study holds ProTherm entries: 5225 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:29 p.m.
|Number of data points||3|
|Proteins||ATP synthase subunit alpha, chloroplastic|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence MATIRADEISKIIRERIEGYNREVKVVNTGTVLQVGDGIARIHGLDEVMAGELVEFEEGTIGIALNLESNNVGVVLMGDGLMIQEGSSVKATGRIAQIPVSEAYLGRVINALAKPIDGRGEITASESRLIESPAPGIMSRRSVYEPLQTGLIAIDAMIPVGRGQRELIIGDRQTGKTAVATDTILNQQGQNVICVYVAIGQKASSVAQVVTNFQERGAMEYTIVVAETADSPATLQYLAPYTGAALAEYFMYRERHTLIIYDDLSKQAQAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKLSSLLGEGSMTALPIVETQAGDVSAYIPTNVISITDGQIFLSADLFNAGIRPAINVGISVSRVGSAAQIKAMKKVAGKLKLELAQFAELEAFAQFASDLDKATQNQLARGQRLRELLKQPQSAPLTVEEQVMTIYTGTNGYLDSLELDQVRKYLVELRTYVKTNKPEFQEIISSTKTFTEEAEALLKEAIQEQMERFLLQEQA|