Thermal stability of membrane-reconstituted yeast cytochrome c oxidase

Abstract

The thermal dependence of the structural stability of membrane-reconstituted yeast cytochrome c oxidase has been studied by using different techniques including high-sensitivity differential scanning calorimetry, differential detergent solubility thermal gel analysis, and enzyme activity measurements. For these studies, the enzyme has been reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) vesicles using detergent dialysis. The phospholipid moiety affects the stability of the enzyme as judged by the dependence of the denaturation temperature on the lipid composition of the bilayer. The enzyme is more stable when reconstituted with the 18-carbon, unsaturated phospholipid (DEPC) than with the 14-carbon saturated phospholipid (DMPC). In addition, the shapes of the calorimetric transition profiles are different in the two lipid systems, indicating that not all of the subunits are affected equally by the lipid moiety. The overall enthalpy change for the enzyme denaturation is essentially the same for the two lipid reconstitutions (405 kcal/mol of protein for the DMPC and 425 kcal/mol for the DEPC-reconstituted enzyme). In both systems, the van't Hoff to calorimetric enthalpy ratios are less than 0.2, indicating that the unfolding of the enzyme cannot be represented as a two-state process. Differential detergent solubility experiments have allowed us to determine individual subunit thermal denaturation profiles. These experiments indicate that the major contributors to the main transition peak observed calorimetrically are subunits I and II and that the transition temperature of subunit III is the most affected by the phospholipid moiety. Experiments performed at different scanning rates indicate that the thermal denaturation of the enzyme is a kinetically controlled process characterized by activation energies on the order of 40 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Irreversible thermal denaturation ofTorpedo californicaacetylcholinesterase

“…This experiment rules out the possibility of overlapping independent transitions. Thus, at a scan rate 59.6 K/h, AChE demonstrates a single thermal transition within a narrow temperature range.A similar asymmetry of the DSC peak has been observed for several proteins that undergo irreversible thermal transition, including thermolysin, cytochrome c oxidase, carboxypeptidase B, phosphoglycerate kinase, G-actin, and 8-kDa sea anemone cytotoxin (Sanchez-Ruiz et al, 1988;Morin et al, 1990;Conejero-Lara et al, 1991a, 1991bGalisteo et al, 1991;Le Bihan & Gicquaud, 1993;Zhadan & Shnyrov, 1994). In all these cases, the behavior of the systems was described as a thermal denaturation process under kinetic control.…”

Biochemical and Biophysical Characterization of the Two Isoforms of cbb3-Type Cytochrome c Oxidase from Pseudomonas stutzeri

“…3). As a rescan of the same protein sample displayed no endothermic signal, this thermal denaturation was found to be irreversible, which is consistent with the results obtained from the yeast and Paracoccus denitrificans aa 3 -CcOs (37,38). Because of the irreversible nature of the thermal denaturation process, the calorimetric data cannot be directly analyzed in terms of equilibrium thermodynamics (39).…”

A pathway for the thermal destabilization of bacteriorhodopsin

“…The kinetically controlled character of irreversible protein denaturation had been deduced from calorimetric studies for this and other membrane proteins [4,5,13]; however, the present paper provides direct structural evidence that this is the case (see mainly Fig. 3).…”

“…The intramembranous regions of those proteins are believed to be stable mainly because of hydrogen bonds formed within the hydrophobic membrane matrix [1][2][3]. Calorimetric studies of membrane protein stability have shown that, at least in some cases, thermal denaturation is a kinetically controlled irreversible process, in which the observed transition temperature is a function of the experimental heating rate [4,5]. Recent data on the thermal denaturation of bovine and Paracoccus cytocrome c oxidases [2,3] suggest that most of the intramembranous secondary structure is maintained even above denaturation temperatures.…”