Kinetic study into the irreversible thermal denaturation of bacteriorhodopsin

Galisteo, Maria L.; Sánchez‐Ruiz, José M.

doi:10.1007/bf00205809

Cited by 42 publications

(51 citation statements)

References 29 publications

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“…Galisteo and Sanchez-Ruiz (1993) have shown that the irreversible thermal denaturation of intact bacteriorhodopsin follows the two-state kinetic model (Sanchez-Ruiz et al, 1988) at pH 9.5, with an activation energy, E, of 361 ( 15 kJ/mol. In our case, apart from native BR, the AB‚CDEFG sample is the only one to show a single denaturation peak, corresponding to a single population of the cleaved protein.…”

Section: Resultsmentioning

confidence: 99%

Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems

et al. 1996

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Differential scanning calorimetry and Fourier-transform infrared spectroscopy have been used to characterize the thermal stability of bacteriorhodopsin (BR) cleaved within different loops connecting the helical rods. The results are compared to those of the native protein. We show that the denaturation temperature and enthalpy of BR cleaved at peptide bond 71-72 or 155-156 are lower than those of the intact protein, and that these values become even lower for the BR cleaved at both peptide bonds. The effect of cleavage on the denaturation temperature and enthalpy values seems to be additive as has been previously suggested [Khan, T. W., Sturtevant, J. M., & Engelman, D. M. (1992) Biochemistry 31, 8829]. The thermal denaturation of all the samples was irreversible and scan-rate dependent. When cleaved at the 71-72 bond BR follows quantitatively the predictions of the two-state kinetic model at pH 9.5, with an activation energy of 374 kJ/mol, similar to that of native BR. Calorimetry experiments with different populations of intact and cleaved BR provide direct evidence for some intermolecular cooperativity upon denaturation. The denatured samples maintain a large proportion of R helices and structure, a fact which seems to be related to their low denaturation enthalpy as compared to that of water-soluble, globular proteins.Thermodynamic-data analysis and energetic characterization of the thermal stability of membrane proteins (SanchezRuiz & Mateo, 1987;Ruiz-Sanz et al., 1992) as well as that of several globular proteins (Sanchez-Ruiz et al., 1988;Conejero-Lara et al., 1991) cannot be undertaken because of their non-equilibrium, irreversible denaturation (SanchezRuiz, 1992). This irreversibility is usually due to nonequilibrium processes taking place on protein unfolding (Klibanov & Ahern, 1987). Even in this case differential scanning calorimetry (DSC) 1 can provide the denaturation enthalpy (∆H) and temperature (T m ) values of the transition for a given scan rate. Thus T m can be used as an operative parameter, at least in comparative, relative terms, to characterize the thermal stability of a protein which unfolds under non-equilibrium conditions. Fourier-transform infrared spectroscopy (FTIR) is a very suitable technique to complement protein DSC studies, particularly in the case of membrane proteins, since it provides structural information concerning the native and denatured states and can also be used to observe the denaturation process itself (Surewicz & Mantsch, 1988;Surewicz et al., 1993;Arrondo et al., 1993;Jackson & Mantsch, 1995).Bacteriorhodopsin (BR), the only protein present in the purple membrane of Halobacterium salinarium, is one of the best known and well-studied intrinsic membrane proteins, both from a structural and a functional point of view (Henderson et al., 1990;Rothschild, 1992;Lanyi, 1993;Grigorieff et al., 1996). Previous denaturation studies carried out by DSC and spectroscopic techniques have thrown light on the importance of several structural features on BR's stability, ...

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Section: Resultsmentioning

confidence: 99%

Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems

et al. 1996

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“…would lead the protein to the final denatured state and could also be related to the complete destruction of the membrane, as reported by Shnyrov et al 171, visualizing by electron microscopy after heating PM to about 100°C. Finally, since BR denaturation is an irreversible, kinetically controlled process, as Galisteo and SanchezRuiz [16] have recently proved, we heated all our samples at the same scan rate, i.e. l"C/min, and at pH 7.5, where the two-state kinetic model does not apply [27].…”

Section: Discussionmentioning

confidence: 99%

Thermal transitions in the purple membrane from Halobacterium halobium

Shnyrov

Mateo

1993

FEBS Letters

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The application of a successive annealing procedure to the scanning calorimetric endotherm of the purple membrane from H&bacterium halobium in phosphate buffer. pH 7.5, leads to five thermal transitions beneath the overall endotherm. Circular dichroism and fluorescence experiments have also been carried out with the natrve membrane heated at the same scan rate as in calorimetric runs (1"Clmin) as well as with previously heated membrane samples. These results, together with others from the literature, have been used to suggest a preliminary explanation of the five thermal transitions.

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“…8O"C, attributed to a structural change in the crystal lattice of the membrane, followed by an irreversible transition at ca. lOO"C, associated with partial thermal unfolding of the membrane protein [16][17][18][19][20][21][22][23]. In the present study the thermal behavior of partially delipidated PM as a function of pH and bound cations removal has been compared to that of native PM.…”

Section: Iutroductionmentioning

confidence: 99%

Thermal stability of lipid‐depleted purple membranes at neutral and low pH values

1994

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Differential scanning calorimetry was used to compare the thermal behavior of native and delipidated purple membrane fragments at pH values corresponding to purple, blue and acid-purple forms. At neutral pH, delipidation results in a 2.5-to 3-times increase in the cooperativity of the denaturational transition, accompanied by a minor increase in its temperature. At pH values below 5 the delipidated membranes exhibit considerably higher thermal stability than the native membranes. The reversible predenaturational transition observed in the native state is not detectable upon delipidation. There is no strict correlation between color changes upon acid&cation and deionization of either native or delipidated purple membranes and their thermal stability.

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Kinetic study into the irreversible thermal denaturation of bacteriorhodopsin

Cited by 42 publications

References 29 publications

Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems

Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems

Thermal transitions in the purple membrane from Halobacterium halobium

Thermal stability of lipid‐depleted purple membranes at neutral and low pH values

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