A pathway for the thermal destabilization of bacteriorhodopsin

Taneva, Stefka G.; Caaveiro, José M. M.; Muga, Arturo; Goñi, Félix M.

doi:10.1016/0014-5793(95)00570-y

Cited by 47 publications

(71 citation statements)

References 18 publications

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“…The spectral changes of each sample are considerably different, indicating a strong effect of lipids and lattice on the thermal stability and melting mechanism. Previous results (22,30,31) have shown that, as the temperature is raised, native bR shows a shift in the amide I ␣-helical frequency from the unusually FIG. 1.…”

Section: Resultsmentioning

confidence: 92%

“…There has been evidence based on x-ray structure analysis that the pre-melting transition may be a breaking of the crystal packing within the membrane into a liquid-like phase (52). Evidence based on CD and FT-IR spectroscopy has pointed to the possibility of a helical transition, in which the bR helices change conformation from predominantly ␣ II to predominantly a I during this transition (30,31,53). It is possible that these two explanations are coupled and that the helix conformation is determined by the lattice arrangement or vice versa.…”

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confidence: 99%

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The Role of the Native Lipids and Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy

Heyes

El‐Sayed

2002

Journal of Biological Chemistry

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We report the effect of partial delipidation and monomerization on the protein conformational changes of bacteriorhodopsin (bR) as a function of temperature. Removal of up to 75% of the lipids is known to have the lattice structure of the purple membrane, albeit as a smaller unit cell, whereas treatment by Triton monomerizes bR into micelles. The effects of these modifications on the protein secondary structure is analyzed by monitoring the protein amide I and amide II bands in the Fourier transform-infrared (FT-IR) spectra. It is found that removal of the first 75% of the lipids has only a slight effect on the secondary structure at physiological temperature, whereas monomerizing bR into micelles alters the secondary structure considerably. Upon heating, the bR monomer is found to have a very low thermal stability compared with the native bR with its melting point reduced from 97 to 65°C, and the premelting transition in which the protein changes conformation in native bR at 80°C could not be observed. Also, the N-H to N-D exchange of the amide II band is effectively complete at room temperature, suggesting that there are no hydrophobic regions that are protected from the aqueous medium, possibly explaining the low thermal stability of the monomer. On the other hand, 75% delipidated bR has its melting temperature close to that of the native bR and does have a pre-melting transition, although the pre-melting transition occurs at significantly higher temperature than that of the native bR (91°C compared with 80°C) and is still reversible. Furthermore, we have also observed that the reversibility of this pre-melting transition of both native and partially delipidated bR is time-dependent and becomes irreversible upon holding at 91°C between 10 and 30 min. These results are discussed in terms of the lipid and lattice contribution to the protein thermal stability of native bR.

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Section: Resultsmentioning

confidence: 92%

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confidence: 99%

The Role of the Native Lipids and Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy

Heyes

El‐Sayed

2002

Journal of Biological Chemistry

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“…Our results for the two other cleaved BR samples, ABCDE‚FG and AB‚CDE‚FG (see Results), lead to the same conclusion. Previous work (Cladera & Padrós, 1992;Cladera et al, 1992b;Taneva et al, 1995) has shown that the thermal denaturation of BR under different conditions leads to a compact denatured state, which retains a large proportion of ordered structure. Comparing the FTIR spectra of the denatured state of ABCDEFG (Cladera & Padrós, 1992) with that of AB‚CDE‚FG ( Figure 6) it appears that both denatured states are similar in the overall content of secondary structure.…”

Section: Discussionmentioning

confidence: 99%

“…In particular, uncleaved BR has been shown to undergo irreversible, scanrate-dependent thermal denaturation at both pH 9.5 and 7.5 (Taneva et al, 1995), following, in the former case, the twostate kinetic model (Sanchez-Ruiz et al, 1988), with an activation energy of 361 ( 15 kJ/mol (Galisteo & SanchezRuiz, 1993). Our results with the ABCDEFG sample agree with those of Galisteo and Sanchez-Ruiz (1993) (results not shown).…”

Section: Discussionmentioning

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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“…This proposal is in line with experimental IR data from unfolding studies. 40,[42][43][44] Theoretical Methods…”

Section: Introductionmentioning

confidence: 99%

Vibrational Coupling between Helices Influences the Amide I Infrared Absorption of Proteins: Application to Bacteriorhodopsin and Rhodopsin

Karjalainen

Barth

2012

J. Phys. Chem. B

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The amide I spectrum of multimers of helical protein segments was simulated using transition dipole coupling (TDC) for long-range interactions between individual amide oscillators and DFT data from dipeptides (la Cour Jansen et al. 2006, J. Chem. Phys. 125, 44312) for nearest neighbor interactions. Vibrational coupling between amide groups on different helices shift the helix absorption to higher wavenumbers. This effect is small for helix dimers (1 cm −1 ) at 10Å distance and only moderately affected by changes in the relative orientation between the helices. However, the effect becomes considerable when several helices are bundled in membrane proteins. Particular examples are the 7-helix membrane proteins bacteriorhodopsin (BR) and rhodopsin, where the upshift is 4.3 and 5.3 cm −1 , respectively, due to inter-helical coupling within a BR monomer. A further upshift of 4.0 cm −1 occurs when BR monomers associate to trimers. We propose that inter-helical vibrational coupling explains the experimentally observed unusually high wavenumber of the amide I band of BR.

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A pathway for the thermal destabilization of bacteriorhodopsin

Cited by 47 publications

References 18 publications

The Role of the Native Lipids and Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy

The Role of the Native Lipids and Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy

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

Vibrational Coupling between Helices Influences the Amide I Infrared Absorption of Proteins: Application to Bacteriorhodopsin and Rhodopsin

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