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

Taneva, Stefka G.; Koynova, Rumiana; Tenchov, Boris

doi:10.1016/0014-5793(94)00426-9

Cited by 7 publications

(7 citation statements)

References 29 publications

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“…In accordance with previous studies, the main irreversible transition temperature (T m ) of the WT is pH-dependent [44]. Furthermore, T m values for WT at acid and alkaline pH ( Table 2) are in good agreement with previously reported data [36,45]. In comparison with WT, the main transitions of unfolding of 3Glu are not downshifted by very much, between 6 and 12°C, depending on pH [45], implying destabilization of the tertiary structure of the mutated protein to some extent (Table 2).…”

Section: Dscsupporting

confidence: 92%

“…Furthermore, T m values for WT at acid and alkaline pH ( Table 2) are in good agreement with previously reported data [36,45]. In comparison with WT, the main transitions of unfolding of 3Glu are not downshifted by very much, between 6 and 12°C, depending on pH [45], implying destabilization of the tertiary structure of the mutated protein to some extent (Table 2). Moreover, in both WT and 3Glu, the transitions from native to unfolded state of the active site occur at lower temperatures than those of tertiary structure ( Table 2).…”

Section: Dscsupporting

confidence: 90%

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The effect of triple glutamic mutations E9Q/E194Q/E204Q on the structural stability of bacteriorhodopsin

et al. 2014

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In the present study, we report on the structural features of the bacteriorhodopsin triple mutant E9Q/E194Q/E204Q (3Glu) of bacteriorhodopsin by combining experimental and molecular dynamics (MD) approaches. In 3Glu mutant, Glu9, Glu194 and Glu204 residues located at the extracellular side of the protein were mutated altogether to glutamines. UV-visible and differential scanning calorimetry experiments served as diagnostic tools for monitoring the resistance against thermal stress of the active site and the tertiary structures of the 3Glu. The analyses of the UV-visible thermal difference spectra demonstrate that the spectral forms at room temperature and the thermal unfolding path differ in the wild-type bacteriorhodopsin and the 3Glu. Even with these spectral differences, the thermal unfolding of the active site occurs at rather similar melting temperatures in both proteins. A noteworthy consequence of the mutations is the altered two-dimensional packing revealed by the lack of the pre-transition peak in differential scanning calorimetry traces of 3Glu mutant, as previously detected in wild-type and the corresponding single mutants. The infrared spectroscopy data agree with the loss of paracrystalinity, illustrating a substantial conversion of a II to a I helical conformation in the 3Glu mutant. Molecular dynamics simulations show higher dynamics flexibility of most of the extracellular regions of 3Glu, which may account for the somewhat lower tertiary structural stability of the mutated protein. Finally, hydrogen bond analysis reveals that the mutated Glu194 and Glu204 residues create~50% less hydrogen bonds with water molecules compared to wild-type bacteriorhodopsin. These results exemplify the role of the water hydrogen-bonding network for structural integrity and conformational flexibility of bacteriorhodopsin.Abbreviations 3Glu, bacteriorhodopsin triple mutant E9Q/E194Q/E204Q; 3GluO, 3Glu opsin; bO, bacterioopsin; bR, bacteriorhodopsin; bRRT, ground state (RT) spectral form; DSC, differential scanning calorimetry; EC, extracellular; MD, molecular dynamics; PDB, Protein Data Bank; rmsf, root mean square fluctuations; SB, Schiff base; TDS, thermal difference spectra; WT, wild-type bacteriorhodopsin.

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Section: Dscsupporting

confidence: 92%

Section: Dscsupporting

confidence: 90%

The effect of triple glutamic mutations E9Q/E194Q/E204Q on the structural stability of bacteriorhodopsin

et al. 2014

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“…1, a and b) (18,38,39). This process was reversible (40,41). The second transition (T max ¼ 371 K) corresponded to the denaturation of the protein (irreversible process), with an E a of 95.15 kcal/mol (see Fig.…”

Section: Membrane Arrangement and Protein Denaturation: Dsc Analysismentioning

confidence: 96%

Influence of Proline on the Thermostability of the Active Site and Membrane Arrangement of Transmembrane Proteins

Perálvarez-Marín

Lórenz-Fonfrı́a

Simón‐Vázquez

et al. 2008

Biophysical Journal

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Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro50, Pro91, and Pro186 in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro91, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371 K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro91 and Pro186 showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8 kcal/mol, respectively, compared to 115.8 kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function.

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“…By using differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), and Fourier transform infrared (FTIR) spectroscopy, the thermal stability of bR has been investigated. − It has been shown that at neutral pH bR has two thermal transitions, a reversible premelting transition with a melting temperature T m ‘ of ∼78 °C, followed by an irreversible main transition with a melting temperature T m of ∼96 °C; During the reversible transition the protein conformation changes from its original unusual α II -helical conformation to a more commonly seen α I -helical conformation. , …”

Section: Introductionmentioning

confidence: 99%

Refolding of Thermally Denatured Bacteriorhodopsin in Purple Membrane

2001

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The change in protein conformational structure and retinal chromophore binding state have been examined by using in situ UV-vis, FTIR, and CD spectroscopies during the thermal denaturation and refolding processes in bacteriorhodopsin (bR) of purple membrane (PM), in its native trimeric and in Triton X-100 solubilized monomeric form. For the trimeric bR, it is found that heating bR through its premelting transition (T > 78 °C, T m ′) does not cause any permanent damage in the protein secondary structure, and a reversible refolding occurs when it cools back to room temperature. For the monomeric bR, it is found that it is less thermally stable than the trimer. There is a significant change in its protein secondary structure and a complete dissociation of retinal occurs irreversibly at a temperature as low as 66 °C. In addition, it is found that heating the trimeric bR through its main molten state (T > 96 °C, T m ) changes the protein secondary structure so that bR does not refold fully into its original secondary structure. Upon cooling back to room temperature, about 90% of the bound retinal in native bR recovers after being heated through its premelting transition, whereas only about 12% of bound retinal recovers if bR is heated above its main melting temperature. It is also found that refolded bR molecules with their retinal chromophore rebound have a photocycle and are capable of pumping protons. Our results also suggest that from its molten state, protein secondary structure refolding precedes retinal rebinding to the Schiff base.

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

Cited by 7 publications

References 29 publications

The effect of triple glutamic mutations E9Q/E194Q/E204Q on the structural stability of bacteriorhodopsin

The effect of triple glutamic mutations E9Q/E194Q/E204Q on the structural stability of bacteriorhodopsin

Influence of Proline on the Thermostability of the Active Site and Membrane Arrangement of Transmembrane Proteins

Refolding of Thermally Denatured Bacteriorhodopsin in Purple Membrane

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