Refolding of Thermally Denatured Bacteriorhodopsin in Purple Membrane

Abstract

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.

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

“…It is shown that, within the first 10 min, there is a slight broadening of the amide I peak, but on the whole there is very little permanent damage. This agrees with our previous report on the transition effects on the CD, FT-IR, and photocycle kinetics (33). However, after about 30 min we see that the reversibility of the pre-melting transition reduces significantly, and the red shift of the ␣-helix peak no longer returns to the original position.…”

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

“…40,[42][43][44]71,72 It has been shown that there are two main temperature dependent transitions, one reversible pre-melting transition around ∼78 • C and an irreversible transition at ∼97 • C. 73 The pre-melting transition has been shown to be associated with gel to liquid transition of the hexagonal BR lattice 74 and a partial unfolding of the helices 69 using X-ray diffraction measurements.…”

Hydrogen/deuterium exchange mass spectrometry and optical spectroscopy as complementary tools for studying the structure and dynamics of a membrane protein

“…The retinal absorption spectrum is sensitive to the protein conformation. Native light-adapted BR in its purple membrane environment has its absorption maximum at 568 nm, indicative of the covalently linked chromophore in a structurally intact environment [51]. For the SDS-solubilized protein a maximum at 392 nm is observed (Fig.…”