7948
J. Phys. Chem. B 2008, 112, 7948–7955
Stability of Integral Membrane Proteins under High Hydrostatic Pressure: The LH2 and LH3 Antenna Pigment-Protein Complexes from Photosynthetic Bacteria Liina Kangur,† Ko˜u Timpmann,† and Arvi Freiberg*,†,‡ Institute of Physics, UniVersity of Tartu, Riia 142, Tartu 51014, Estonia, and Institute of Molecular and Cell Biology, UniVersity of Tartu, Riia 23, Tartu 51010, Estonia ReceiVed: March 5, 2008; ReVised Manuscript ReceiVed: April 15, 2008
The bacteriochlorophyll a-containing LH2 and LH3 antenna complexes are the integral membrane proteins that catalyze the photosynthetic process in purple photosynthetic bacteria. The LH2 complex from Rhodobacter sphaeroides shows characteristic strong absorbance at 800 and 850 nm due to the pigment molecules confined in two separate areas of the protein. In the LH3 complex from Rhodopesudomonas acidophila the corresponding bands peak at 800 and 820 nm. Using the bacteriochlorophyll a cofactors as intrinsic probes to monitor local changes in the protein structure, we investigate spectral responses of the antenna complexes to very high hydrostatic pressures up to 2.5 GPa when embedded into natural membrane environment or extracted with detergent. We first demonstrate that high pressure does induce significant alterations to the tertiary structure of the proteins not only in proximity of the 800 nm-absorbing bacteriochlorophyll a molecules known previously (Gall, A.; et al. Biochemistry 2003, 42, 13019) but also of the 850 nm- and 820 nm-absorbing molecules, including breakage of the hydrogen bond they are involved in. The membrane-protected complexes appear more resilient to damaging effects of the compression compared with the complexes extracted into mixed detergent-buffer environment. Increased resistance of the isolated complexes is observed at high protein concentration resulting aggregation as well as when cosolvent (glycerol) is added into the solution. These stability variations correlate with ability of penetration of the surrounding polar solvent (water) into the hydrophobic protein interiors, being thus the principal reason of the pressure-induced denaturation of the proteins. Considerable variability of elastic properties of the isolated complexes was also observed, tentatively assigned to heterogeneous protein packing in detergent micelles. While a number of the isolated complexes release most of their bacteriochlorophyll a content under high pressure, quite some of them remain apparently intact. The pigmented photosynthetic antenna complexes thus constitute a suitable model system for studying in detail the stability of integral membrane proteins. 1. Introduction Integral membrane proteins are ubiquitous in many lifesupporting functions, such as selective transmission of information and matter across the membrane, immune response, photosynthesis, and respiration (see ref 1 for a review). However, physicochemical properties of these important proteins are still largely unknown, because of the many difficulties met upon their study either in native lipid environments or in aqueous liquid solutions.2–5 The membrane proteins, which display large conserved hydrophobic regions, are essentially insoluble in water. Specific detergents (surfactants) are, therefore, used to extract the proteins from the lipid bilayer membrane and to dissolve them in mixed detergent-protein micelles. In the micelles the hydrophobic surfaces of the protein are covered with the detergent molecules. The mechanism of membrane solubilization is unique to an individual detergent. Also the phase behavior of the solubilized membrane proteins depends on subtle equilibrium between surfactant aggregation and protein-surfactant specific interactions. The most common reason of the isolated protein malfunctioning is that the detergent layer is more mobile and less adherent than the native lipid environment. Pressure modulation is an established technique to study protein structures and functions (see refs 6 and 7 for recent * To whom correspondence should be addressed. E-mail: freiberg@ fi.tartu.ee. † Institute of Physics, University of Tartu. ‡ Institute of Molecular and Cell Biology, University of Tartu.
reviews). Working with the pigmented membrane proteins rather than with pure (cofactor free) proteins is beneficial since the pigments could serve as sensitive intrinsic probes to monitor local pressure-induced modifications in the protein structure. The photosynthetic membrane pigment-protein complexes, especially the bacterial light-harvesting complexes, frequently demonstrate clear-cut structure related to different spatial arrangements of the pigments. Due to this advantage, a number of bacterial antenna complexes have been tried under high pressure.8–17 These early works have shown that hydrostatic pressure almost universally leads to a monotonous low-energy shift (red-shift for short) and broadening of the antenna absorption bands. For the peripheral antenna complex from purple photosynthetic bacteria called light-harvesting complex 2 (LH2), which is one of the best-characterized integral membrane proteins, a loss of the 800 nm-absorbing bacteriochlorophyll a (Bchl) molecules in purified LH2 complexes from Rhodobacter (Rb.) sphaeroides 2.4.1 and Rhodopesudomonas (Rps.) acidophila 10050 has been observed suggesting pressureinduced alterations to the tertiary structure of the protein in proximity of the membrane/cytosol interface. No perturbation of the oligomerization of the polypeptides was, however, found at hydrostatic pressures up to 0.6 GPa. That the oligomeric protein such as LH2 does apparently not dissociate at pressures as high as 0.6 GPa is surprising. From the literature, it is known that most of the multichain soluble proteins unfold at room temperature already below 0.2 GPa.7
10.1021/jp801943w CCC: $40.75 2008 American Chemical Society Published on Web 06/07/2008
Pressure Stability of LH2 This process is driven by a decrease in volume, which could result from both the release of intramolecular voids and the expose of the interior of the protein to solvent. Thus motivated, in this work, we methodically investigate the relative structural roles played by in vitro detergent micelles and in vivo membrane in the high-pressure stability of LH2 and LH3 complexes (structural and spectroscopic properties of these complexes are briefly characterized below). In the mixed LH2/LDAO (lauryldimethylaminoxide)/water system three types of phases have been identified, depending on the protein/detergent molar ratio.18 At low protein concentration and low detergent concentration below the critical micelle concentration, small aggregates of densely packed LH2 complexes were found. High protein concentration at relatively low detergent concentration above the critical micelle concentration favored large aggregates of LH2 complexes within LDAO lamellae. Fully solubilized isolated LH2 complexes were only observed at relatively low protein concentration in the micellar phase of LDAO. Therefore, our basic experimental strategy was to record spectral responses of the purified LH2 and LH3 complexes to elevated pressures at varying concentrations of the protein complexes, detergent, and/or glycerol, a protein stabilizing cosolvent. Unusually high pressures for the proteins up to 2.5 GPa were applied. Comparison was made with the spectra of LH2 complexes in membrane vesicles (chromatophores). The experiments firmly established that distortions induced by high hydrostatic pressures involve the whole bulk of the antenna proteins, not only the binding sites of the 800 nm-absorbing Bchl molecules demonstrated earlier.8–17 Hydrogen bond brakeage in the B850 pigment system occurs at pressures above 0.5 GPa, depending on relative concentration of the protein and LDAO. Preliminary results of this work have been reported.19 While little is known about the structure of antenna complexes in native membranes, the crystal structure of the isolated LH2 and LH3 complexes is well studied. The LH2 complex from Rps. acidophila strain 10050,20 for example, reveals a highly symmetric ring of nine pigment-protein subunits, each containing two (R and β) helical membrane-spanning polypeptides, three noncovalently bound Bchl molecules, and a carotenoid pigment. The core of the LH2 complex is highly hydrophobic. In detergent-isolated complexes, it is filled with detergent.21 A striking feature of the organization of the 27 Bchl molecules in LH2 is their partition into two concentric rings. The first ring consists of a group of 18 closely coupled (intermolecular separation
