The in vitro synergistic denaturation effect of heat and surfactant on photosystem I isolated from Arthrospira Platensis.

Photosystem I (PSI) generates the most negative redox potential found in nature, and the performance of solar energy conversion into alternative energy sources in artificial systems highly depends on the thermal stability of PSI. Thus, understanding thermal denaturation is an important prerequisite for the use of PSI at elevated temperatures. To assess the thermal stability of surfactant-solubilized PSI from cyanobacteria Arthrospira Platensis, the synergistic denaturation effect of heat and surfactant was studied. At room temperature, surfactant n-dodecyl-ß-D-maltoside solubilized PSI trimer gradually disassembles into PSI monomers and free pigments over long time. In the solubilizing process of PSI particles, surfactant can uncouple pigments of PSI, and the high concentration of surfactant causes the pigment to uncouple more; after the surfactant-solubilizing process, the uncoupling is relatively slow. During the heating process, changes were monitored by transmittance T800nm, ellipticity θ686nm and θ222nm, upon slow heating (1.5 °C per minute) of samples in Tris buffer (20 mM, pH 7.8) from 20 to 95 °C. The thermal denaturation of surfactant-solubilized PSI is a much more complicated process, which includes the uncoupling of pigments by surfactants, the disappearance of surrounding surfactants, and the unfolding of PSI α-helices. During the heating process, the uncoupling chlorophyll a (Chla) and converted pheophytin (Pheo) can form excitons of Chla-Pheo. The secondary structure α-helix of PSI proteins is stable up to 87-92 °C in the low-concentration surfactant solubilized PSI, and high-concentration surfactant and pigments uncoupling can accelerate the α-helical unfolding.

Effect of surfactants on apparent oxygen consumption of photosystem I isolated from Arthrospira platensis.

Surfactants play a significant role in solubilization of photosystem I (PSI) in vitro. Triton X-100 (TX), n-Dodecyl-ß-D-maltoside (DDM), and sodium dodecyl sulfate (SDS) were employed to solubilize PSI particles in MES buffer to compare the effect of surfactant and its dosage on the apparent oxygen consumption rate of PSI. Through a combined assessment of sucrose density gradient centrifugation, Native PAGE and 77 K fluorescence with the apparent oxygen consumption, the nature of the enhancement of the apparent oxygen consumption activity of PSI by surfactants has been analyzed. Aggregated PSI particles can be dispersed by surfactant molecules into micelles, and the apparent oxygen consumption rate is higher for surfactant-solubilized PSI than for integral PSI particles. For DDM, PSI particles are solubilized mostly as the integral trimeric form. For TX, PSI particles are solubilized as incomplete trimeric and some monomeric forms. For the much harsher surfactant, SDS, PSI particles are completely solubilized as monomeric and its subunit forms. The enhancement of the oxygen consumption rate cannot be explained only by the effects of surfactant on the equilibrium between monomeric and trimeric forms of solubililized PSI. Care must be taken when the electron transfer activity of PSI is evaluated by methods based on oxygen consumption because the apparent oxygen consumption rate is influenced by uncoupled chlorophyll (Chl) from PSI, i.e., the larger the amount of uncoupled Chl, the higher the rate of apparent oxygen consumption. 77 K fluorescence spectra can be used to ensure that there is no uncoupled Chl present in the system. In order to eliminate the effect of trace uncoupled Chl, an efficient physical quencher of (1)O2, such as 1 mM NaN3, may be added into the mixture.

Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis.

Surfactants play important roles in the preparation, structural, and functional research of membrane proteins, and solubilizing and isolating membrane protein, while keeping their structural integrity and activity intact is complicated. The commercial n-Dodecyl-ß-D-maltoside (DDM) and Triton X-100 (TX) were used as solubilizers to extract and purify trimeric photosystem I (PSI) complex, an important photosynthetic membrane protein complex attracting broad interests. With an optimized procedure, TX can be used as an effective surfactant to isolate and purify PSI, as a replace of the much more expensive DDM. A mechanism was proposed to interpret the solubilization process at surfactant concentrations lower than the critical solubilization concentration. PSI-TX and PSI-DDM had identical polypeptide bands, pigment compositions, oxygen consumption, and photocurrent activities. This provides an alternative procedure and paves a way for economical and large-scale trimeric PSI preparation.

Solubilization and stabilization of isolated photosystem I complex with lipopeptide detergents.

It is difficult to maintain a target membrane protein in a soluble and functional form in aqueous solution without biological membranes. Use of surfactants can improve solubility, but it remains challenging to identify adequate surfactants that can improve solubility without damaging their native structures and biological functions. Here we report the use of a new class of lipopeptides to solubilize photosystem I (PS-I), a well known membrane protein complex. Changes in the molecular structure of these surfactants affected their amphiphilicity and the goal of this work was to exploit a delicate balance between detergency and biomimetic performance in PS-I solubilization via their binding capacity. Meanwhile, the effects of these surfactants on the thermal and structural stability and functionality of PS-I in aqueous solution were investigated by circular dichroism, fluorescence spectroscopy, SDS-PAGE analysis and O2 uptake measurements, respectively. Our studies showed that the solubility of PS-I depended on both the polarity and charge in the hydrophilic head of the lipopeptides and the length of its hydrophobic tail. The best performing lipopeptides in favour of PS-I solubility turned out to be C14DK and C16DK, which were comparable to the optimal amphiphilicity of the conventional chemical surfactants tested. Lipopeptides showed obvious advantages in enhancing PS-I thermostability over sugar surfactant DDM and some full peptide amphiphiles reported previously. Fluorescence spectroscopy along with SDS-PAGE analysis demonstrated that lipopeptides did not undermine the polypeptide composition and conformation of PS-I after solubilization; instead they showed better performance in improving the structural stability and integrity of this multi-subunit membrane protein than conventional detergents. Furthermore, O2 uptake measurements indicated that PS-I solubilized with lipopeptides maintained its functionality. The underlying mechanism for the favorable actions of lipopeptide in PS-I solubilization and stabilization is discussed.