A method for detergent-free isolation of membrane proteins in their local lipid environment.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins

Solubilization of lipids and membrane proteins into nanodiscs : Mode of action and applications of SMA copolymers

Cell membranes separate the inside and outside of cells. Membrane proteins in the cell membrane control the traffic of molecules across the membrane and are therefore targets for a lot of drugs: about 50 % of all approved drugs target a membrane protein! Unfortunately, scientists only know little about membrane proteins as compared to, for example, water soluble proteins. That is because membrane proteins are hard to study since they reside in the hydrophobic patch of the membrane. To study membrane proteins, they need to be isolated and detergents (soap) are required to do so. The problem is: soap molecules wash away and replace the native membrane environment. In that way, many membrane proteins are destabilized by detergents. In this thesis, a novel method is described that overcomes the issues that detergents bring to the study of lipid membranes and membrane proteins. And help comes unexpectedly. It is the synthetic industrial polymer called styrene-maleic acid, which is often used in the car industry, that is able to isolate membrane protein in a very soft manner. Upon the addition of SMA to membranes, the membranes solubilize in nanodisc particles in which a membrane protein is captured along with a small piece of its native lipid environment. This thesis uses a photosynthetic protein named ‘reaction center’ (RC) form the purple bacterium Rhodobacter sphaeroides as model to validate SMA polymers as alternative to soap molecules. The results show that the RC in SMA nanodiscs maintains the native lipid environment, which is in contrast to soaps such as LDAO and DDM. Furthermore, RCs in a nanodisc are more heat and light stable than RCs in soap and in the native membrane. That opens options to use RCs in SMA nanodiscs in bio-solar cells for which protein stability is key. More results give insight in the molecular action of membrane solubilization by SMA polymers. For example, SMA is a very efficient solubilizing agent, much more than for example membrane scaffold proteins (MSPS, which is the conventional way to produce nanodiscs. Also, the SMA variant with a 2:1 styrene-to-maleic acid ratio is the best SMA variant to use. That is because this polymer has the optimal balance in hydrophobic and hydrophilic groups to attack and solubilize lipid membranes, while stabilizing nanodiscs in water. SMA was also found to solubilize the exact membrane lipid composition in nanodiscs, i.e. SMA has no preference to solubilize certain lipid species. This is a very important result, because thanks to this, SMA is the first way to study lipid-lipid and lipid-protein interactions in a direct and biochemical way. The SMA technique is still young (since 2009 reported in the scientific literature) and many concepts need still to be explored. But due to the potential of SMA to solubilize and stabilize membrane proteins, it is possible that purification of membrane proteins using SMA could become the standard tool for biophysical studies of membrane proteins, and that the polymer will greatly facilitate the wider use of SMA nanodiscs in biohybrid devices

Photophysics in single light-harvesting complexes II: from micelle to native nanodisks

Most photosynthetic pigment-protein complexes of algae and higher plants are integral membrane proteins and are thus usually isolated in the presence of detergent to provide a hydrophobic interface and prevent aggregation. It was recently shown that the styrene maleic acid (SMA) copolymer can be used instead to solubilize and isolate protein complexes with their native lipid environment into nanodisk particles. We isolated LHCII complexes in SMA and compared their photophysics with trimeric LHCII complexes in β-DM detergent micelles to understand the effect of the native environment on the function of light-harvesting antennae. The triplet state kinetics and the calculated relative absorption cross section of single complexes indicate the successful isolation of trimeric complexes in SMA nanodisks, confirming the trimeric structure as the likely native configuration. The survival time of complexes before they photobleach is increased in SMA compared to detergent which might be explained by a stabilizing effect of the co-purified lipids in nanodisks. We furthermore find an unquenched fluorescence lifetime of 3.5 ns for LHCII in SMA nanodisks which coincides with detergent isolated complexes and notably differs from 2 ns typically found in native thylakoid structures. A large dynamic range of partially quenched complexes both in detergent micelles and lipid nanodisks is demonstrated by correlating the fluorescence lifetime with the intensity and likely reflects the conformational freedom of these complexes. This further supports the hypothesis that fluorescence intermittency is an intrinsic property of LHCII that may be involved in excess energy dissipation in native light-harvesting

Energy transfer mechanism for downconversion in the (Pr3+, Yb3+) couple

Downconversion of one visible photon into two infrared photons has been reported for the lanthanide ion couple (Pr3+, Yb3+) in a variety of host lattices. The mechanism responsible for downconversion is controversial and has been reported to be either a two-step energy transfer process (via two first-order transfer steps, the first being cross relaxation) or cooperative energy transfer from Pr3+ to two Yb3+ ions (a second-order process). Here we report experiments on downconversion for the (Pr3+, Yb3+) in LiYF4. Luminescence decay curves of the Pr3+ emission are recorded as a function of the Yb3+ concentration and analyzed using Monte Carlo simulations for both cooperative energy transfer and energy transfer through cross relaxation. We obtain a good agreement between experiment and simulations for energy transfer by cross relaxation but not for cooperative energy transfer. The observation that cross relaxation is more efficient than cooperative energy transfer is consistent with Judd-Ofelt calculations for the transition probabilities involved in the two energy transfer processes and the lower probability for the second-order cooperative transfer.Process and EnergyMechanical, Maritime and Materials Engineerin

Energy transfer mechanism for downconversion in the (Pr3+, Yb3+) couple

Downconversion of one visible photon into two infrared photons has been reported for the lanthanide ion couple ( Pr3+ , Yb3+ ) in a variety of host lattices. The mechanism responsible for downconversion is controversial and has been reported to be either a two-step energy transfer process (via two first-order transfer steps, the first being cross relaxation) or cooperative energy transfer from Pr3+ to two Yb3+ ions (a second-order process). Here we report experiments on downconversion for the ( Pr3+ , Yb3+ ) in LiYF4 . Luminescence decay curves of the Pr3+ emission are recorded as a function of the Yb3+ concentration and analyzed using Monte Carlo simulations for both cooperative energy transfer and energy transfer through cross relaxation. We obtain a good agreement between experiment and simulations for energy transfer by cross relaxation but not for cooperative energy transfer. The observation that cross relaxation is more efficient than cooperative energy transfer is consistent with Judd-Ofelt calculations for the transition probabilities involved in the two energy transfer processes and the lower probability for the second-order cooperative transfe