BackgroundThe reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored.ResultsThe styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes.ConclusionsWe report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.
The solubilization of large, unilamellar egg phosphatidylcholine vesicles by the nonionic detergent octyl glucoside (OG) was investigated by nuclear magnetic resonance (NMR), fluorescence anisotropy, turbidity, electron microscopy, and centrifugation followed by compositional analysis. The solubilization process is well described by the three-stage model previously proposed for other detergents. In stage I, the OG partitions between the bilayer and aqueous phases with a molar partition coefficient of 59 +/- 6. The presence of OG in the bilayers produces a small "fluidizing" effect, as indicated by changes in the NMR and fluorescence anisotropy parameters. A rearrangement that forms large mixed bilayers occurs in the latter part of stage I. Stage II, the conversion of detergent-saturated bilayers into mixed micelles, begins at a ratio of total OG concentration minus the critical micelle concentration to total phosphatidylcholine concentration of approximately 1.5 and continues until this ratio reaches about 3.0. The correction for the critical micelle concentration of the OG is necessary for comparison of experimental results obtained at different lipid concentrations. The mixed bilayer-mixed micelle interconversion is quantified by the centrifugation experiments and by 31P NMR. The agreement between the two methods is excellent. Advantages of the NMR method are discussed. In stage III, which was not studied in detail here, all of the phosphatidylcholine is present as mixed micelles. Evidence is presented that the various structures present in the dispersions are in equilibrium with one another during these experiments.
The photoreceptor rod outer segment (ROS) provides a unique system in which to investigate the role of cholesterol, an essential membrane constituent of most animal cells. The ROS is responsible for the initial events of vision at low light levels. It consists of a stack of disk membranes surrounded by the plasma membrane. Light capture occurs in the outer segment disk membranes that contain the photopigment, rhodopsin. These membranes originate from evaginations of the plasma membrane at the base of the outer segment. The new disks separate from the plasma membrane and progressively move up the length of the ROS over the course of several days. Thus the role of cholesterol can be evlauated in two distinct membranes. Furthermore, because the disk membranes vary in age it can also be investigated in a membrane as a function of the membrane age. The plasma membrane is enriched in cholesterol and in saturated fatty acids species relative to the disk membrane. The newly formed disk membranes have 6-fold more cholesterol than disks at the apical tip of the ROS. The partitioning of cholesterol out of disk membranes as they age and are apically displaced is consistent with the high PE content of disk membranes relative to the plasma membrane. The cholesterol composition of membranes has profound consequences on the major protein, rhodopsin. Biophysical studies in both model membranes and in native membranes have demonstrated that cholesterol can modulate the activity of rhodopsin by altering the membrane hydrocarbon environment. These studies suggest that mature disk membranes initiate the visual signal cascade more effectively than the newly synthesized, high cholesterol basal disks. Although rhodopsin is also the major protein of the plasma membrane, the high membrane cholesterol content inhibits rhodopsin participation in the visual transduction cascade. In addition to its effect on the hydrocarbon region, cholesterol may interact directly with rhodopsin. While high cholesterol inhibits rhodopsin activation, it also stabilizes the protein to denaturation. Therefore the disk membrane must perform a balancing act providing sufficient cholestrol to confer stability but without making the membrane too restrictive to receptor activation. Within a given disk membrane, it is likely that cholesterol exhibits an asymmetric distribution between the inner and outer bilayer leaflets. Furthremore, there is some evidence of cholesterol microdomains in the disk membranes. The availability of the disk protein, rom-1 may be sensitive to membrane cholesterol. The effects exerted by cholesterol on rhodopsin function have far-reaching implications for the study of G-protein coupled receptors as a whole. These studies show that the function of a membrane receptor can be modulated by modification of HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript the lipid bilayer, particularly cholesterol. This provides a powerful means of fine-tuning the activity of a membrane protein without resorting ...
Rhodopsin is a G protein receptor from a many-membered family of membrane receptors. No high-resolution structure exists for any member of this family due to the insolubility of membrane proteins and the difficulty in crystallizing membrane proteins. Two new approaches to the structure of rhodopsin are described that circumvent these limitations: (1) individual solution structures of the four cytoplasmic domains of rhodopsin are fitted with the transmembrane domain; (2) the solution structure of a complex of the four cytoplasmic domains is determined from nuclear magnetic resonance data. The two structures are similar. To test the validity of these structures, specific site-to-site distances measured on intact membrane-bound rhodopsin are compared to the same distances on the structures reported here. Excellent agreement is obtained. Furthermore, the agreement is obtained with distances measured on the activated form of teh receptor and not with distances on the dark-adapted form of rhodopsin. This approach may prove to have general applicability for the determination of the structure for membrane proteins.
Activation of G-protein coupled receptors (GPCR) is not yet understood. A recent structure showed most of rhodopsin in the ground (not activated) state of the GPCR, but the cytoplasmic face, which couples to the G protein in signal transduction, was not well-defined. We have determined an experimental three-dimensional structure for rhodopsin in the unactivated state, which shows good agreement with the crystal structure in the transmembrane domain. This new structure defines the cytoplasmic face of rhodopsin. The G-protein binding site can be mapped. The same experimental approach yields a preliminary structure of the cytoplasmic face in the activated (metarhodopsin II) receptor. Differences between the two structures suggest how the receptor is activated to couple with transducin.
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