Protein-detergent complexes

T,he stoichiometric characterization of detergent-protein complexes has been the object of many studies over the past 30 years (6). Recent studies have placed more emphasis upon developing a molecular-kinetic description of the complex (2, 8). The importance of such descriptions lies in the fact that detergent-protein complexes can be considered as lipoprotein model systems. Indeed, virtually all conceptions of the microscopic nature of lipid-protein interactions are based on the properties of detergent-protein complexes (3). 

The experimental procedure consisted first of generating a detergent-protein complex monolayer of fixed area at the air-water interface, followed by varying the substrate pH. The experimental parameters were film surface pressure and the subphase pH. 

An essential caveat must be observed, however the molecular weight estimation measures the size of the detergent-protein complex, and different proteins bind different amounts of detergent. All empirical methods, accordingly, are fraught with possible error, and the results reported or reviewed now should be looked upon as preliminary and indicative, rather than definitive. 

The cytochrome b559 content was found to be 1 per 2 pheophytin a (19) and subsequent analyses have shown the same ratio in larger PS2 preparations (20). The fatty acid content has been analysed and indicates that there is always less than 1.0 mole dlacyl glycerolipid 2 pheophytin a. This low level probably arises from non-specific interaction with the detergent-protein complex. 

Detergent treatment of a suspension of thylakoids dissolves the membranes, releasing complexes containing both chlorophyll and protein. These chlorophyll-protein complexes represent integral components of the thylakoid membrane, and their organization reflects their roles as either light-harvesting com-... 

In the case of PS II membrane proteins, as discussed above, the hydrophobic and hydrophilic pairs of attached lipids can partially support the protein complex at the air-water interface, despite their large size and density. However, in the case of PS II core complex, the detergent strips the attached lipids and some extrinsic proteins. The remaining protein complex is water soluble. It is very difficult to prepare a stable monolayer of water-soluble proteins with the Langmuir method. Indeed, it is hard to directly prepare a stable monolayer of PS II core complex because of its water solubility as well as density. One possible solution is to change the density and ionic strength of the subphase [9]. 

Law and Reisine [73] reported that the cloned 8 receptor physically associated with G0. They solubilized the 8 receptor with a mild detergent which allowed solubilized 8 receptors to remain associated with G proteins. They then showed that antisera directed against G0 co-immunoprecipitated 8 receptor/G protein complexes. 

Photosystem I is a membrane pigment-protein complex in green plants, algae as well as cyanobacteria, and undergoes redox reactions by using the electrons transferred from photosystem II (PS II) [1], These membrane proteins are considered to be especially interesting in the study of monomolecular assemblies, because their structure contains hydrophilic area that can interact with the subphase as well as hydrophobic domains that can interact either with each other or with detergent and lipids [2], Moreover, studies with such proteins directly at the air-water interface are expected to be a valuable approach for their two-dimensional crystallization. 

The tissue or cell sample is firstly homogenized in a buffer containing a detergent such as Triton X-100 and sodium deodecyl sulphate (SDS), which disrupts the cell and dissociates DNA-protein complexes. Protein and RNA are then removed by sequential incubations with a proteolytic enzyme (usually proteinase K) and ribonuclease. Finally the DNA is extracted into ethanol. Ethanol only precipitates long chain nucleic acids and so leaves the single nucleotides from RNA digestion in the aqueous layer. 

A relatively simple and quick procedure for the isolation of Photosystem I-enriched particles from the thermophilic cyanobacterium Phormidium laminosum, without the use of detergents for solubilization, is described. The procedure involves sonication of cells, centrifugation and DEAE-cellulose chromatography. The particles had an 02 uptake activity of up to 200 pmol 02. mg chlorophyll h 1 and appeared as vesicles of 200 100 nm diameter when observed under electron microscopy. The analysis of the chlorophyll-protein complexes by polyacrylamide gel electrophoresis showed that these particles are enriched in the complexes associated with Photosystem I and partially depleted in those associated with Photosystem II. The particles did not contain ferredoxin and were active in NADP-photoreduction only in the presence of added ferredox in. They were also able to photoreduce externally added electron mediators using ascorbate as electron donor, the reduced mediators can be coupled to hydrogenase for the production of H2 or for the activation of cyanobacterial phosphoribulokinase using a ferredoxin/thioredoxin system. 

The detergent treatment conditions of samples drastically affected the SDS-PAGE of the Chl-protein complexes. Thus, when samples were SDS-treated for different time lengths the A-2 band was affected faster than the other green bands, and no A-2 band could be observed after incubation of samples for 90 min with SDS at a final SDS Chi ratio=20 l (data not shown). However, the effect of the LDAO pretreatment of samples was less apparent... 

Huang, C. and Bems, D.S. 1983. Partial characterization of six chlorophyll a-protein complexes isolated from a blue-green alga by a non-detergent method. Arch. Biochem. Biophys., 220.145-154. 

In PS I, as in PS II, there are a number of Chi alb protein complexes having lightharvesting and energy-transfer functions. Such complexes most probably exist in direct contact with the RC (part of the core complex), and certainly exist as peripheral LHC I antenna complexes further removed from the RC. A native PS I complex (80-180 Chi per RC, 100 kDa) with at least 6 polypeptides was isolated by solubilization of the thylakoid membrane with nonionic detergents (for example, Triton X-100) [177,178]. With further detergent treatment, the PS I complex dissociated into the core complex (CC I with the RC) and the peripheral antenna complex (LHC I) (spinach, barley, pea, Chlamydomonas reinhardtii [179-183]. The peripheral antenna complex (pea, spinach Chi alb ratio 4.0 1, typical fluorescence at 730 nm) contains 3-4 antenna polypeptides (19-25 kDa) [181,184,185]. This complex was also dissociated into two different antenna complexes - LHC la (2 polypeptides of 22 and 23 kDa) and LHC Ib (1 polypeptide, 20 kDa) - which differ in their fluorescence characteristics (680 nm and 730 nm) [184]. No structural data on these polypeptides are available at present. It was postulated that in C. reinhardtii, in addition to the peripheral antenna complex, an antenna system (with 4 polypeptides) exists, which connects the peripheral antenna energetically with the core complex CC 1 [183]. 

Zhang H, Kurisu G, Smith JL, Cramer WA. A defined protein- 66. detergent-lipid complex for crystallization of integral membrane proteins The cytochrome b6f complex of oxygenic photosynthesis. Proc. Natl. Acad. Sci. U.S.A. 2003 100 5160-5163. 

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