Untitled

Abstract

We have recently reported the crystallization of the reaction center of Photosystem II in the presence of detergent mixtures [Adir N (1999) Acta Crystallogr D Biol Crystallogr D55: 891-894]. We have used high performance liquid chromatography, dynamic light scattering, native gel electrophoresis and thermoluminescence measurements to characterize the interaction between these detergent mixtures and RC II, to try and understand their role in the crystallization process. Size exclusion HPLC and dynamic light scattering confirmed that the isolated RC II used for crystallization was exclusively monomeric. Dynamic light scattering measurements show that the detergent mixtures formed single micelles within a limited range of hydrodynamic radii. Both size exclusion HPLC and dynamic light scattering were used to follow the interaction between the detergent mixtures and monomeric RC II. These techniques revealed a decrease in the detergent mixture treated RC II particle size (with respect with the untreated RC II), and that RC II from solubilized crystals contained particles of the same size. Native gel electrophoresis showed that this change in apparent size is not due to the disintegration of the internal structure of the RC II complex. Thermoluminescence measurements of solubilized RC II crystals showed charge recombination from the S(2,3)Q(A) (-) state, indicating that RC II remains functionally viable following detergent mixture treatment and crystallization. The role of the detergent mixtures in the crystallization of RC II is discussed.

Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures

“…As seen in Fig 2C , a competitive effect governs the process: at 50μM of DCMU the photocurrent was inhibited by about 60%, while 500μM DCMU entirely blocked the photocurrent. Finally, to verify that PSII was the electron source, PSII purified from spinach in β-dodecyl-maltoside [ 23 , 24 ], was analyzed, as presented in Fig 2D . As with intact thylakoids, a direct photocurrent could not be detected in the presence or absence of DCMU, indicating that PSII purified from spinach did not transfer electrons directly to the graphite.…”

“…Grana stacks enriched thylakoid preparations, namely BBY membranes, were extracted using the detergent Triton-X (Sigma) described by Berthold et al [ 19 ]. Highly active photosystem II was purified from spinach or tobacco using the detergent n-octyl-glycoside (Anatrace), followed by solubilization of the isolate in micelles of the detergent n-Dodecyl-β-D-Maltoside (Thermo Scientific) as previously published [ 23 , 24 ]. The chlorophyll content in thylakoid preparations was determined according to Arnon [ 36 ].…”

Screening of detergents for solubilization, purification and crystallization of membrane proteins: a case study on succinate:ubiquinone oxidoreductase fromEscherichia coli

“…In conclusion, we show that the phospholipid content of purified SQR depends greatly on the detergent used for solubilization and purification and that the enzyme with the fewest bound phospholipid molecules was successfully crystallized in two crystal forms using mixtures of C n E m and C n M. The reaction centre of photosystem II has also been crystallized using detergent mixtures and the role of detergent mixtures in crystallization has been discussed by Rukhman et al (2000). On the basis of the knowledge obtained in this study, we have succeeded in the crystallization of two membrane proteins, quinol:fumarate reductase from Ascaris suum mitochondria and recombinant cyanide-insensitive alternative oxidase from Trypanosoma brucei, using different mixtures of detergents (details will be published elsewhere).…”

Toward the Crystallization of Photosystem II Core Complex from Pisum sativum L.

“…The ∼35 chlorophyll a molecules, over 10 carotenoids, quinone molecules (Q A and Q B ) and a host of inorganic ions (Ca 2+ , Cl − , Mn 3+ , Mg 2+ , Fe 2+ , HCO 3− ) coordinated by specific residues of the PSII proteins along with redox-active groups (e.g., D1-Tyr161), serve for light harvesting, light transformation, electron transport, and water splitting. It is expected that the PSII CC is also associated with numerous lipid molecules originating from thylakoid membrane that will play various structural and functional roles akin to those proposed for the PSII of cyanobacteria. ,, …”