The Association of Different Detergents with the Photosynthetic Reaction Center Protein of Rhodobacter sphaeroides R26 and the Effects on its Photochemistry

Abstract

Detergent-free reaction centers from Rhodobacter sphaeroides R26 were used to study the solubilization of reaction centers in various detergents and their effects on reaction center photochemistry. 500Z 100 n-octyl-P-D-glucopyranoside or 51 i 5 Triton X-100 molecules were associated with one reaction center. For N,N-alkylamine N-oxide detergents with chain lengths in the range from 8-13 carbon atoms, the number of detergent molecules associated with the reaction centers increased with decreasing chain length. The amount of detergent molecules associated with the reaction centers decreased almost tenfold if the pH was increased from pH 6 to pH 10. The addition of 5 % 1,2,3-heptanetriol to various detergents lowered the detergentheaction center ratio by a factor of two compared to that for the pure detergent. The detergent concentration at which solubilization of the reaction center occurs was close to the critical micelle concentration for all detergents studied. The absorption band at 865 nm of the primary donor in the reaction center shifts to 846nm when detergent was removed from the reaction center; upon resolubilization with various detergents, this band shifts back to 865 nm. In 80-90% of the detergent-free reaction centers, the secondary electron transfer from QA to QB was inhibited; this electron transfer was restored after re-addition of detergent.Keywords: photosynthesis ; membrane protein ; detergent: crystallization; Rhodobacter sphaeroides.Crystallization of detergent-solubilized membrane proteins is complicated by the randomly ordered detergent molecules surrounding the proteins. The success of crystallization of membrane proteins depends on optimizing hydrophobic and hydrophilic interactions between the protein molecules. The type of detergent plays an important role in this process. The length of the detergent alkyl chain determines the thickness of the detergent belt around the protein. This belt should be as thin as possible to increase the number of contact sites between the membrane proteins ; however, the detergent belt should not be too small, so that the detergents can form bridges between the proteins inside the crystal structure [I]. Additionally, the polar head group of the detergent should be small to ensure optimal hydrophilic interactions between the protein molecules [l].The first successful crystallization of a membrane protein was achieved with the photosynthetic reaction center (RC) protein from the purple bacterium Rhodopseudomonas viridis [2]. The addition of 1-3% of the amphiphillic molecule 1,2,3-heptanetriol was essential for the formation of crystals. This led Michel [3] to postulate that the dimensions of the micellar disk surrounding the protein are fine-tuned by 1,2,3-heptanetriol so as to fit into the crystal structure and to increase proteidprotein interactions. Measurements on the micellar radius of N,Ndimethyl dodecylamine N-oxide (DodNOMe, or LDAO) in solution have shown a decrease of the radius upon addition of 1,2,3-heptanetriol The size of this detergent belt is d...

Cofactor-specific photochemical function resolved by ultrafast spectroscopy in photosynthetic reaction center crystals

“…S4. We note that many experimental parameters can cause shifts in the absorption peaks for the RC cofactors (45,46). In the case of the crystal used in Fig.…”

Kinetics of the quinone binding reaction at the QB site of reaction centers from the purple bacteria Rhodobacter sphaeroides reconstituted in liposomes

“…This explains the slow exchange process of the quinone at its binding site. Conversely, the dimensions of Triton X-100 micelles [35] are larger than those formed by LDAO, thereby allowing a larger quinone pool size as well as higher ligand mobility.…”

Modelling the bacterial photosynthetic reaction center. V. Assignment of the electronic transition observed at 2200 cm−1 in the special-pair radical-cation as a second-highest occupied molecular orbital to highest occupied molecular orbital transition

“…Two site effects has been observed in bacterial reaction centers that cause significant modulation of the properties of the reaction center; these are the detergent effect on Rh. sphaeroides 74,75 and the effects of some large-scale mutations on Rb. capsulatus, 76 with spectral similarities 76 suggesting similar origins for these two effects.…”