Methods for Studying Interactions of Detergents and Lipids with α‐Helical and β‐Barrel Integral Membrane Proteins

Hasan, S. Saif; Baniulis, Danas; Yamashita, Eiki; Zhalnina, Mariya V.; Zakharov, Stanisłav D.; Stofleth, Jason T.

doi:10.1002/0471140864.ps2907s74

Cited by 3 publications

(3 citation statements)

References 47 publications

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“…1B). These spectra are consistent with the well-documented precedent for the spectrum for light-induced oxidation of cytochrome f (Cramer and Hasan, 2016; Hasan et al, 2018), and also provide a spectrophotometric reference for the additional studies reported here on the amplitude and kinetics of the light-induced redox turnover of b-cytochromes in intact cyanobacteria. The amplitude and time course of 'dark reduction,' the redox change in the dark to light-induced oxidation of cytochrome f in spinach thylakoid membranes, measured by the absorbance change at 554 nm relative to a reference change measured at 540 nm, is shown (Fig.…”

Section: Resultssupporting

confidence: 90%

“…Synechococcus sp. PCC 7002, wild-type, were grown in medium A (Hasan et al, 2018) under optimal growth conditions, 38°C with an incident light intensity of 100-150 micro Einsteins•m − 2 •s − 1 in 2% (v/v) CO 2 /air. The cell titer was assayed using a Cary 3 spectrophotometer through the optical density of the cell culture measured at 660 nm relative to that at a reference wavelength of 730 nm, using a spectrophotometer with the light collection and detector geometry designed for a high e ciency of collection of light scattered from the turbid cell suspension.…”

Section: Methodsmentioning

confidence: 99%

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Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria

Prabhu,

Cramer

2023

Preprint

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A working concept for the regulation of electron transport in oxygenic photosynthesis is that the electron transfer rate between the two photosystems, PSI and PSII, is governed by a ‘Q-cycle’ pathway operating in the electron transport chain which connects the two ‘reaction center’ complexes. The ‘Q-cycle’ concept was initially inferred from studies on mitochondrial electron transport. This concept has been assumed to be relevant to the electron transport pathways operating in oxygenic photosynthesis, with the majority of studies done on chloroplasts or thylakoid membranes The present study examines the existence and properties of a putative ‘Q-cycle’ in cyanobacteria. Light-induced spectral changes associated with cytochrome redox reactions in intact cells of the cyanobacterium Synechococcus sp. corresponded to the oxidation-reduction of cytochrome f. A correlated reduction of heme b6 was, however, not observed. The absence of significant cytochrome b reduction might be considered inconsistent with the set of electron transfer events associated conceptually with a ‘Q-cycle’ model of the electron transfer events in the chain. However, because heme b6 in the intact cyanobacteria is mostly reduced, it is not observable as a net electron acceptor of the plastoquinol or semiquinone formed by electron transfer from photosystem II. The redox environment of intact cyanobacteria in the dark resting state has an ambient potential sufficiently reducing that the ‘Q-cycle’ pathway for electron transport, well studied and characterized for function in isolated thylakoid membranes or chloroplasts, is not observed. This apparent quandary’ is a consequence of the reducing (negative potential) intracellular redox environment of cyanobacteria, which imposes a reduced state on the b-hemes, thereby preventing observation of their light-induced reduction.

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Section: Resultssupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria

Prabhu,

Cramer

2023

Preprint

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“…Orwick‐Rydmark, Arnold, & Linke () presents an extensive review of detergent diversity and focuses on their uses for MP solubilization. Additionally, Saif Hasan et al., () presents a case study that discusses specific strategies for selecting a detergent for purification and crystallization of two different integral MP complexes. Briefly, detergents are amphipathic molecules.…”

Section: Introductionmentioning

confidence: 99%

Screening of Detergents for Stabilization of Functional Membrane Proteins

et al. 2018

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Membrane protein studies usually require use of detergents to extract and isolate proteins from membranes and manipulate them in a soluble context for their functional or structural characterization. However, solubilization with detergent may interfere with MP stability and may directly affect MP function or structure. Moreover, detergent properties can be affected such as critical micellar concentration (CMC) can be affected by the experimental conditions. Consequently, the experimenter must pay attention to both the protein and the behavior of the detergent. This article provides a convenient protocol for estimating the CMC of detergents in given experimental conditions. Then, it presents two protocols aimed at monitoring the function of a membrane protein in the presence of detergent. Such experiments may help to test various detergents for their inactivating or stabilizing effects on long incubation times, ranging from few hours to some days. © 2018 by John Wiley & Sons, Inc.

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Role of Domain Swapping in the Hetero-Oligomeric Cytochrome b₆f Lipoprotein Complex

et al. 2015

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Domain swapping that contributes to the stability of biologically crucial multisubunit complexes has been implicated in protein oligomerization. In the case of membrane protein assemblies, domain swapping of the iron–sulfur protein (ISP) subunit occurs in the hetero-oligomeric cytochrome b6f and bc1 complexes, which are organized as symmetric dimers that generate the transmembrane proton electrochemical gradient utilized for ATP synthesis. In these complexes, the ISP C-terminal predominantly β-sheet extrinsic domain containing the redox-active [2Fe-2S] cluster resides on the electrochemically positive side of each monomer in the dimeric complex. This domain is bound to the membrane sector of the complex through an N-terminal transmembrane α-helix that is “swapped’ to the other monomer of the complex where it spans the complex and the membrane. Detailed analysis of the function and structure of the b6f complex isolated from the cyanobacterium Fremyella diplosiphon SF33 shows that the domain-swapped ISP structure is necessary for function but is not necessarily essential for maintenance of the dimeric structure of the complex. On the basis of crystal structures of the cytochrome complex, the stability of the cytochrome dimer is attributed to specific intermonomer protein–protein and protein–lipid hydrophobic interactions. The geometry of the domain-swapped ISP structure is proposed to be a consequence of the requirement that the anchoring helix of the ISP not perturb the heme organization or quinone channel in the conserved core of each monomer.

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Methods for Studying Interactions of Detergents and Lipids with α‐Helical and β‐Barrel Integral Membrane Proteins

Cited by 3 publications

References 47 publications

Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria

Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria

Screening of Detergents for Stabilization of Functional Membrane Proteins

Role of Domain Swapping in the Hetero-Oligomeric Cytochrome b₆f Lipoprotein Complex

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Methods for Studying Interactions of Detergents and Lipids with α‐Helical and β‐Barrel Integral Membrane Proteins

Cited by 3 publications

References 47 publications

Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria

Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria

Screening of Detergents for Stabilization of Functional Membrane Proteins

Role of Domain Swapping in the Hetero-Oligomeric Cytochrome b6f Lipoprotein Complex

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Product

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Role of Domain Swapping in the Hetero-Oligomeric Cytochrome b₆f Lipoprotein Complex