Studying the stoichiometries of membrane proteins by mass spectrometry: microbial rhodopsins and a potassium ion channel

Hoffmann, Jan; Aslimovska, Lubica; Bamann, Christian; Glaubitz, Clemens; Bamberg, Ernst; Brutschy, Bernd

doi:10.1039/b924630d

Cited by 58 publications

(54 citation statements)

References 32 publications

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“…We therefore tentatively conclude that PR is predominantly present in a hexameric organization, both in the thylakoid and in the cytoplasmic membranes of Synechocystis. The small amount of monomeric PR may actually be caused by detergent solubilization (63,78).…”

Section: Discussionmentioning

confidence: 99%

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

Chen

Steen

Dekker

et al. 2016

Metabolic Engineering

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Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10 5 molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize alltrans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a nonpumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.

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Section: Discussionmentioning

confidence: 99%

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

Chen

Steen

Dekker

et al. 2016

Metabolic Engineering

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“…With respect to elucidating the stoichiometry of the minimum functional ion channel unit, mass spectrometry provides one useful approach [ 29 , 109 ]. However, it has become apparent that in vivo the pore-forming subunits of ion channels are also in contact with multiple regulatory proteins that serve to infl uence ion channel expression, localization, gating, and internalization [ 110 ]. The best known example being the routine co-assembly of voltage-gated ion channel α subunits with regulatory β subunits [ 111 , 112 ].…”

Section: Protein-protein Interactionsmentioning

confidence: 99%

Applications for Mass Spectrometry in the Study of Ion Channel Structure and Function

Samways

2014

Advances in Experimental Medicine and Biology

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Ion channels are intrinsic membrane proteins that form gated ion-permeable pores across biological membranes. Depending on the type, ion channels exhibit sensitivities to a diverse range of stimuli including changes in membrane potential, binding by diffusible ligands, changes in temperature and direct mechanical force. The purpose of these proteins is to facilitate the passive diffusion of ions down their respective electrochemical gradients into and out of the cell, and between intracellular compartments. In doing so, ion channels can affect transmembrane potentials and regulate the intracellular homeostasis of the important second messenger, Ca(2+). The ion channels of the plasma membrane are of particular clinical interest due to their regulation of cell excitability and cytosolic Ca(2+) levels, and the fact that they are most amenable to manipulation by exogenously applied drugs and toxins. A critical step in improving the pharmacopeia of chemicals available that influence the activity of ion channels is understanding how their three-dimensional structure imparts function. Here, progress has been slow relative to that for soluble protein structures in large part due to the limitations of applying conventional structure determination methods, such as X-ray crystallography, nuclear magnetic resonance imaging, and mass spectrometry, to membrane proteins. Although still an underutilized technique in the assessment of membrane protein structure, recent advances have pushed mass spectrometry to the fore as an important complementary approach to studying the structure and function of ion channels. In addition to revealing the subtle conformational changes in ion channel structure that accompany gating and permeation, mass spectrometry is already being used effectively for identifying tissue-specific posttranslational modifications and mRNA splice variants. Furthermore, the use of mass spectrometry for high-throughput proteomics analysis, which has proven so successful for soluble proteins, is already providing valuable insight into the functional interactions of ion channels within the context of the macromolecular-signaling complexes that they inhabit in vivo. In this chapter, the potential for mass spectrometry as a complementary approach to the study of ion channel structure and function will be reviewed with examples of its application.

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“…In particular, large noncovalent assemblies have been studied using matrix-assisted laser desorption/ionization (MALDI) and laser-induced liquid bead ion desorption (LILBID) (32)(33)(34). In the case of MALDI, the analyte is embedded in a dry matrix that, upon laser excitation, ionizes, desorbs, and transfers charge to the analyte.…”

Section: Alternative Ionization Methodsmentioning

confidence: 99%

Analytical Approaches for Size and Mass Analysis of Large Protein Assemblies

Snijder¹,

Heck

2014

Annual Rev. Anal. Chem.

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Analysis of the size and mass of nanoparticles, whether they are natural biomacromolecular or synthetic supramolecular assemblies, is an important step in the characterization of such molecular species. In recent years, electrospray ionization (ESI) has emerged as a technology through which particles with masses up to 100 MDa can be ionized and transferred into the gas phase, preparing them for accurate mass analysis. Here we review currently used methodologies, with a clear focus on native mass spectrometry (MS). Additional complementary methodologies are also covered, including ion-mobility analysis, nanomechanical mass sensors, and charge-detection MS. The literature discussed clearly demonstrates the great potential of ESI-based methodologies for the size and mass analysis of nanoparticles, including very large naturally occurring protein assemblies. The analytical approaches discussed are powerful tools in not only structural biology, but also nanotechnology. 43

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Studying the stoichiometries of membrane proteins by mass spectrometry: microbial rhodopsins and a potassium ion channel

Cited by 58 publications

References 32 publications

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

Applications for Mass Spectrometry in the Study of Ion Channel Structure and Function

Analytical Approaches for Size and Mass Analysis of Large Protein Assemblies

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