Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer: implications for in vitro studies of amphipol-stabilized membrane proteins

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep membrane proteins (MPs) water-soluble while stabilizing them biochemically. We have examined the factors that determine the size and dispersity of MP/APol complexes and studied the dynamics of the association, taking as a model system the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) trapped by A8-35, a polyacrylate-based APol. Molecular sieving indicates that the solution properties of the APol largely determine those of tOmpA/APol complexes. Achieving monodispersity depends on using amphipols that themselves form monodisperse particles, on working in neutral or basic solutions, and on the presence of free APols. In order to investigate the role of the latter, a fluorescently labeled version of A8-35 has been synthesized. F?rster resonance energy transfer measurements show that extensive dilution of tOmpA/A8-35 particles into an APol-free medium does not entail any detectable desorption of A8-35, even after extended periods of time (hours-days). The fluorescent APol, on the other hand, readily exchanges for other surfactants, be they detergent or unlabeled APol. These findings are discussed in the contexts of sample optimization for MP solution studies and of APol-mediated MP functionalization.     

Labeling and Functionalizing Amphipols for Biological Applications

Amphipols (APols) are short amphipathic polymers developed as an alternative to detergents for handling membrane proteins (MPs) in aqueous solution. MPs are, as a rule, much more stable following trapping with APols than they are in detergent solutions. The best-characterized APol to date, called A8-35, is a mixture of short-chain sodium polyacrylates randomly derivatized with octylamine and isopropylamine. Its solution properties have been studied in detail, and it has been used extensively for biochemical and biophysical studies of MPs. One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or otherwise, without affecting their physical-chemical properties. Furthermore, several variously modified APols can be mixed, achieving multiple functionalization of MP/APol complexes in the easiest possible manner. Labeled or tagged APols are being used to study the solution properties of APols, their miscibility, their biodistribution upon injection into living organisms, their association with MPs and the composition, structure and dynamics of MP/APol complexes, examining the exchange of surfactants at the surface of MPs, labeling MPs to follow their distribution in fractionation experiments or to immobilize them, increasing the contrast between APols and solvent or MPs in biophysical experiments, improving NMR spectra, etc. Labeling or functionalization of APols can take various courses, each of which has its specific constraints and advantages regarding both synthesis and purification. The present review offers an overview of the various derivatives of A8-35 and its congeners that have been developed in our laboratory and discusses the pros and cons of various synthetic routes.     

Sulfonated amphipols: synthesis, properties, and applications

Amphipols (APols) are amphiphatic polymers that keep membrane proteins (MPs) water-soluble. The best characterized and most widely used APol to date, A8-35, comprises a polyacrylate backbone grafted with octyl- and isopropylamine side chains. The nature of its hydrophilic moieties prevents its use at the slightly acidic pH that is desirable to slow down the rate of amide proton exchange in solution NMR studies. We describe here the synthesis and properties of pH-insensitive APols obtained by replacing isopropyles with taurine. Sulfonated APols (SAPols) can be used to trap MPs in the form of small complexes, to stabilize them, and to keep them water-soluble even at low pH. [(15) N,(1) H]-transverse relaxation-optimized spectroscopy NMR spectra obtained at pH 6.8 of a bacterial outer MP folded in SAPols show that the protein is correctly folded. The spectra have a resolution similar to that achieved with A8-35 and reveal water-exposed amide and indole protons whose resonance peaks are absent at pH 8.0.     

High-Resolution Structure of a Membrane Protein Transferred from Amphipol to a Lipidic Mesophase

Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2 Å. The structure of BR was solved to 2 Å and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs.     

Isolation of Escherichia coli Mannitol Permease,EIImtl, Trapped in Amphipol A8-35 and Fluorescein-Labeled A8-35

Amphipols (APols) are short amphipathic polymers that keep integral membrane proteins water-soluble while stabilizing them as compared to detergent solutions. In the present work, we have carried out functional and structural studies of a membrane transporter that had not been characterized in APol-trapped form yet, namely EII^mtl, a dimeric mannitol permease from the inner membrane of Escherichia coli. A tryptophan-less and dozens of single-tryptophan (Trp) mutants of this transporter are available, making it possible to study the environment of specific locations in the protein. With few exceptions, the single-Trp mutants show a high mannitol-phosphorylation activity when in membranes, but, as variance with wild-type EII^mtl, some of them lose most of their activity upon solubilization by neutral (PEG- or maltoside-based) detergents. Here, we present a protocol to isolate these detergent-sensitive mutants in active form using APol A8-35. Trapping with A8-35 keeps EII^mtl soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EII^mtl mutant was found to be ~3× higher than the activity of the same protein in dodecylmaltoside. The preparations are suitable both for functional and for fluorescence spectroscopy studies. A fluorescein-labeled version of A8-35 has been synthesized and characterized. Exploratory studies were conducted to examine the environment of specific Trp locations in the transmembrane domain of EII^mtl using Trp fluorescence quenching by water-soluble quenchers and by the fluorescein-labeled APol. This approach has the potential to provide information on the transmembrane topology of MPs.     

Role of the Twin Arginine Protein Transport Pathway in the Assembly of the Streptomyces coelicolor Cytochrome bc1 Complex

The cytochrome bc₁-cytochrome aa₃ complexes together comprise one of the major branches of the bacterial aerobic respiratory chain. In actinobacteria, the cytochrome bc₁ complex shows a number of unusual features in comparison to other cytochrome bc₁ complexes. In particular, the Rieske iron-sulfur protein component of this complex, QcrA, is a polytopic rather than a monotopic membrane protein. Bacterial Rieske proteins are usually integrated into the membrane in a folded conformation by the twin arginine protein transport (Tat) pathway. In this study, we show that the activity of the Streptomyces coelicolor M145 cytochrome bc₁ complex is dependent upon an active Tat pathway. However, the polytopic Rieske protein is still integrated into the membrane in a ΔtatC mutant strain, indicating that a second protein translocation machinery also participates in its assembly. Difference spectroscopy indicated that the cytochrome c component of the complex was correctly assembled in the absence of the Tat machinery. We show that the intact cytochrome bc₁ complex can be isolated from S. coelicolor M145 membranes by affinity chromatography. Surprisingly, a stable cytochrome bc₁ complex containing the Rieske protein can be isolated from membranes even when the Tat system is inactive. These findings strongly suggest that the additional transmembrane segments of the S. coelicolor Rieske protein mediate hydrophobic interactions with one or both of the cytochrome subunits.     

FRET measurement of cytochrome bc1 and reaction centre complex proximity in live Rhodobacter sphaeroides cells

In the model purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides, solar energy is converted via coupled electron and proton transfer reactions within the intracytoplasmic membranes (ICMs), infoldings of the cytoplasmic membrane that form spherical ‘chromatophore’ vesicles. These bacterial ‘organelles’ are ideal model systems for studying how the organisation of the photosynthetic complexes therein shape membrane architecture. In Rba. sphaeroides, light-harvesting 2 (LH2) complexes transfer absorbed excitation energy to dimeric reaction centre (RC)-LH1-PufX complexes. The PufX polypeptide creates a channel that allows the lipid soluble electron carrier quinol, produced by RC photochemistry, to diffuse to the cytochrome bc₁ complex, where quinols are oxidised to quinones, with the liberated protons used to generate a transmembrane proton gradient and the electrons returned to the RC via cytochrome c₂. Proximity between cytochrome bc₁ and RC-LH1-PufX minimises quinone/quinol/cytochrome c₂ diffusion distances within this protein-crowded membrane, however this distance has not yet been measured. Here, we tag the RC and cytochrome bc₁ with yellow or cyan fluorescent proteins (YFP/CFP) and record the lifetimes of YFP/CFP Förster resonance energy transfer (FRET) pairs in whole cells. FRET analysis shows that that these complexes lie on average within 6 nm of each other. Complementary high-resolution atomic force microscopy (AFM) of intact, purified chromatophores verifies the close association of cytochrome bc₁ complexes with RC-LH1-PufX dimers. Our results provide a structural basis for the close kinetic coupling between RC-LH1-PufX and cytochrome bc₁ observed by spectroscopy, and explain how quinols/quinones and cytochrome c₂ shuttle on a millisecond timescale between these complexes, sustaining efficient photosynthetic electron flow.     

Effect of integral membrane proteins on the lateral mobility of plastoquinone in phosphatidylcholine proteoliposomes

Pyrene fluorescence quenching by plastoquinone was used to estimate the rate of plastoquinone lateral diffusion in soybean phosphatidylcholine proteoliposomes containing the following integral membrane proteins: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc₁, and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 · 10^-7 cm² s^-1 in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc₁, and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration.     

Superoxide anion generation by the cytochrome bc1 complex

We have measured the rates of superoxide anion generation by cytochrome bc₁ complexes isolated from bovine heart and yeast mitochondria and by cytochrome bc₁ complexes from yeast mutants in which the midpoint potentials of the cytochrome b hemes and the Rieske iron-sulfur cluster were altered by mutations in those proteins. With all of the bc₁ complexes the rate of superoxide anion production was greatest in the absence of bc₁ inhibitor and ranged from 3% to 5% of the rate of cytochrome c reduction. Stigmatellin, an inhibitor that binds to the ubiquinol oxidation site in the bc₁ complex, eliminated superoxide anion formation, while myxothiazol, another inhibitor of ubiquinol oxidation, allowed superoxide anion formation at a low rate. Antimycin, an inhibitor that binds to the ubiquinone reduction site in the bc₁ complex, also allowed superoxide anion formation and at a slightly greater rate than myxothiazol. Changes in the midpoint potentials of the cytochrome b hemes had no significant effect on the rate of cytochrome c reduction and only a small effect on the rate of superoxide anion formation. A mutation in the Rieske iron-sulfur protein that lowers its midpoint potential from +285 to +220 mV caused the rate of superoxide anion to decline in parallel with a decline in cytochrome c reductase activity. These results indicate that superoxide anion is formed by similar mechanisms in mammalian and yeast bc₁ complexes. The results also show that changes in the midpoint potentials of the redox components that accept electrons during ubiquinol oxidation have only small effects on the formation of superoxide anion, except to the extent that they affect the activity of the enzyme.     

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH₂) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc₁ (cytbc₁) complexes that oxidise QH₂ to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc₁ complexes with gold nanobeads, each attached by a Histidine₁₀ (His₁₀)-tag to the C-terminus of cytc₁. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc₁ complexes occur as dimers in the membrane. The cytbc₁ complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC–LH1–PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc₁ complexes were retrieved from chromatophores partially solubilised by detergent; RC–LH1–PufX complexes tended to co-purify with cytbc₁ whereas LH2 complexes became detached, consistent with clusters of cytbc₁ complexes close to RC–LH1–PufX arrays, but not with a fixed, stoichiometric cytbc₁–RC–LH1–PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc₁, ATP synthase, cytaa₃ and cytcbb₃ membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1–RC–PufX dimers & 2 RC–LH1–PufX monomers, 4 cytbc₁ dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.     

Solubilization of V-ATPase transmembrane peptides by amphipol A8-35.

Two transmembrane peptides encompassing the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae were studied as complexes with APols A8-35 by CD and fluorescence spectroscopy, with the goal to use APols to provide a membrane-mimicking environment for the peptides. CD spectroscopy was used to obtain the overall secondary structure of the peptides, whereas fluorescence spectroscopy provided information about the local environment of their tryptophan residues. The fluorescence results indicate that both peptides are trapped by APols and the CD results that they adopt a beta-sheet conformation. This result is in contrast with previous work that showed that the same peptides are alpha-helical in SDS micelles and organic solvents. These observations are discussed in the context of APol physical-chemical properties and transmembrane peptide structural propensity.     

Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc₁ complex (cytbc₁) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc₁ complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc₁ complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc₁ to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc₁ complexes retain their native dimeric structure and co-purify with 56 ± 6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b₆f complex (cytb₆f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc₁/cytb₆f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q₀ and Q_i sites.     

Intrinsic curvature properties of photosynthetic proteins in chromatophores

In purple bacteria, photosynthesis is carried out on large indentations of the bacterial plasma membrane termed chromatophores. Acting as primitive organelles, chromatophores are densely packed with the membrane proteins necessary for photosynthesis, including light harvesting complexes LH1 and LH2, reaction center (RC), and cytochrome bc₁. The shape of chromatophores is primarily dependent on species, and is typically spherical or flat. How these shapes arise from the protein-protein and protein-membrane interactions is still unknown. Now, using molecular dynamics simulations, we have observed the dynamic curvature of membranes caused by proteins in the chromatophore. A membrane-embedded array of LH2s was found to relax to a curved state, both for LH2 from Rps. acidophila and a homology-modeled LH2 from Rb. sphaeroides. A modeled LH1-RC-PufX dimer was found to develop a bend at the dimerizing interface resulting in a curved shape as well. In contrast, the bc₁ complex, which has not been imaged yet in native chromatophores, did not induce a preferred membrane curvature in simulation. Based on these results, a model for how the different photosynthetic proteins influence chromatophore shape is presented.     

Amphipols for Each Season

Amphipols (APols) are short amphipathic polymers that can substitute for detergents at the transmembrane surface of membrane proteins (MPs) and, thereby, keep them soluble in detergent free aqueous solutions. APol-trapped MPs are, as a rule, more stable biochemically than their detergent-solubilized counterparts. APols have proven useful to produce MPs, most noticeably by assisting their folding from the denatured state obtained after solubilizing MP inclusion bodies in either SDS or urea. They facilitate the handling in aqueous solution of fragile MPs for the purpose of proteomics, structural and functional studies, and therapeutics. Because APols can be chemically labeled or functionalized, and they form very stable complexes with MPs, they can also be used to functionalize those indirectly, which opens onto many novel applications. Following a brief recall of the properties of APols and MP/APol complexes, an update is provided of recent progress in these various fields.     

Timing of dimerization of the bc1 complex during mitochondrial respiratory chain assembly

The mitochondrial bc₁ complex plays an important role in mitochondrial respiration. It transfers electrons from ubiquinol to the soluble electron shuttle cytochrome c and thereby contributes to the proton motive force across the inner mitochondrial membrane. In the yeast Saccharomyces cerevisiae, each monomer consists of three catalytic and seven accessory subunits. The bc₁ complex is an obligate homo-dimer in all systems. It is currently not known when exactly during the assembly dimerization occurs. In this study, we determined that the dimer formation is an early event. Specifically, dimerization is mediated by the interaction of a stable tetramer formed by the two Cor subunits, Cor1 and Cor2, that joins assembly intermediate II, containing the fully hemylated cytochrome b and the two small accessory proteins, Qcr7 and Qcr8. Addition of cytochrome c₁ and Qcr6 can either occur concomitantly or independently of dimerization. These results reveal a strict order in assembly, where dimerization occurs after stabilization of co-factor acquisition by cytochrome b. Finally, assembly is completed by addition of the remaining subunits.     

Effect of Mutations of Arginine 94 on Proton Pumping,Electron Transfer,and Superoxide Anion Generation in Cytochrome b of the bc1 Complex from Rhodobacter sphaeroides

Proton transfer involving internal water molecules that provide hydrogen bonds and facilitate proton diffusion has been identified in some membrane proteins. Arg-94 in cytochrome b of the Rhodobacter sphaeroides bc₁ complex is fully conserved and is hydrogen-bonded to the heme propionate and a chain of water molecules. To further elucidate the role of Arg-94, we generated the mutations R94A, R94D, and R94N. The wild-type and mutant bc₁ complexes were purified and then characterized. The results show that substitution of Arg-94 decreased electron transfer activity and proton pumping capability and increased O₂^˙̄ production, suggesting the importance of Arg-94 in the catalytic mechanism of the bc₁ complex in R. sphaeroides. This also suggests that the transport of H⁺, O₂, and O₂^˙̄ in the bc₁ complex may occur by the same pathway.     

In Vivo Characterization of the Biodistribution Profile of Amphipol A8–35

Amphipols (APols) are polymeric surfactants that keep membrane proteins (MPs) water-soluble in the absence of detergent, while stabilizing them. They can be used to deliver MPs and other hydrophobic molecules in vivo for therapeutic purposes, e.g., vaccination or targeted delivery of drugs. The biodistribution and elimination of the best characterized APol, a polyacrylate derivative called A8–35, have been examined in mice, using two fluorescent APols, grafted with either Alexa Fluor 647 or rhodamine. Three of the most common injection routes have been used, intravenous (IV), intraperitoneal (IP), and subcutaneous (SC). The biodistribution has been studied by in vivo fluorescence imaging and by determining the concentration of fluorophore in the main organs. Free rhodamine was used as a control. Upon IV injection, A8–35 distributes rapidly throughout the organism and is found in most organs but the brain and spleen, before being slowly eliminated (10–20 days). A similar pattern is observed after IP injection, following a brief latency period during which the polymer remains confined to the peritoneal cavity. Upon SC injection, A8–35 remains essentially confined to the point of injection, from which it is only slowly released. An interesting observation is that A8–35 tends to accumulate in fat pads, suggesting that it could be used to deliver anti-obesity drugs.     

Parameters determining the relative efficacy of hydroxy-naphthoquinone inhibitors of the cytochrome bc1 complex

Hydroxy-naphthoquinones are competitive inhibitors of the cytochrome bc₁ complex that bind to the ubiquinol oxidation site between cytochrome b and the iron-sulfur protein and presumably mimic a transition state in the ubiquinol oxidation reaction catalyzed by the enzyme. The parameters that affect efficacy of binding of these inhibitors to the bc₁ complex are not well understood. Atovaquone®, a hydroxy-naphthoquinone, has been used therapeutically to treat Pneumocystis carinii and Plasmodium infections. As the pathogens have developed resistance to this drug, it is important to understand the molecular basis of the drug resistance and to develop new drugs that can circumvent the drug resistance. We previously developed the yeast and bovine bc₁ complexes as surrogates to model the interaction of atovaquone with the bc₁ complexes of the target pathogens and human host. As a first step to identify new cytochrome bc₁ complex inhibitors with therapeutic potential and to better understand the determinants of inhibitor binding, we have screened a library of 2-hydroxy-naphthoquinones with aromatic, cyclic, and non-cyclic alkyl side-chain substitutions at carbon-3 on the hydroxy-quinone ring. We found a group of compounds with alkyl side-chains that effectively inhibit the yeast bc₁ complex. Molecular modeling of these into the crystal structure of the yeast cytochrome bc₁ complex provides structural and quantitative explanations for their binding efficacy to the target enzyme. In addition we also identified a 2-hydroxy-naphthoquinone with a branched side-chain that has potential for development as an anti-fungal and anti-parasitic therapeutic.     

Bacteriorhodopsin/amphipol complexes: structural and functional properties

The membrane protein bacteriorhodopsin (BR) can be kept soluble in its native state for months in the absence of detergent by amphipol (APol) A8-35, an amphiphilic polymer. After an actinic flash, A8-35-complexed BR undergoes a complete photocycle, with kinetics intermediate between that in detergent solution and that in its native membrane. BR/APol complexes form well defined, globular particles comprising a monomer of BR, a complete set of purple membrane lipids, and, in a peripheral distribution, ∼2 g APol/g BR, arranged in a compact layer. In the absence of free APol, BR/APol particles can autoassociate into small or large ordered fibrils.     

Quinone Pathways in Entire Photosynthetic Chromatophores of Rhodospirillum photometricum

In photosynthetic organisms, membrane pigment-protein complexes [light-harvesting complex 1 (LH1) and light-harvesting complex 2 (LH2)] harvest solar energy and convert sunlight into an electrical and redox potential gradient (reaction center) with high efficiency. Recent atomic force microscopy studies have described their organization in native membranes. However, the cytochrome (cyt) bc₁ complex remains unseen, and the important question of how reduction energy can efficiently pass from core complexes (reaction center and LH1) to distant cyt bc₁ via membrane-soluble quinones needs to be addressed. Here, we report atomic force microscopy images of entire chromatophores of Rhodospirillum photometricum. We found that core complexes influence their molecular environment within a critical radius of ∼ 250 Å. Due to the size mismatch with LH2, lipid membrane spaces favorable for quinone diffusion are found within this critical radius around cores. We show that core complexes form a network throughout entire chromatophores, providing potential quinone diffusion pathways that will considerably speed the redox energy transfer to distant cyt bc₁. These long-range quinone pathway networks result from cooperative short-range interactions of cores with their immediate environment.