Intestinal permeation enhancers enable oral delivery of macromolecules up to 70 kDa in size.

The decades-long effort to deliver peptide drugs orally has resulted in several clinically successful formulations. These formulations are enabled by the inclusion of permeation enhancers that facilitate the intestinal absorption of peptides. Thus far, these oral peptide drugs have been limited to peptides less than 5 kDa, and it is unclear whether there is an upper bound of protein size that can be delivered with permeation enhancers. In this work, we examined two permeation enhancers, 1-phenylpiperazine (PPZ) and sodium deoxycholate (SDC), for their ability to increase intestinal transport of a model macromolecule (FITC-Dextran) as a function of its size. Specifically, the permeability of dextrans with molecular weights of 4, 10, 40, and 70 kDa was assessed in an in vitro and in vivo model of the intestine. In Caco-2 monolayers, both PPZ and SDC significantly increased the permeability of only FD4 and FD10. However, in mice, PPZ and SDC behaved differently. While SDC improved the absorption of all tested sizes of dextrans, PPZ was effective only for FD4 and FD10. This work is the first report of PPZ as a permeation enhancer in vivo, and it highlights the ability of permeation enhancers to improve the absorption of macromolecules across a broad range of sizes relevant for protein drugs.

New structural insights into the recognition of undamaged splayed-arm DNA with a single pair of non-complementary nucleotides by human nucleotide excision repair protein XPA.

XPA (Xeroderma pigmentosum complementation group A) is a core scaffold protein that plays significant roles in DNA damage verification and recruiting downstream endonucleases in the nucleotide excision repair (NER) pathway. Here, we present the 2.81 Å resolution crystal structure of the DNA-binding domain (DBD) of human XPA in complex with an undamaged splayed-arm DNA substrate with a single pair of non-complementary nucleotides. The structure reveals that two XPA molecules bind to one splayed-arm DNA with a 10-bp duplex recognition motif in a non-sequence-specific manner. XPA molecules bind to both ends of the DNA duplex region with a characteristic ß-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic ß-hairpin. Upon DNA binding, the C-terminal last helix of XPA would shift towards the minor groove of the DNA substrate for better interaction. Notably, human XPA is able to bind to the undamaged DNA duplex without any kinks, and XPA-DNA binding does not bend the DNA substrate obviously. This study provides structural basis for the binding mechanism of XPA to the undamaged splayed-arm DNA with a single pair of non-complementary nucleotides.

Natural Variation Reveals a Key Role for Rhamnogalacturonan I in Seed Outer Mucilage and Underlying Genes.

On imbibition, Arabidopsis (Arabidopsis thaliana) seeds release polysaccharides from their epidermal cells that form a two-layered hydrogel, termed mucilage. Analysis of a publicly available data set of outer seed mucilage traits of over 300 accessions showed little natural variation in composition. This mucilage is almost exclusively made up of rhamnogalacturonan I (RGI), highlighting the importance of this pectin for outer mucilage function. In a genome-wide association study, observed variations in polymer amount and macromolecular characteristics were linked to several genome polymorphisms, indicating the complexity of their genetic regulation. Natural variants with high molar mass were associated with a gene encoding a putative glycosyltransferase called MUCILAGE-RELATED70 (MUCI70). muci70 insertion mutants produced many short RGI polymers that were highly substituted with xylan, confirming that polymorphism in this gene can affect RGI polymer size. A second gene encoding a putative copper amine oxidase of clade 1a (CuAOα1) was associated with natural variation in the amount of RGI present in the outer mucilage layer; cuaoα1 mutants validated its role in pectin production. As the mutant phenotype is unique, with RGI production only impaired for outer mucilage, this indicates that CuAOα1 contributes to a further mechanism controlling mucilage synthesis.

Spectral comparisons of mammalian cells and intact organelles by solid-state NMR.

Whole-cell protein profiling, spatial localization, and quantification of activities such as gene transcription and protein translation are possible with modern biochemical and biophysical techniques. Yet, addressing questions of overall compositional changes within a cell - capturing the relative amounts of protein and ribosomal RNA levels and lipid content simultaneously - would require extractions and purifications with caveats due to isolation yields and detection methods. A holistic view of cellular composition would aid in the study of cellular composition and function. Here, solid state NMR is used to identify 13C NMR signatures for cellular organelles in HeLa cells without the use of any isotopic labeling. Comparisons are made with carbon spectra of subcellular assemblies including DNA, lipids, ribosomes, nuclei and mitochondria. Whole-cell comparisons are made with different mammalian cells lines, with red blood cells that lack nuclei and organelles, and with Gram-negative and Gram-positive bacteria. Furthermore, treatment of mammalian cells with cycloheximide, a commonly used protein synthesis inhibitor, revealed unanticipated changes consistent with a significant increase in protein glycosylation, obvious at the whole cell level. Thus, we demonstrate that solid-state NMR serves as a unique analytical tool to catalog and compare the ratios of distinct carbon types in cells and serves as a discovery tool to reveal the workings of inhibitors such as cycloheximide on whole-cell biochemistry.

Near-infrared excited cooperative upconversion in luminescent Ytterbium(ΙΙΙ) bioprobes as light-responsive theranostic agents.

Near-infrared (NIR) Ytterbium(ΙΙΙ) complexes namely [Yb(dpq)(DMF)2Cl3] (1), [Yb(dppz)(DMF)2Cl3] (2), [Yb(dpq)(ttfa)3] (3) and [Yb(dppz)(ttfa)3] (4) based on photosensitizing antenna: dipyrido-[3,2-d:2',3'-f]-quinoxaline (dpq), dipyrido-[3,2-a:2',3'-c]-phenazine (dppz) and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (Httfa), were designed as NIR bioimaging agents utilizing cooperative upconversion luminescence (CUCL) of Yb(III). Their structures, detailed photophysical properties, biological interactions, photo-induced DNA cleavage, NIR photocytotoxicity and cellular internalization and bioimaging properties were examined. Discrete mononuclear complexes adopt a seven-coordinated {LnN2O2Cl3} mono-capped octahedron (1, 2) and eight-coordinated {LnN2O6} distorted square antiprism geometry (3, 4) with bidentate N, N-donor dpq, dppz and O,O-donor ttfa ligands. The designed Yb(III) probes (3, 4) having advantages of dual sensitizing antennae (dpq/dppz and Httfa) to modulate the desirable optical properties in NIR region for bioimaging in biologically transparent window and light-responsive intracellular damage with spatiotemporal control. The lack of inner-sphere water (q = 0), remarkable photostability, large Stokes' shifts, presence of energetically rightly poised ligand 3T states allows efficient energy transfer (ET) to the emissive 2F5/2 state of Yb(ΙΙΙ). The unique cooperative upconversion luminescence (CUCL) of Yb(III) was observed in 1-4 in the visible blue region (λem = 490 nm) upon NIR excitation at 980 nm, makes them special candidates for NIR-to-visible or NIR-to-NIR cellular imaging probes. The CUCL property of Yb(III) were observed in the discrete mononuclear complexes both in solid state and solution. We elegantly utilized this remarkable property of Yb(III) for cellular imaging application for the first time to the our knowledge including potential uses in CUC/multiphoton excitation microscopy. The complexes exhibit significant binding propensity to DNA, HSA and BSA (K ∼ 105 M-1). They effectively cleave supercoiled (SC) DNA to its nicked circular (NC) form at 365 nm via photoredox pathways. The cellular uptake studies evidently displayed cytosolic and nuclear localization of the complexes. Finally, the capability of Yb(III) complexes usage for PDT were demonstrated through significant near-IR photocytotoxicity at 980 nm CW laser. The results depicted here offers an intelligent strategy towards developing light-responsive highly photostable Yb(III) probes for NIR theranostic application in the biologically transparent phototherapeutic window.

A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation.

Biomineralization, including shell formation, is dedicatedly regulated by matrix proteins. PfY2, a matrix protein detected in the ethylene diamine tetraacetic acid (EDTA)-soluble fraction from both prismatic layer and nacreous layer, was discovered by our group using microarray. It may play dual roles during biomineralization. However, the molecular mechanism is still unclear. In this research, we studied the function of PfY2 on crystallization in vivo and in vitro, revealing that it might be a negative regulator during shell formation. Notching experiment indicated that PfY2 was involved in shell repairing and regenerating process. Repression of PfY2 gene affected the structure of prismatic and nacreous layer simultaneously, confirming its dual roles in shell formation. Recombinant protein rPfY2 significantly suppressed CaCO3 precipitation rate, participated in the crystal nucleation process, changed the morphology of crystals and inhibited the transformation of amorphous calcium carbonate (ACC) to stable calcite or aragonite in vitro. Our results may provide new evidence on the biomineralization inhibition process.

High intensity focused ultrasound hyperthermia for enhanced macromolecular delivery.

Mild hyperthermia has been used in combination with polymer therapeutics to further increase delivery to solid tumors and enhance efficacy. An attractive method for generating heat is through non-invasive high intensity focused ultrasound (HIFU). HIFU is often used for ablative therapies and must be adapted to produce uniform mild hyperthermia in a solid tumor. In this work a magnetic resonance imaging guided HIFU (MRgHIFU) controlled feedback system was developed to produce a spatially uniform 43°C heating pattern in a subcutaneous mouse tumor. MRgHIFU was employed to create hyperthermic conditions that enhance macromolecular delivery. Using a mouse model with two subcutaneous tumors, it was demonstrated that MRgHIFU enhanced delivery of both Evans blue dye (EBD) and Gadolinium-chelated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. The EBD accumulation in the heated tumors increased by nearly 2-fold compared to unheated tumors. The Gadolinium-chelated HPMA copolymers also showed significant enhancement in accumulation over control as evaluated through MRI T1-mapping measurements. Results show the potential of HIFU-mediated hyperthermia for enhanced delivery of polymer therapeutics.

Case control study: magnetic resonance spectroscopy of brain in HIV infected patients.

BACKGROUND: In vivo proton magnetic resonance spectroscopy ((1)H-MRS) studies on brain in HIV infected patients have shown significant alteration in neuro-biochemicals. METHODS: In this study, we measured the neuro-biochemical metabolites from the left frontal white matter (FWM) and left basal ganglia (BG) caudate head nucleus in 71 subjects that include 30 healthy controls, 20 asymptomatic HIV and 21 HIV patients with CNS lesion. Proton MR spectra were acquired at 3 T MRI system and the concentration (institutional units) of tNAA (N-acetylaspartate, NAA + N-acetylaspartylglutamate, NAAG), tCr (Creatine, Cr + phosphocreatine, PCr), choline containing compounds (tCho), glutamate + glutamine (Glx) and lipid and macromolecules at 0.9 ppm were determined using LC Model. RESULTS: In BG, the concentration of tNAA (6.71 ± 0.64) was decreased and in FWM, the concentration of Glx (20.4 ± 7.8), tCr (9.14 ± 3.04) and lipid and macromolecules at 0.9 ppm (8.69 ± 2.96) were increased in HIV patients with CNS lesion. In healthy controls, the concentration of tNAA in BG was 7.31 ± 0.47 and concentration of Glx, tCr and lipid and macromolecules in FWM were 15.0 ± 6.06, 6.95 ± 2.56, 5.59 ± 1.56, respectively. CONCLUSION: Reduced tNAA in BG suggests neuronal loss in HIV patients with CNS lesion while increased Glx in FWM may suggest excito-toxicity. In addition, increased levels of tCr in FWM of HIV patients were observed. The study indicates region specific metabolic changes in tNAA, tCr and Glx in brain of HIV infected patients.

Impaired macromolecular protein pools in fronto-striato-thalamic circuits in type 2 diabetes revealed by magnetization transfer imaging.

Previous research has shown that type 2 diabetes mellitus (T2DM) is associated with white matter microstructural changes, cognitive impairment, and decreased resting-state functional connectivity and spontaneous brain activity. This study used magnetization transfer imaging to examine, for the first time, the integrity of macromolecular protein pools in fronto-striato-thalamic circuits and its clinical and cognitive correlates in patients with T2DM. T2DM patients without mood disorders (n = 20, aged 65.05 ± 11.95 years) and healthy control subjects (HCs; n = 26, aged 62.92 ± 12.71 years) were recruited. Nodes of fronto-striato-thalamic circuits-head of the caudate nucleus (hCaud), putamen, globus pallidus, thalamus-and four cortical regions-rostral and dorsal anterior cingulate cortex, dorsolateral prefrontal cortex, and lateral orbitofrontal cortex-were examined. Compared with HCs, patients with T2DM had significantly lower magnetization transfer ratio (MTR) in bilateral anterior cingulate and hCaud. Reduced MTRs in the above regions showed correlations with T2DM-related clinical measures, including hemoglobin A1c level and vascular risk factors, and neuropsychological task performance in the domains of learning and memory, executive function, and attention and information processing. The impaired biophysical integrity of brain macromolecular protein pools and their local microenvironments in T2DM patients may provide insights into the neurological pathophysiology underlying diabetes-associated clinical and cognitive deficits.

Production of a bioflocculant from Aspergillus niger using palm oil mill effluent as carbon source.

This study evaluated the potential of bioflocculant production from Aspergillus niger using palm oil mill effluent (POME) as carbon source. The bioflocculant named PM-5 produced by A. niger showed a good flocculating capability and flocculating rate of 76.8% to kaolin suspension could be achieved at 60 h of culture time. Glutamic acid was the most favorable nitrogen source for A. niger in bioflocculant production at pH 6 and temperature 35 °C. The chemical composition of purified PM-5 was mainly carbohydrate and protein with 66.8% and 31.4%, respectively. Results showed the novel bioflocculant (PM-5) had high potential to treat river water from colloids and 63% of turbidity removal with the present of Ca(2+) ion.

Recent advances toward a general purpose linear-scaling quantum force field.

Conspectus There is need in the molecular simulation community to develop new quantum mechanical (QM) methods that can be routinely applied to the simulation of large molecular systems in complex, heterogeneous condensed phase environments. Although conventional methods, such as the hybrid quantum mechanical/molecular mechanical (QM/MM) method, are adequate for many problems, there remain other applications that demand a fully quantum mechanical approach. QM methods are generally required in applications that involve changes in electronic structure, such as when chemical bond formation or cleavage occurs, when molecules respond to one another through polarization or charge transfer, or when matter interacts with electromagnetic fields. A full QM treatment, rather than QM/MM, is necessary when these features present themselves over a wide spatial range that, in some cases, may span the entire system. Specific examples include the study of catalytic events that involve delocalized changes in chemical bonds, charge transfer, or extensive polarization of the macromolecular environment; drug discovery applications, where the wide range of nonstandard residues and protonation states are challenging to model with purely empirical MM force fields; and the interpretation of spectroscopic observables. Unfortunately, the enormous computational cost of conventional QM methods limit their practical application to small systems. Linear-scaling electronic structure methods (LSQMs) make possible the calculation of large systems but are still too computationally intensive to be applied with the degree of configurational sampling often required to make meaningful comparison with experiment. In this work, we present advances in the development of a quantum mechanical force field (QMFF) suitable for application to biological macromolecules and condensed phase simulations. QMFFs leverage the benefits provided by the LSQM and QM/MM approaches to produce a fully QM method that is able to simultaneously achieve very high accuracy and efficiency. The efficiency of the QMFF is made possible by partitioning the system into fragments and self-consistently solving for the fragment-localized molecular orbitals in the presence of the other fragment's electron densities. Unlike a LSQM, the QMFF introduces empirical parameters that are tuned to obtain very accurate intermolecular forces. The speed and accuracy of our QMFF is demonstrated through a series of examples ranging from small molecule clusters to condensed phase simulation, and applications to drug docking and protein-protein interactions. In these examples, comparisons are made to conventional molecular mechanical models, semiempirical methods, ab initio Hamiltonians, and a hybrid QM/MM method. The comparisons demonstrate the superior accuracy of our QMFF relative to the other models; nonetheless, we stress that the overarching role of QMFFs is not to supplant these established computational methods for problems where their use is appropriate. The role of QMFFs within the toolbox of multiscale modeling methods is to extend the range of applications to include problems that demand a fully quantum mechanical treatment of a large system with extensive configurational sampling.

Arsenic and selenium toxicity and their interactive effects in humans.

Arsenic (As) and selenium (Se) are unusual metalloids as they both induce and cure cancer. They both cause carcinogenesis, pathology, cytotoxicity, and genotoxicity in humans, with reactive oxygen species playing an important role. While As induces adverse effects by decreasing DNA methylation and affecting protein 53 expression, Se induces adverse effects by modifying thioredoxin reductase. However, they can react with glutathione and S-adenosylmethionine by forming an As-Se complex, which can be secreted extracellularly. We hypothesize that there are two types of interactions between As and Se. At low concentration, Se can decrease As toxicity via excretion of As-Se compound [(GS3)2AsSe](-), but at high concentration, excessive Se can enhance As toxicity by reacting with S-adenosylmethionine and glutathione, and modifying the structure and activity of arsenite methyltransferase. This review is to summarize their toxicity mechanisms and the interaction between As and Se toxicity, and to provide suggestions for future investigations.

Downregulation of the Werner syndrome protein induces a metabolic shift that compromises redox homeostasis and limits proliferation of cancer cells.

The Werner syndrome protein (WRN) is a nuclear protein required for cell growth and proliferation. Loss-of-function mutations in the Werner syndrome gene are associated with the premature onset of age-related diseases. How loss of WRN limits cell proliferation and induces replicative senescence is poorly understood. Here, we show that WRN depletion leads to a striking metabolic shift that coordinately weakens the pathways that generate reducing equivalents for detoxification of reactive oxygen species and increases mitochondrial respiration. In cancer cells, this metabolic shift counteracts the Warburg effect, a defining characteristic of many malignant cells, resulting in altered redox balance and accumulation of oxidative DNA damage that inhibits cell proliferation and induces a senescence-like phenotype. Consistent with these findings, supplementation with antioxidant rescues at least in part cell proliferation and decreases senescence in WRN-knockdown cancer cells. These results demonstrate that WRN plays a critical role in cancer cell proliferation by contributing to the Warburg effect and preventing metabolic stress.

Characterization of the polyadenylation activity in a replicase complex from Bamboo mosaic virus-infected Nicotiana benthamiana plants.

Bamboo mosaic virus (BaMV) has a positive-sense single-stranded RNA genome with a 5' cap and a 3' poly(A) tail. To characterize polyadenylation activity in the BaMV replicase complex, we performed the in vitro polyadenylation with various BaMV templates. We conducted a polyadenylation activity assay for BaMV RNA by using a partially purified BaMV replicase complex. The results showed that approximately 200 adenylates at the 3' end of the RNA were generated on the endogenous RNA templates. Specific fractions derived from uninfected Nicotiana benthamiana plants enhanced the polyadenylation activity, implying that host factors are involved in polyadenylation. Furthermore, polyadenylation can be detected in newly synthesized plus-strand RNA in vitro when using the exogenous BaMV minus-strand minigenome. For polyadenylation on the exogenous plus-strand minigenome, the 3' end requires at least 4A to reach 22% polyadenylation activity. The results indicate that the BaMV replicase complex recognizes the 3' end of BaMV for polyadenylation.

p-Sulfonato-calix[n]arenes inhibit staphylococcal bicomponent leukotoxins by supramolecular interactions.

PVL (Panton-Valentine leukocidin) and other Staphylococcus aureus ß-stranded pore-forming toxins are important virulence factors involved in various pathologies that are often necrotizing. The present study characterized leukotoxin inhibition by selected SCns (p-sulfonato-calix[n]arenes): SC4, SC6 and SC8. These chemicals have no toxic effects on human erythrocytes or neutrophils, and some are able to inhibit both the activity of and the cell lysis by leukotoxins in a dose-dependent manner. Depending on the type of leukotoxins and SCns, flow cytometry revealed IC50 values of 6-22 µM for Ca2+ activation and of 2-50 µM for cell lysis. SCns were observed to affect membrane binding of class S proteins responsible for cell specificity. Electrospray MS and surface plasmon resonance established supramolecular interactions (1:1 stoichiometry) between SCns and class S proteins in solution, but not class F proteins. The membrane-binding affinity of S proteins was Kd=0.07-6.2 nM. The binding ability was completely abolished by SCns at different concentrations according to the number of benzenes (30-300 µM; SC8>SC6≫SC4). The inhibitory properties of SCns were also observed in vivo in a rabbit model of PVL-induced endophthalmitis. These calixarenes may represent new therapeutic avenues aimed at minimizing inflammatory reactions and necrosis due to certain virulence factors.

Screening of probiotic lactobacilli for inhibition of Shigella sonnei and the macromolecules involved in inhibition.

A total of 91 lactobacilli were screened for antimicrobial activity against Shigella sonnei. Agar-well assay showed that 16 lactobacilli displayed strong antibacterial activity against S. sonnei. The nature of these antimicrobial agents were investigated and shown to be dependent on their production of organic acids. Adhesion tests showed that 6 lactobacilli demonstrated good adherence to HT-29 cells, of these Lactobacillus johnsonii F0421 were selected for acid and bile salt tolerance properties. We further research on L. johnsonii F0421 inhibition of S. sonnei adhesion to HT-29 cells. The result showed that L. johnsonii F0421 exhibited significant inhibitory activity and excluded, competed and displaced adhered S. sonnei by 48%, 38% and 33%, respectively. In order to elucidate the inhibitory functions of macromolecules involved in L. johnsonii F0421, the cells were treated with 5 M LiCl, 0.05 M sodium metaperiodate and heating and assayed for inhibition activity. The results suggested a role of S-layer proteins on L. johnsonii F0421 cells in inhibition of the adhesion process, but carbohydrates do not seem to be involved. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant bands of approximately 40 kDa. In addition, 100 µg/well of S-layer proteins from L. johnsonii F0421 cells were effective in inhibiting adhesion of S. sonnei to HT-29 cells. These findings suggest that L. johnsonii F0421 possesses the capacity for inhibition of S. sonnei activity as well as probiotic properties, which could serve as a potential novel and effective probiotic strain for use in the food industry.

Macromolecular and small-molecule modulation of intracellular Aß42 aggregation and associated toxicity.

Aß (amyloid ß-peptide) has a central role in AD (Alzheimer's disease) where neuronal toxicity is linked to its extracellular and intracellular accumulation as oligomeric species. Searching for molecules that attenuate Aß aggregation could uncover novel therapies for AD, but most studies in mammalian cells have inferred aggregation indirectly by assessing levels of secreted Aß peptide. In the present study we establish a mammalian cell system for the direct visualization of Aß formation by expression of an Aß(42)-EGFP (enhanced green fluorescent protein) fusion protein in the human embryonic kidney cell line T-REx293, and use this to identify both macromolecules and small molecules that reduce aggregation and associated cell toxicity. Thus a molecular shield protein AavLEA1 [Aphelenchus avenae LEA (late embryogenesis abundant) protein 1], which limits aggregation of proteins with expanded poly(Q) repeats, is also effective against Aß(42)-EGFP when co-expressed in T-REx293 cells. A screen of polysaccharide and small organic molecules from medicinal plants and fungi reveals one candidate in each category, PS5 (polysaccharide 5) and ganoderic acid DM respectively, with activity against Aß. Both PS5 and ganoderic acid DM probably promote Aß aggregate clearance indirectly through the proteasome. The model is therefore of value to study the effects of intracellular Aß on cell physiology and to identify reagents that counteract those effects.

Synthesis, characterization, nucleic acid binding, and cytotoxic activity of a Ru(II) polypyridyl complex.

The binding properties of [Ru(bpy)(2)(H(2)IIP)](2+) (1) {bpy=2,2'-bipyridine, H(2)IIP=2-(indole-3-yl)-imidazolo[4,5-f][1,10]phenanthroline} with calf thymus DNA (CT-DNA) and yeast tRNA have been investigated comparatively by different spectroscopic and viscosity measurements. The results suggest that the affinity of complex 1 binding with yeast tRNA is stronger than that of complex 1 binding with CT-DNA, and complex 1 is a better enantioselective binder to yeast tRNA than to CT-DNA. The toxicity of complex 1 was concentration dependent, and HL-60 cells are more sensitive to complex 1 than Hep-G2 cells; complex 1 could induce Hep-G2 cell apoptosis.

Macromolecular organization of ATP synthase and complex I in whole mitochondria.

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F(1) heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.