Unusual Architecture Research Articles

Nature in the city

On May 32, 1773 the Duke from my favourite film “That Very Munchhausen” said the significant words: “It is amazing… It is amazing how our people harmonize with nature!” A bit later almost his fellow-countryman Friedensreich Hundertwasser said that happiness is merging with nature. It is possible to live happily without money. It is impossible to do it without nature. Merging with nature was crucial in the life of this wonderful man, and even in his death. We have almost learnt to live without money. But we do not want to live without nature, and we cannot. We asked ourselves a psychoanalytical question: “Would you like to talk about it?” and then answered like honest patients: “Yes, we would”. In the context of the crisis the time is ripe for reflection, which is better to do alone with nature. This is how we approached the subject of the latest festival “Zodchestvo of Eastern Siberia” (pp. 13-46, 72-81). The cross-cutting theme concerning the city for people is continued in this issue. It should not be thought that architects create only buildings. Architects’ materials are neither stone with its modern substitutes nor even the ivy covering construction failures. The latest Pritsker Prize laureate Alejandro Aravena says that the most important thing in urbanism is public spaces. Changeable, diverse, filled with sense and visual events, they constitute a special nature of the city. How to bring together the elements of this ‘second nature’ with living biocenoses of the primary nature? Can we always differ one from the other? By the 355th anniversary of Irkutsk, its leading architects had worked out more than twenty different designs of squares (pp. 92-112) by the mayor’s order. Besides the future squares and pocket parks of Irkutsk, several green spaces of Moscow, Tokyo, London, Paris and German towns are presented in the issue, as well as competitive designs of the New York Aquarium and the Horse Park in Korea. The object of the issue is a wonderful museum on the Kulikovo Field designed by Sergey Gnedovsky. The unusual architecture of the new museum makes it akin to the earth’s tectonic fault caused by an intolerable tension in expectation of the battle (pp. 113-123). Oleg Yavein and Ilya Lezhava bring us back to the classic of the 20th century Mies van der Rohe and his unique dialogue with nature (pp. 162-175). Relationship between architecture and nature is a rich and limitless theme, like both of these substances. That is why the new Project Baikal issue contains, as usual, more questions than answers. Such is the nature of our journal.

Mechanistic signs of double-barreled structure in a fluoride ion channel.

The Fluc family of F(-) ion channels protects prokaryotes and lower eukaryotes from the toxicity of environmental F(-). In bacteria, these channels are built as dual-topology dimers whereby the two subunits assemble in antiparallel transmembrane orientation. Recent crystal structures suggested that Fluc channels contain two separate ion-conduction pathways, each with two F(-) binding sites, but no functional correlates of this unusual architecture have been reported. Experiments here fill this gap by examining the consequences of mutating two conserved F(-)-coordinating phenylalanine residues. Substitution of each phenylalanine specifically extinguishes its associated F(-) binding site in crystal structures and concomitantly inhibits F(-) permeation. Functional analysis of concatemeric channels, which permit mutagenic manipulation of individual pores, show that each pore can be separately inactivated without blocking F(-) conduction through its symmetry-related twin. The results strongly support dual-pathway architecture of Fluc channels.

A quintuple star system containing two eclipsing binaries

We present a quintuple star system that contains two eclipsing binaries. The unusual architecture includes two stellar images separated by 11" on the sky: EPIC 212651213 and EPIC 212651234. The more easterly image (212651213) actually hosts both eclipsing binaries which are resolved within that image at 0.09", while the westerly image (212651234) appears to be single in adaptive optics (AO), speckle imaging, and radial velocity (RV) studies. The 'A' binary is circular with a 5.1-day period, while the 'B' binary is eccentric with a 13.1-day period. The gamma velocities of the A and B binaries are different by ~10 km/s. That, coupled with their resolved projected separation of 0.09", indicates that the orbital period and separation of the 'C' binary (consisting of A orbiting B) are ~65 years and ~25 AU, respectively, under the simplifying assumption of a circular orbit. Motion within the C orbit should be discernible via future RV, AO, and speckle imaging studies within a couple of years. The C system (i.e., 212651213) has a radial velocity and proper motion that differ from that of 212651234 by only ~1.4 km/s and ~3 mas/yr. This set of similar space velocities in 3 dimensions strongly implies that these two objects are also physically bound, making this at least a quintuple star system.

Deletion of the N- or C-Terminal Helix of Apolipophorin III To Create a Four-Helix Bundle Protein.

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein found in insects and plays an important function in lipid transport. The protein has an unusual five-helix bundle architecture, deviating from the common four-helix bundle motif. To understand the role of the additional helix in apoLp-III, the N-terminal or C-terminal helix was deleted to create a putative four-helix bundle protein. While the protein lacking helix-1 could be expressed in bacteria albeit at reduced yields, apoLp-III lacking helix-5 could not be produced. Mutational analysis by truncating helix-5 showed that a minimum segment of approximately one-third of the C-terminal helix is required for protein expression. The variant lacking helix-5 was produced by inserting a methionine residue between helix-4 and -5; subsequent cyanogenbromide cleavage generated the four-helix variant. Both N- and C-terminal helix deletion variants displayed significantly reduced helical content, protein stability, and tertiary structure. Despite the significantly altered structure, the variants were still fully functional. The rate of dimyristoylphosphatidylcholine vesicle solubilization was enhanced 4-5-fold compared to the wild-type protein, and the deletion variants were effective in binding to lipolyzed low density lipoprotein thereby preventing lipoprotein aggregation. These results show that the additional helix of apoLp-III is not essential for lipid binding but is required for proper folding to keep the protein into a stable conformation.

Leader Peptide-Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease-Targeted Libraries.

Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases.

Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies.

A subset of bovine antibodies have an exceptionally long third heavy-chain complementarity determining region (CDR H3) that is highly variable in sequence and includes multiple cysteines. These long CDR H3s (up to 69 residues) fold into a long stalk atop which sits a knob domain that is located far from the antibody surface. Three new bovine Fab crystal structures have been determined to decipher the conserved and variable features of ultralong CDR H3s that lead to diversity in antigen recognition. Despite high sequence variability, the stalks adopt a conserved β-ribbon structure, while the knob regions share a conserved β-sheet that serves as a scaffold for two connecting loops of variable length and conformation, as well as one conserved disulfide. Variation in patterns and connectivity of the remaining disulfides contribute to the knob structural diversity. The unusual architecture of these ultralong bovine CDR H3s for generating diversity is unique in adaptive immune systems.

Leader Peptide‐Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease‐Targeted Libraries

AbstractMicroviridins are a family of ribosomally synthesized and post‐translationally modified peptides with a highly unusual architecture featuring non‐canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP‐grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT‐type N‐acetyltransferase. The method facilitates the efficient in vitro one‐pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo‐enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure–activity relationships for respective target proteases.

Hiding in plain sight: Koshicola spirodelophila gen. et sp. nov. (Chaetopeltidales, Chlorophyceae), a novel green alga associated with the aquatic angiosperm Spirodela polyrhiza.

Discovery and morphological characterization of a novel epiphytic aquatic green alga increases our understanding of Chaetopeltidales, a poorly known order in Chlorophyceae. Chloroplast genomic data from this taxon reveals an unusual architecture previously unknown in green algae. Using light and electron microscopy, we characterized the morphology and ultrastructure of a novel taxon of green algae. Bayesian phylogenetic analyses of nuclear and plastid genes were used to test the hypothesized membership of this taxon in order Chaetopeltidales. With next-generation sequence data, we assembled the plastid genome of this novel taxon and compared its gene content and architecture to that of related species to further investigate plastid genome traits. The morphology and ultrastructure of this alga are consistent with placement in Chaetopeltidales (Chlorophyceae), but a distinct trait combination supports recognition of this alga as a new genus and species-Koshicola spirodelophila gen. et sp. nov. Its placement in the phylogeny as a descendant of a deep division in the Chaetopeltidales is supported by analysis of molecular data sets. The chloroplast genome is among the largest reported in green algae and the genes are distributed on three large (rather than a single) chromosome, in contrast to other studied green algae. The discovery of Koshicola spirodelophila gen. et sp. nov. highlights the importance of investigating even commonplace habitats to explore new microalgal diversity. This work expands our understanding of the morphological and chloroplast genomic features of green algae, and in particular those of the poorly studied Chaetopeltidales.

Chimeric Sex-Determining Chromosomal Regions and Dysregulation of Cell-Type Identity in a Sterile Zygosaccharomyces Allodiploid Yeast.

Allodiploidization is a fundamental yet evolutionarily poorly characterized event, which impacts genome evolution and heredity, controlling organismal development and polyploid cell-types. In this study, we investigated the sex determination system in the allodiploid and sterile ATCC 42981 yeast, a member of the Zygosaccharomyces rouxii species complex, and used it to study how a chimeric mating-type gene repertoire contributes to hybrid reproductive isolation. We found that ATCC 42981 has 7 MAT-like (MTL) loci, 3 of which encode α-idiomorph and 4 encode a-idiomorph. Two phylogenetically divergent MAT expression loci were identified on different chromosomes, accounting for a hybrid a/α genotype. Furthermore, extra a-idimorph-encoding loci (termed MTLa copies 1 to 3) were recognized, which shared the same MATa1 ORFs but diverged for MATa2 genes. Each MAT expression locus was linked to a HML silent cassette, while the corresponding HMR loci were located on another chromosome. Two putative parental sex chromosome pairs contributed to this unusual genomic architecture: one came from an as-yet-undescribed taxon, which has the NCYC 3042 strain as a unique representative, while the other did not match any MAT-HML and HMR organizations previously described in Z. rouxii species. This chimeric rearrangement produces two copies of the HO gene, which encode for putatively functional endonucleases essential for mating-type switching. Although both a and α coding sequences, which are required to obtain a functional cell-type a1-α2 regulator, were present in the allodiploid ATCC 42981 genome, the transcriptional circuit, which regulates entry into meiosis in response to meiosis-inducing salt stress, appeared to be turned off. Furthermore, haploid and α-specific genes, such as MATα1 and HO, were observed to be actively transcribed and up-regulated under hypersaline stress. Overall, these evidences demonstrate that ATCC 42981 is unable to repress haploid α-specific genes and to activate meiosis in response to stress. We argue that sequence divergence within the chimeric a1-α2 heterodimer could be involved in the generation of negative epistasis, contributing to the allodiploid sterility and the dysregulation of cell identity.

The high mobility group protein HMO1 functions as a linker histone in yeast.

BackgroundEukaryotic chromatin consists of nucleosome core particles connected by linker DNA of variable length. Histone H1 associates with the linker DNA to stabilize the higher-order chromatin structure and to modulate the ability of regulatory factors to access their nucleosomal targets. In Saccharomyces cerevisiae, the protein with greatest sequence similarity to H1 is Hho1p. However, during vegetative growth, hho1∆ cells do not show any discernible cell growth defects or the changes in bulk chromatin structure that are characteristic of chromatin from multicellular eukaryotes in which H1 is depleted. In contrast, the yeast high mobility group (HMGB) protein HMO1 has been reported to compact chromatin, as evidenced by increased nuclease sensitivity in hmo1∆ cells. HMO1 has an unusual domain architecture compared to vertebrate HMGB proteins in that the HMG domains are followed by a lysine-rich extension instead of an acidic domain. We address here the hypothesis that HMO1 serves the role of H1 in terms of chromatin compaction and that this function requires the lysine-rich extension.ResultsWe show here that HMO1 fulfills this function of a linker histone. For histone H1, chromatin compaction requires its basic C-terminal domain, and we find that the same pertains to HMO1, as deletion of its C-terminal lysine-rich extension renders chromatin nuclease sensitive. On rDNA, deletion of both HMO1 and Hho1p is required for significantly increased nuclease sensitivity. Expression of human histone H1 completely reverses the nuclease sensitivity characteristic of chromatin isolated from hmo1∆ cells. While chromatin remodeling events associated with repair of DNA double-strand breaks occur faster in the more dynamic chromatin environment created by the hmo1 deletion, expression of human histone H1 results in chromatin remodeling and double-strand break repair similar to that observed in wild-type cells.ConclusionOur data suggest that S. cerevisiae HMO1 protects linker DNA from nuclease digestion, a property also characteristic of mammalian linker histone H1. Notably, association with HMO1 creates a less dynamic chromatin environment that depends on its lysine-rich domain. That HMO1 has linker histone function has implications for investigations of chromatin structure and function as well as for evolution of proteins with roles in chromatin compaction.

Elucidating the pH Dependent Mechanism of OmpG Gating

Protein pores are steadily emerging as valuable stochastic sensors for single- molecule detection. Over the decade, developments in stochastic sensing have been based on single channel recording in planar lipid bilayer. Historically, nanopores with rigid structures have been frequently used for sensing molecules and biological species. However, we have chosen a nanopore with a flexible structure for our protein nanopore: monomeric β-barrel porin outer membrane protein G (OmpG). This monomeric porin is composed of 14- s- strands connected by seven flexible extracellular loops. Wild-type OmpG spontaneously gates during an applied potential as revealed by planar bilayer studies. Strong evidence suggests that the dynamic movement of OmpG's loop 6 is responsible for the gating mechanism of the porin. The unusual flexible architecture of this porin not only successfully binds protein targets, but also provides unique detection. Our engineered OmpG sensor is sensitive enough to provide, for the first time, the ability to resolve homologous protein analytes.The ability of our biosensor to discriminate between homologous proteins reveals the important need in better understanding the underlying mechanism of OmpG gating. This understanding will allow us to further engineer OmpG and tailor it for sensing an array of protein analytes. In addition, this knowledge will aid in a deeper understanding of the biology of this membrane protein itself. In this work, we highlight the pH-dependent gating mechanism of OmpG by investigating repulsive forces of OmpG's extracellular loops.

Extensive Mitochondrial mRNA Editing and Unusual Mitochondrial Genome Organization in Calcaronean Sponges

One of the unusual features of DNA-containing organelles in general and mitochondria in particular is the frequent occurrence of RNA editing [1]. The term "RNA editing" refers to a variety of mechanistically unrelated biochemical processes that alter RNA sequence during or after transcription [2]. The editing can be insertional, deletional, or substitutional and has been found in all major types of RNAs [3, 4]. Although mitochondrial mRNA editing is widespread in some eukaryotic lineages [5-7], it is rare in animals, with reported cases limited both in their scope and in phylogenetic distribution [8-11] (see also [12]). While analyzing genomic data from calcaronean sponges Sycon ciliatum and Leucosolenia complicata, we were perplexed by the lack of recognizable mitochondrial coding sequences. Comparison of genomic and transcriptomic data from these species revealed the presence of mitochondrial cryptogenes whose transcripts undergo extensive editing. This editing consisted of single or double uridylate (U) insertionsin pre-existing short poly(U) tracts. Subsequent analysis revealed the presence of similar editing inSycon coactum and the loss of editing in Petrobiona massiliana, a hypercalcified calcaronean sponge. In addition, mitochondrial genomes of at least some calcaronean sponges were found to have a highly unusual architecture, with nearly all genes located on individual and likely linear chromosomes. Phylogenetic analysis of mitochondrial coding sequences revealed accelerated rates of sequence evolution in this group. The latter observation presents a challenge for the mutational-hazard hypothesis [13], which posits that mRNA editing should not occur in lineages with an elevated mutation rate.

DNA recognition for virus assembly through multiple sequence-independent interactions with a helix-turn-helix motif.

The helix-turn-helix (HTH) motif features frequently in protein DNA-binding assemblies. Viral pac site-targeting small terminase proteins possess an unusual architecture in which the HTH motifs are displayed in a ring, distinct from the classical HTH dimer. Here we investigate how such a circular array of HTH motifs enables specific recognition of the viral genome for initiation of DNA packaging during virus assembly. We found, by surface plasmon resonance and analytical ultracentrifugation, that individual HTH motifs of the Bacillus phage SF6 small terminase bind the packaging regions of SF6 and related SPP1 genome weakly, with little local sequence specificity. Nuclear magnetic resonance chemical shift perturbation studies with an arbitrary single-site substrate suggest that the HTH motif contacts DNA similarly to how certain HTH proteins contact DNA non-specifically. Our observations support a model where specificity is generated through conformational selection of an intrinsically bent DNA segment by a ring of HTHs which bind weakly but cooperatively. Such a system would enable viral gene regulation and control of the viral life cycle, with a minimal genome, conferring a major evolutionary advantage for SPP1-like viruses.

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.

The Cystine Knot Is Responsible for the Exceptional Stability of the Insecticidal Spider Toxin ω-Hexatoxin-Hv1a.

The inhibitor cystine knot (ICK) is an unusual three-disulfide architecture in which one of the disulfide bonds bisects a loop formed by the two other disulfide bridges and the intervening sections of the protein backbone. Peptides containing an ICK motif are frequently considered to have high levels of thermal, chemical and enzymatic stability due to cross-bracing provided by the disulfide bonds. Experimental studies supporting this contention are rare, in particular for spider-venom toxins, which represent the largest diversity of ICK peptides. We used ω-hexatoxin-Hv1a (Hv1a), an insecticidal toxin from the deadly Australian funnel-web spider, as a model system to examine the contribution of the cystine knot to the stability of ICK peptides. We show that Hv1a is highly stable when subjected to temperatures up to 75 °C, pH values as low as 1, and various organic solvents. Moreover, Hv1a was highly resistant to digestion by proteinase K and when incubated in insect hemolymph and human plasma. We demonstrate that the ICK motif is essential for the remarkable stability of Hv1a, with the peptide’s stability being dramatically reduced when the disulfide bonds are eliminated. Thus, this study demonstrates that the ICK motif significantly enhances the chemical and thermal stability of spider-venom peptides and provides them with a high level of protease resistance. This study also provides guidance to the conditions under which Hv1a could be stored and deployed as a bioinsecticide.

DOPPLER MONITORING OF THE WASP-47 MULTIPLANET SYSTEM

We present precise Doppler observations of WASP-47, a transiting planetary system featuring a hot Jupiter with both inner and outer planetary companions. This system has an unusual architecture and also provides a rare opportunity to measure planet masses in two different ways: the Doppler method, and the analysis of transit-timing variations (TTV). Based on the new Doppler data, obtained with the Planet Finder Spectrograph on the Magellan/Clay 6.5m telescope, the mass of the hot Jupiter is $370 \pm 29~M_{\oplus}$. This is consistent with the previous Doppler determination as well as the TTV determination. For the inner planet WASP-47e, the Doppler data lead to a mass of $12.2\pm 3.7~ M_{\oplus}$, in agreement with the TTV-based upper limit of $<$22~$M_{\oplus}$ ($95\%$ confidence). For the outer planet WASP-47d, the Doppler mass constraint of $10.4\pm 8.4~M_{\oplus}$ is consistent with the TTV-based measurement of $15.2^{+6.7}_{-7.6}~ M_{\oplus}$.

Modular Design of the Selectivity Filter Pore Loop in a Novel Family of Prokaryotic 'Inward Rectifier' (NirBac) channels.

Potassium channels exhibit a modular design with distinct structural and functional domains; in particular, a highly conserved pore-loop sequence that determines their ionic selectivity. We now report the functional characterisation of a novel group of functionally non-selective members of the prokaryotic ‘inward rectifier’ subfamily of K+ channels. These channels share all the key structural domains of eukaryotic and prokaryotic Kir/KirBac channels, but instead possess unique pore-loop selectivity filter sequences unrelated to any other known ionic selectivity filter. The strikingly unusual architecture of these ‘NirBac’ channels defines a new family of functionally non-selective ion channels, and also provides important insights into the modular design of ion channels, as well as the evolution of ionic selectivity within this superfamily of tetrameric cation channels.

Biofilm architecture in a novel pressurized biofilm reactor

A novel pure-oxygen pressurized biofilm reactor was operated at different organic loading, mechanical shear and hydrodynamic conditions to understand the relationships between biofilm architecture and its operation. The ultimate goal was to improve the performance of the biofilm reactor. The biofilm was labeled with seven stains and observed with confocal laser scanning microscopy. Unusual biofilm architecture of a ribbon embedded between two surfaces with very few points of attachment was observed. As organic loading increased, the biofilm morphology changed from a moderately rough layer into a locally smoother biomass with significant bulging protuberances, although the chemical oxygen demand (COD) removal efficiency remained unchanged at about 75%. At higher organic loadings, biofilms contained a larger fraction of active cells distributed uniformly within a proteinaceous matrix with decreasing polysaccharide content. Higher hydrodynamic shear in combination with high organic loading resulted in the collapse of biofilm structure and a substantial decrease in reactor performance (a COD removal of 16%). Moreover, the important role of proteins for the spatial distribution of active cells was demonstrated quantitatively.

Two-sided block of a dual-topology F− channel

The Fluc family is a set of small membrane proteins forming F(-)-specific electrodiffusive ion channels that rescue microorganisms from F(-) toxicity during exposure to weakly acidic environments. The functional channel is built as a dual-topology homodimer with twofold symmetry parallel to the membrane plane. Fluc channels are blocked by nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries. The unusual symmetrical antiparallel dimeric architecture of Flucs demands that the two chemically equivalent monobody-binding epitopes reside on opposite ends of the channel, a double-sided blocking situation that has never before presented itself in ion channel biophysics. However, it is not known if both sites can be simultaneously occupied, and if so, whether monobodies bind independently or cooperatively to their transmembrane epitopes. Here, we use direct monobody-binding assays and single-channel recordings of a Fluc channel homolog to reveal a novel trimolecular blocking behavior that reveals a doubly occupied blocked state. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity between the two blocking sites.

Weird Ion…Weird Ion Channels Mediating Resistance to Fluoride Toxicity

F‐ specific ion channels of the newly discovered “Fluc” family act as F‐ exporters protect many microorganisms from the toxic effects of this environmentally pervasive halide. The channel is constructed as an four‐TM helix dual‐topology homodimer, in which the two subunits adopt antiparallel orientation within the membrane. We previously used fibronectin domain‐based “monobodies” selected by phage display methods to establish this unusual membrane architecture, showing that these small proteins block single Fluc channels from either side of the membrane. We now ask whether the two blocking sites on opposite sides of the membrane can both be occupied simultaneously by monobodies. Our analysis examines the dependence of single‐channel block‐times as a function of monobody concentration varied symmetrically on the two sides of the channel. If both sides can be blocked symmetrically, a linear blocking scheme should be followed:&#x0D;Open ‐‐‐‐ Block(1) ‐‐‐‐Block(2),Where both blocked states are nonconducting. This scheme makes quantitative predictions for the increase in block‐times with monobody concentration that provide information about any cooperativity, negative or positive, that may operate between the two sites.