Invariant Positions Research Articles

Comparison of physical and chemical vapor deposition for magnesium intercalation underneath epitaxial graphene

Intercalation of Mg into epitaxial graphene was investigated using chemical vapor deposition (CVD) and physical vapor deposition (PVD) for the formation of n-type doped graphene. Scanning electron microscopy and atomic force microscopy indicate PVD has significantly greater metal deposition and surface roughness compared to the CVD process. Findings from x-ray photoelectron spectroscopy infer the formation of Mg intercalated quasi-freestanding epitaxial graphene indicated by shifting of the SiC peak in C 1s spectra. This is substantiated by visual evidence from scanning tunneling electron microscopy of one or more layers of Mg residing underneath EG. Furthermore, an invariant 2D peak position in Raman spectra taken over a 7-day period in ambient conditions support arguments of the stability of electron doped Mg-intercalated epitaxial graphene. CVD offers a cleaner and smoother surface compared to PVD due to improved control over when the substrate is subjected to Mg exposure, but PVD allows for greater Mg concentrations per minute that may increase the amount of intercalated metal.

Structural basis for the unique molecular properties of broad-range phospholipase C from Listeria monocytogenes

Listeriosis is one of the most serious foodborne diseases caused by the intracellular bacterium Listeria monocytogenes. Its two major virulence factors, broad-range phospholipase C (LmPC-PLC) and the pore-forming toxin listeriolysin O (LLO), enable the bacterium to spread in the host by destroying cell membranes. Here, we determine the crystal structure of LmPC-PLC and complement it with the functional analysis of this enzyme. This reveals that LmPC-PLC has evolved several structural features to regulate its activity, including the invariant position of the N-terminal tryptophan (W1), the structurally plastic active site, Zn2+-dependent activity, and the tendency to form oligomers with impaired enzymatic activity. We demonstrate that the enzymatic activity of LmPC-PLC can be specifically inhibited by its propeptide added in trans. Furthermore, we show that the phospholipase activity of LmPC-PLC facilitates the pore-forming activity of LLO and affects the morphology of LLO oligomerization on lipid membranes, revealing the multifaceted synergy of the two virulence factors.

Automated identification and tracking of cells in Cytometry of Reaction Rate Constant (CRRC).

Cytometry of Reaction Rate Constant (CRRC) is a method for studying cell-population heterogeneity using time-lapse fluorescence microscopy, which allows one to follow reaction kinetics in individual cells. The current and only CRRC workflow utilizes a single fluorescence image to manually identify cell contours which are then used to determine fluorescence intensity of individual cells in the entire time-stack of images. This workflow is only reliable if cells maintain their positions during the time-lapse measurements. If the cells move, the original cell contours become unsuitable for evaluating intracellular fluorescence and the CRRC experiment will be inaccurate. The requirement of invariant cell positions during a prolonged imaging is impossible to satisfy for motile cells. Here we report a CRRC workflow developed to be applicable to motile cells. The new workflow combines fluorescence microscopy with transmitted-light microscopy and utilizes a new automated tool for cell identification and tracking. A transmitted-light image is taken right before every fluorescence image to determine cell contours, and cell contours are tracked through the time-stack of transmitted-light images to account for cell movement. Each unique contour is used to determine fluorescence intensity of cells in the associated fluorescence image. Next, time dependencies of the intracellular fluorescence intensities are used to determine each cell's rate constant and construct a kinetic histogram "number of cells vs rate constant." The new workflow's robustness to cell movement was confirmed experimentally by conducting a CRRC study of cross-membrane transport in motile cells. The new workflow makes CRRC applicable to a wide range of cell types and eliminates the influence of cell motility on the accuracy of results. Additionally, the workflow could potentially monitor kinetics of varying biological processes at the single-cell level for sizable cell populations. Although our workflow was designed ad hoc for CRRC, this cell-segmentation/cell-tracking strategy also represents an entry-level, user-friendly option for a variety of biological assays (i.e., migration, proliferation assays, etc.). Importantly, no prior knowledge of informatics (i.e., training a model for deep learning) is required.

Evolution of an extreme hemoglobin phenotype contributed to the sub-Arctic specialization of extinct Steller's sea cows.

The extinct Steller's sea cow (Hydrodamalis gigas; †1768) was a whale-sized marine mammal that manifested profound morphological specializations to exploit the harsh coastal climate of the North Pacific. Yet despite first-hand accounts of their biology, little is known regarding the physiological adjustments underlying their evolution to this environment. Here, the adult-expressed hemoglobin (Hb; a2β/δ2) of this sirenian is shown to harbor a fixed amino acid replacement at an otherwise invariant position (β/δ82Lys→Asn) that alters multiple aspects of Hb function. First, our functional characterization of recombinant sirenian Hb proteins demonstrate that the Hb-O2 affinity of this sub-Arctic species was less affected by temperature than those of living (sub)tropical sea cows. This phenotype presumably safeguarded O2 delivery to cool peripheral tissues and largely arises from a reduced intrinsic temperature sensitivity of the H. gigas protein. Additional experiments on H. gigas β/δ82Asn→Lys mutant Hb further reveal this exchange renders Steller's sea cow Hb unresponsive to the potent intraerythrocytic allosteric effector 2,3-diphosphoglycerate, a radical modification that is the first documented example of this phenotype among mammals. Notably, β/δ82Lys→Asn moreover underlies the secondary evolution of a reduced blood-O2 affinity phenotype that would have promoted heightened tissue and maternal/fetal O2 delivery. This conclusion is bolstered by analyses of two Steller's sea cow prenatal Hb proteins (Hb Gower I; z2e2 and HbF; a2g2) that suggest an exclusive embryonic stage expression pattern, and reveal uncommon replacements in H. gigas HbF (g38Thr→Ile and g101Glu→Asp) that increased Hb-O2 affinity relative to dugong HbF. Finally, the β/δ82Lys→Asn replacement of the adult/fetal protein is shown to increase protein solubility, which may have elevated red blood cell Hb content within both the adult and fetal circulations and contributed to meeting the elevated metabolic (thermoregulatory) requirements and fetal growth rates associated with this species cold adaptation.

Testing the gravitational redshift with an inner Solar System probe: The VERITAS case

The NASA Discovery-class mission VERITAS, selected in June 2021, will be launched toward Venus after 2027. In addition to the science instrumentation that will build global foundational geophysical datasets, VERITAS proposed to conduct a technology demonstration for the Deep Space Atomic Clock (DSAC-2). A first DSAC successfully operated in low-Earth orbit for more than two years, demonstrated the trapped ion atomic clock technology, and established a new level of performance for clocks in space. DSAC-2 would have further improvements in size, power, and performance. It would host a $1\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}13}$ grade USO to produce a frequency output with short-term stability of less than $2\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}13}/\sqrt{\ensuremath{\tau}}$ (where $\ensuremath{\tau}$ is the averaging time). However, due to funding shortfalls, DSAC-2, had to be canceled. The initially foreseen presence of an atomic clock on board the probe, however, raised the question whether this kind of instrumentation could be useful not only for navigation and time transfer but also for fundamental physics tests. In this work, we consider the DSAC-2 atomic clock and VERITAS mission as a specific example to measure possible discrepancies in the redshift predicted by general relativity by using an atomic clock onboard an interplanetary spacecraft. In particular we investigate the possibility of measuring possible violations of the local Lorentz invariance and local position invariance principles. We perform accurate simulations of the experiment during the VERITAS cruise phase. We consider different parametrizations of the possible violations of the general relativity, different operational conditions, and several different assumptions on the expected measurement performance. We show that DSAC-2 onboard VERITAS would provide new and improved constraints with respect to the current knowledge. Our analysis shows the scientific value of atomic clocks like DSAC-2 hosted onboard interplanetary spacecraft.

FEATURES OF THE FIELD WEAKENING MODE IN А SWITCHED RELUCTANCE MACHINE

The magnetic field weakening mode of electric machines is used in modern drives to extend the drive operational speed range when the power supply voltage is limited. In conventional electric drives, a real or fictitious flux control loop, established in a vector control system by using coordinate and phase transformations of corresponding variables, is used for attenuating the motor magnetic flux. Switched reluctance machines have certain design advantages over conventional electric machines; they feature high reliability and wide control possibilities. However, to realize these possibilities, appropriate control outputs should be generated, the choice of which depends on the operational speed range. Another peculiarity of these machines is that there is no separate magnetic flux stabilization or control circuit. The article considers the specific features pertinent to the switched reluctance machine operation in various speed ranges for adequately taking them into account in control algorithms. The studies were carried out using the electric drive simulation model in the MATLAB Simulink environment. It is shown that during the electric drive operation at its limit mechanical characteristic in the low-speed zone under the speed variation conditions, the phase flux linkage average value can be considered almost unchanged. However, control of the average torque carried out by changing the current limitation level is performed at the magnetic flux weakening background. It has been determined that during operation in a high-speed zone at invariable phase switching positions with the speed growing in a natural manner (i.e., without applying control actions), there is an excessive decrease in the flux linkage average value, which leads to a dramatic drop in the phase average torque. This drop can be compensated by progressively shifting the phase engagement position in the leading direction.

Technical note: Characteristics of the energy spread kernels of scattered radiation in an x-ray room.

To describe the scattered radiation spectra inside an x-ray room for different scatteringconditions. Monte Carlo simulations of an x-ray room using phantoms of different size, varying field sizes, and a range of mono-energetic beams were carried out. For each energy, the particle fluence spectrum of scattered photons was collected at different spherical zones to describe the radiation reaching the different boundaries of the x-ray room. The effect on the scattered spectrum of the room floor was alsoconsidered. The scattered spectra for mono-energetic primary beams at a given spherical zone give rise to oriented energy spread kernels (OESKs) that can be used to calculate the scattered spectrum for any poly-energetic beam at that zone. Despite the large differences, which can be seen in the OESKs when the scattering conditions vary, an important invariance is also observed: the position of the broad scatter peak for a given primary energy andzone. The result of breaking down the calculation of the scattered radiation spectrum into the different factors that influence it allows estimating the spectrum in a wide range of situations. The invariant position of the broad scatter peak can be used to estimate the highest energy of the scattered photons for a given primary energy and zone, which may determine radiation shieldingneeds.

Evaluation of HIV-1 integrase variability by combining computational and probabilistic approaches.

This study aimed at updating previous data on HIV-1 integrase variability, by using effective bioinformatics methods combining different statistical instruments from simple entropy and mutation rate to more specific approaches such as Hellinger distance. A total of 2133 HIV-1 integrase sequences were analyzed in: i) 1460 samples from drug-naïve [DN] individuals; ii) 386 samples from drug-experienced but INI-naïve [IN] individuals; iii) 287 samples from INI-experienced [IE] individuals. Within the three groups, 76 amino acid positions were highly conserved (≤0.2% variation, Hellinger distance: <0.25%), with 35 fully invariant positions; while, 80 positions were conserved (>0.2% to <1% variation, Hellinger distance: <1%). The H12-H16-C40-C43 and D64-D116-E152 motifs were all well conserved. Some residues were affected by dramatic changes in their mutation distributions, especially between DN and IE samples (Hellinger distance ≥1%). In particular, 15 positions (D6, S24, V31, S39, L74, A91, S119, T122, T124, T125, V126, K160, N222, S230, C280) showed a significant decrease of mutation rate in IN and/or IE samples compared to DN samples. Conversely, 8 positions showed significantly higher mutation rate in samples from treated individuals (IN and/or IE) compared to DN. Some of these positions, such as E92, T97, G140, Y143, Q148 and N155, were already known to be associated with resistance to integrase inhibitors; other positions including S24, M154, V165 and D270 are not yet documented to be associated with resistance. Our study confirms the high conservation of HIV-1 integrase and identified highly invariant positions using robust and innovative methods. The role of novel mutations located in the critical region of HIV-1 integrase deserves further investigation.

Graph Matching Based Image Registration for Multi-View Through-the-Wall Imaging Radar

Among various types of sensors, through-the-wall imaging radar (TWIR) is widely used in concealed targets detection and urban environment perception. Especially, multi-view TWIR has been received more and more attention in recent years for its ability to provide more accurate position estimation and reduce the blind areas. However, when exploiting the multi-view TWIR to detect targets in the complicated indoor environment with an unavailable global positioning system, the relative position of the radars is hard to be obtained and the acquired non-uniform images can not be aligned automatically. To deal with these problems, an image registration approach based on graph matching for multi-view TWIR is proposed in this paper. In this approach, we exploit an important feature of multi-view TWIR images: the invariant relative position of targets in different views. Based on this characteristic, the acquired images are modeled as complete graphs, and graph matching is employed to acquire the relative position parameters of the radars and register the images. Compared with the classic method, the proposed method can overcome the critical challenges of invalid local features as well as high-intensity ghosts, and achieve better performance for multi-view TWIR image registration. The effectiveness of the proposed method is evaluated via both simulations and real data tests.

Digital orbitals: connecting the tight-binding and plane-wave approaches through sampled-real-space models

Discrete approximations to continuous systems have proven valuable in many areas of physics and engineering. Discrete real-space calculations are often used in electronic structure calculations for molecules, and have been used in electronic structure calculations for crystals. Besides their computational utility, they can be used to connect the plane-wave and tight-binding methods of crystalline electronic structure calculations. A fully discrete-space approach results in exactly equivalent, finite, plane-wave and tight-binding bases for the same crystal because the two bases therefore span exactly the same space. Beyond connecting the two methods, the fully discrete approach addresses important issues in tight-binding. First, it results in a fully gauge invariant position operator capable of sub-cell spatial resolution. Second, it explains the observation in tight-binding that a small basis with long-range couplings and a large basis with short-range couplings can often reproduce technologically relevant bands with comparable accuracy.

Anatomical Comparison of Antennal Lobes in Two Sibling Ectropis Moths: Emphasis on the Macroglomerular Complex.

Ectropis obliqua and Ectropis grisescens are two sibling moth species of tea plantations in China. The male antennae of both species can detect shared and specific sex pheromone components. Thus, the primary olfactory center, i.e., the antennal lobe (AL), plays a vital role in distinguishing the sex pheromones. To provide evidence for the possible mechanism allowing this distinction, in this study, we compared the macroglomerular complex (MGC) of the AL between the males of the two species by immunostaining using presynaptic antibody and propidium iodide (PI) with antennal backfills, and confocal imaging and digital 3D-reconstruction. The results showed that MGC of both E. obliqua and E. grisescens contained five glomeruli at invariant positions between the species. However, the volumes of the anterior-lateral glomerulus (ALG) and posterior-ventral (PV) glomerulus differed between the species, possibly related to differences in sensing sex pheromone compounds and their ratios between E. obliqua and E. grisescens. Our results provide an important basis for the mechanism of mating isolation between these sibling moth species.

3D axial-attention for lung nodule classification

In recent years, Non-Local-based methods have been successfully applied to lung nodule classification. However, these methods offer 2D attention or limited 3D attention to low-resolution feature maps. Moreover, they still depend on a convenient local filter such as convolution as full 3D attention is expensive to compute and requires a big dataset, which might not be available. We propose to use 3D Axial-Attention, which requires a fraction of the computing power of a regular Non-Local network (i.e., self-attention). Unlike a regular Non-Local network, the 3D Axial-Attention network applies the attention operation to each axis separately. Additionally, we solve the invariant position problem of the Non-Local network by proposing to add 3D positional encoding to shared embeddings. We validated the proposed method on 442 benign nodules and 406 malignant nodules, extracted from the public LIDC-IDRI dataset by following a rigorous experimental setup using only nodules annotated by at least three radiologists. Our results show that the 3D Axial-Attention model achieves state-of-the-art performance on all evaluation metrics, including AUC and Accuracy. The proposed model provides full 3D attention, whereby every element (i.e., pixel) in the 3D volume space attends to every other element in the nodule effectively. Thus, the 3D Axial-Attention network can be used in all layers without the need for local filters. The experimental results show the importance of full 3D attention for classifying lung nodules.

Lewis Structures from Open Quantum Systems Natural Orbitals: Real Space Adaptive Natural Density Partitioning.

Building chemical models from state-of-the-art electronic structure calculations is not an easy task, since the high-dimensional information contained in the wave function needs to be compressed and read in terms of the accepted chemical language. We have already shown (Phys. Chem. Chem. Phys.2018, 20, 2136830095829) how to access Lewis structures from general wave functions in real space by reformulating the adaptive natural density partitioning (AdNDP) method proposed by Zubarev and Boldyrev (Phys. Chem. Chem. Phys.2008, 10, 520718728862). This provides intuitive Lewis descriptions from fully orbital invariant position space descriptors but depends on not immediately accessible higher order cumulant density matrices. By using an open quantum systems (OQS) perspective, we here show that the rigorously defined OQS fragment natural orbitals can be used to build a consistent real space adaptive natural density partitioning based only on spatial information and the system’s one-particle density matrix. We show that this rs-AdNDP approach is a cheap, efficient, and robust technique that immerses electron counting arguments fully in the real space realm.

An updated antennal lobe atlas for the yellow fever mosquito Aedes aegypti.

The yellow fever mosquito Aedes aegypti is a prolific vector of arboviral and filarial diseases that largely relies on its sense of smell to find humans. To facilitate in-depth analysis of the neural circuitry underlying Ae. aegypti olfactory-driven behaviors, we generated an updated in vitro atlas for the antennal lobe olfactory brain region of this disease vector using two independent neuronal staining methods. We performed morphological reconstructions with replicate fixed, dissected and stained brain samples from adult male and female Ae. aegypti of the LVPib12 genome reference strain and determined that the antennal lobe in both sexes is comprised of approximately 80 discrete glomeruli. Guided by landmark features in the antennal lobe, we found 63 of these glomeruli are stereotypically located in spatially invariant positions within these in vitro preparations. A posteriorly positioned, mediodorsal glomerulus denoted MD1 was identified as the largest spatially invariant glomerulus in the antennal lobe. Spatial organization of glomeruli in a recently field-derived strain of Ae. aegypti from Puerto Rico was conserved, despite differences in antennal lobe shape relative to the inbred LVPib12 strain. This model in vitro atlas will serve as a useful community resource to improve antennal lobe annotation and anatomically map projection patterns of neurons expressing target genes in this olfactory center. It will also facilitate the development of chemotopic maps of odor representation in the mosquito antennal lobe to decode the molecular and cellular basis of Ae. aegypti attraction to human scent and other chemosensory cues.

Topological atlas of the hypothalamus in adult rhesus monkey

The prosomeric model explains the embryological development of the central nervous system (CNS) shared by all vertebrates as a Bauplan. As a primary event, the early neural plate is patterned by intersecting longitudinal plates and transverse segments, forming a mosaic of progenitor units. The hypothalamus is specified by three prosomeres (hp1, hp2, and the acroterminal domain) of the secondary prosencephalon with corresponding alar and basal plate parts, which develop apart from the diencephalon. Mounting evidence suggests that progenitor units within alar and basal plate parts of hp1 and hp2 give rise to distinct hypothalamic nuclei, which preserve their relative invariant positioning (topology) in the adult brain. Nonetheless, the principles of the prosomeric model have not been applied so far to the hypothalamus of adult primates. We parcellated hypothalamic nuclei in adult rhesus monkeys (Macaca mulatta) using various stains to view architectonic boundaries. We then analyzed the topological relations of hypothalamic nuclei and adjacent hypothalamic landmarks with homology across rodent and primate species to trace the origin of adult hypothalamic nuclei to the alar or basal plate components of hp1 and hp2. We generated a novel atlas of the hypothalamus of the adult rhesus monkey with developmental ontologies for each hypothalamic nucleus. The result is a systematic reinterpretation of the adult hypothalamus whose prosomeric ontology can be used to study relationships between the hypothalamus and other regions of the CNS. Further, our atlas may serve as a tool to predict causal patterns in physiological and pathological pathways involving the hypothalamus.

The perceived level of physical condition in young people aged 20-22 years

In the current context, the specific activities of youth in technical university education are focused on professional training, which consists in learning all particularities arising from the specific occupational field. The students’ working regime is dominated by sedentary lifestyles, reduced mobility and invariable positions for 8 to 10 hours per day. This issue is also highlighted by the fact that there has been a decrease in the number of physical education and sport classes in technical university education. Concerned to understand the social importance of the sporting phenomenon that is directly influenced by the physical education process, we wanted to know the level of the most studied factor, namely physical condition. In this regard, we started a study by which we investigated the level of physical condition on a sample of 70 students aged 20-22 from the Politehnica University of Bucharest. The research involved conducting a questionnaire survey, which provided information on the health status of students, their preferences regarding the form of organization of the physical education activity in technical higher education, as well as the frequency of their participation in these activities or in leisure activities. The results of the research gave us argumentative answers to the interrogative assertions that preceded the study, highlighting on the one hand the worrying level of students’ physical condition/fitness and on the other hand that they were much more aware of the importance of movement in their lives.

MLH1 intronic variants mapping to + 5 position of splice donor sites lead to deleterious effects on RNA splicing.

Germline pathogenic variants in the DNA mismatch repair genes (MMR): MLH1, MSH2, MSH6, and PMS2, are causative of Lynch syndrome (LS). However, many of the variants mapping outside the invariant splice site positions (IVS ± 1, IVS ± 2) are classified as variants of unknown significance (VUS). Three such variants (MLH1 c.588+5G>C, c.588+5G>T and c.677+5G>A) were identified in 8 unrelated LS families from Argentina, Brazil and Chile. Herein, we collected clinical information on these families and performed segregation analysis and RNA splicing studies to assess the implication of these VUS in LS etiology. Pedigrees showed a clear pattern of variant co-segregation with colorectal cancer and/or other LS-associated malignancies. Tumors presented deficient expression of MLH1-PMS2 proteins in 7/7 of the LS families, and MSI-high status in 3/3 cases. Moreover, RNA analyses revealed that c.588+5G>C and c.588+5G>T induce skipping of exon 7 whereas c.677+5G>A causes skipping of exon 8. In sum, we report that the combined clinical findings in the families and the molecular studies provided the evidences needed to demonstrate that the three MLH1 variants are causative of LS and to classify c.588+5G>C and c.677+5G>A as class 5 (pathogenic), and c.588+5G>T as class 4 (likely-pathogenic). Our findings underline the importance of performing clinical and family analyses, as well as RNA splicing assays in order to determine the clinical significance of intronic variants, and contribute to the genetic counseling and clinical management of patients and their relatives.

Distribution and development of the external sense organ pattern on the appendages of postembryonic and adult stages of the spider Parasteatoda tepidariorum

Spiders are equipped with a large number of innervated cuticular specializations, which respond to various sensory stimuli. The physiological function of mechanosensory organs has been analysed in great detail in some model spider species (e.g. Cupiennius salei); however, much less is known about the distribution and function of chemosensory organs. Furthermore, our knowledge on how the sense organ pattern develops on the spider appendages is limited. Here we analyse the development of the pattern and distribution of six different external mechano- and chemosensory organs in all postembryonic stages and in adult male and female spiders of the species Parasteatoda tepidariorum. We show that except for small mechanosensory setae, external sense organs appear in fixed positions on the pedipalps and first walking legs, arranged in longitudinal rows along the proximal-distal axis or in invariable positions relative to morphological landmarks (joints, distal tarsal tip). A comparison to other Entelegynae spiders shows that these features are conserved. We hope that this study lays the foundation for future molecular analysis to address the question how this conserved pattern is generated.

Exon size and sequence conservation improves identification of splice-altering nucleotides.

Pre-mRNA splicing is regulated through multiple trans-acting splicing factors. These regulators interact with the pre-mRNA at intronic and exonic positions. Given that most exons are protein coding, the evolution of exons must be modulated by a combination of selective coding and splicing pressures. It has previously been demonstrated that selective splicing pressures are more easily deconvoluted when phylogenetic comparisons are made for exons of identical size, suggesting that exon size–filtered sequence alignments may improve identification of nucleotides evolved to mediate efficient exon ligation. To test this hypothesis, an exon size database was created, filtering 76 vertebrate sequence alignments based on exon size conservation. In addition to other genomic parameters, such as splice-site strength, gene position, or flanking intron length, this database permits the identification of exons that are size- and/or sequence-conserved. Highly size-conserved exons are always sequence-conserved. However, sequence conservation does not necessitate exon size conservation. Our analysis identified evolutionarily young exons and demonstrated that length conservation is a strong predictor of alternative splicing. A published data set of approximately 5000 exonic SNPs associated with disease was analyzed to test the hypothesis that exon size–filtered sequence comparisons increase detection of splice-altering nucleotides. Improved splice predictions could be achieved when mutations occur at the third codon position, especially when a mutation decreases exon inclusion efficiency. The results demonstrate that coding pressures dominate nucleotide composition at invariable codon positions and that exon size–filtered sequence alignments permit identification of splice-altering nucleotides at wobble positions.

Instability in a coiled-coil signaling helix is conserved for signal transduction in soluble guanylyl cyclase.

How nitric oxide (NO) activates its primary receptor, α1/β1 soluble guanylyl cyclase (sGC or GC-1), remains unknown. Likewise, how stimulatory compounds enhance sGC activity is poorly understood, hampering development of new treatments for cardiovascular disease. NO binding to ferrous heme near the N-terminus in sGC activates cyclase activity near the C-terminus, yielding cGMP production and physiological response. CO binding can also stimulate sGC, but only weakly in the absence of stimulatory small-molecule compounds, which together lead to full activation. How ligand binding enhances catalysis, however, has yet to be discovered. Here, using a truncated version of sGC from Manduca sexta, we demonstrate that the central coiled-coil domain, the most highly conserved region of the ~150,000 Da protein, not only provides stability to the heterodimer but is also conformationally active in signal transduction. Sequence conservation in the coiled coil includes the expected heptad-repeating pattern for coiled-coil motifs, but also invariant positions that disfavor coiled-coil stability. Full-length coiled coil dampens CO affinity for heme, while shortening of the coiled coil leads to enhanced CO binding. Introducing double mutation αE447L/βE377L, predicted to replace two destabilizing glutamates with leucines, lowers CO binding affinity while increasing overall protein stability. Likewise, introduction of a disulfide bond into the coiled coil results in reduced CO affinity. Taken together, we demonstrate that the heme domain is greatly influenced by coiled-coil conformation, suggesting communication between heme and catalytic domains is through the coiled coil. Highly conserved structural imperfections in the coiled coil provide needed flexibility for signal transduction.