Search

Background: In all organisms, the ribosome performs the unique and essential function of translating mRNA into protein. Since deletion of any ribosomal protein (RP) is embryonic lethal in complex eukaryotes, the ribosome is believed to be structurally uniform throughout the organism, with all RPs essential for cell viability. Results: We tested the structural homogeneity of RP use in ribosomes for normal and cancer tissues and cell lines using RP mRNA, ribosome profiling, and protein data. Analysis of RP mRNA data from 11,688 normal samples for 53 human tissues from 714 subjects and 10,363 tumor samples for 33 human cancers, normalized by total RP mRNA level per sample, showed that both normal (non-diseased) and tumor samples cluster by tissue type in humans. Matrix factorization showed that at least 3 RP mRNA signatures are necessary to describe normal blood and brain tissues, and a minimum of 16 RP mRNA signatures are necessary to describe data from 53 different normal tissues. A pan-cancer analysis of copy number variation (CNV) in 10,845 tumor samples for 33 human cancers showed that loss of one or both copies of RP genes was prevalent in every cancer type. Furthermore, there was no association between the number of double-deleted RP genes in tumors and patient survival, showing that RP loss does not reduce tumor fitness. CRISPR-Cas9 deletion of RP genes shows that many RPs are not essential in many cell lines. In several cancers, multiple RP-subtypes exist, with significant survival and genomic differences among them. These RP-subtypes often map to known molecular subtypes, and various RP genes are often deleted in one or both subtypes. This suggests that when genomic landscapes are modified in tumors, genes coding RPs are often lost, but these losses do not affect tumor viability. Analysis of mRNA data and ribosome profiling data of RP genes for cells and tissues from human, mouse and rat showed that these are highly correlated, showing that transcripts encoding ribosomal proteins are being translated into ribosomal proteins at rates proportional to their mRNA levels. Consistently, both mRNA data and ribosome profiling data of RP genes, normalized by total level per sample, showed tissue specific and development-stage specific clusters. Finally, analysis of RP protein levels in human adult and fetal tissues, standardized per sample, showed both development-stage and tissue specificity, showing that there is both tissue and development-stage specific heterogeneity of RP protein usage. Conclusions: These results suggest that there are multiple ribosome types in complex eukaryotes, with different RP composition which are regulated in a tissue and development-stage specific manner by some novel, yet unknown mechanism. Citation Format: Anshuman Panda, Anupama Yadav, Huwate Yeerna, Amartya Singh, Michael Biehl, Markus Lux, Alexander Schulz, Tyler Klecha, Sebastian Doniach, Hossein Khiabanian, Shridar Ganesan, Pablo Tamayo, Gyan Bhanot. The composition of the human ribosome varies significantly in different normal and malignant tissues [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5865.

We give results from a detailed analysis of human Ribosomal Protein (RP) levels in normal and cancer samples and cell lines from large mRNA, copy number variation and ribosome profiling datasets. After normalizing total RP mRNA levels per sample, we find highly consistent tissue specific RP mRNA signatures in normal and tumor samples. Multiple RP mRNA-subtypes exist in several cancers, with significant survival and genomic differences. Some RP mRNA variations among subtypes correlate with copy number loss of RP genes. In kidney cancer, RP subtypes map to molecular subtypes related to cell-of-origin. Pan-cancer analysis of TCGA data showed widespread single/double copy loss of RP genes, without significantly affecting survival. In several cancer cell lines, CRISPR-Cas9 knockout of RP genes did not affect cell viability. Matched RP ribosome profiling and mRNA data in humans and rodents stratified by tissue and development stage and were strongly correlated, showing that RP translation rates were proportional to mRNA levels. In a small dataset of human adult and fetal tissues, RP protein levels showed development stage and tissue specific heterogeneity of RP levels. Our results suggest that heterogeneous RP levels play a significant functional role in cellular physiology, in both normal and disease states. © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

International audience; Small Angle X-ray Scattering (SAXS) techniques are becoming more and more useful for structural biologists and biochemists, thanks to better access to dedicated synchrotron beamlines, better detectors and the relative easiness of sample preparation. The ability to compute the theoretical SAXS profile of a given structural model, and to compare this profile with the measured scattering intensity, yields crucial structural informations about the macromolecule under study and/or its complexes in solution. An important contribution to the profile, besides the macromolecule itself and its solvent-excluded volume, is the excess density due to the hydration layer. AquaSAXS takes advantage of recently developed methods, such as AquaSol, that give the equilibrium solvent density map around macromolecules, to compute an accurate SAXS/WAXS profile of a given structure and to compare it to the experimental one. Here, we describe the interface architecture and capabilities of the AquaSAXS web server (http://lorentz.dynstr.pasteur.fr/aquasaxs.php).

Myosin VI has challenged the lever arm hypothesis of myosin movement because of its ability to take approximately 36-nm steps along actin with a canonical lever arm that seems to be too short to allow such large steps. Here we demonstrate that the large step of dimeric myosin VI is primarily made possible by a medial tail in each monomer that forms a rare single alpha-helix of approximately 10 nm, which is anchored to the calmodulin-bound IQ domain by a globular proximal tail. With the medial tail contributing to the approximately 36-nm step, rather than dimerizing as previously proposed, we show that the cargo binding domain is the dimerization interface. Furthermore, the cargo binding domain seems to be folded back in the presence of the catalytic head, constituting a potential regulatory mechanism that inhibits dimerization.

International audience; Tools to study disordered systems with local structural order, such as proteins in solution, remain limited. Such understanding is essential for e.g. rational drug design. Correlated X-ray scattering (CXS) has recently attracted new interest as a way to leverage next-generation light sources to study such disordered matter. The CXS experiment measures angular correlations of the intensity caused by the scattering of X-rays from an ensemble of identical particles, with disordered orientation and position. Averaging over 15 496 snapshot images obtained by exposing a sample of silver nanoparticles in solution to a micro-focused synchrotron radiation beam, we report on experimental efforts to obtain CXS signal from an ensemble in three dimensions. A correlation function was measured at wide angles corresponding to atomic resolution that matches theoretical predictions. These preliminary results suggest that other CXS experiments on disordered ensembles\textemdashsuch as proteins in solution\textemdashmay be feasible in the future.

Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2–1.5 A° per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ,16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.

We measured the distance between fluorescent-labeled DNA loci of various interloci contour lengths in Caulobacter crescentus swarmer cells to determine the in vivo configuration of the chromosome. For DNA segments less than about 300 kb, the mean interloci distances, 〈 r 〉, scale as n 0.22 , where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n 0.22 with broad cell-to-cell variability. For DNA segments greater than about 300 kb, the mean interloci distances scale as n , in agreement with previous observations. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. Predictions from Brownian dynamics simulations of the packing of supercoiled DNA polymers in an elongated cell-like confinement are also consistent with a branched DNA structure, and simulated interloci distance distributions predict that confinement leads to “freezing” of the supercoiled configuration. Lateral positions of labeled loci at comparable positions along the length of the cell are strongly correlated when the longitudinal locus positions differ by parS centromere to the distal cell pole may arise from the release at the polar region of potential energy within the supercoiled DNA.

Short single-stranded nucleic acids are ubiquitous in biological processes; understanding their physical properties provides insights to nucleic acid folding and dynamics. We used small-angle x-ray scattering to study 8-100 residue homopolymeric single-stranded DNAs in solution, without external forces or labeling probes. Poly-T's structural ensemble changes with increasing ionic strength in a manner consistent with a polyelectrolyte persistence length theory that accounts for molecular flexibility. For any number of residues, poly-A is consistently more elongated than poly-T, likely due to the tendency of A residues to form stronger base-stacking interactions than T residues.

We investigate the Josephson current in a graphene superconductor/normal/superconductor junction, where superconductivity is induced by means of the proximity effect from external contacts. We take into account the possibility of anisotropic pairing by also including singlet nearest-neighbor interactions, and investigate how the transport properties are affected by the symmetry of the superconducting order parameter. This corresponds to an extension of the usual on-site interaction assumption, which yields an isotropic s-wave order parameter near the Dirac points. Here, we employ a full numerical solution as well as an analytical treatment, and show how the proximity effect may induce exotic types of superconducting states near the Dirac points, e.g. $p_x$- and $p_y$-wave pairing or a combination of s-wave and $p+\i p$-wave pairing. We find that the Josephson current exhibits a weakly-damped, oscillatory dependence on the length of the junction when the graphene sheet is strongly doped. The analytical and numerical treatments are found to agree well with each other in the s-wave case when calculating the critical current and current-phase relationship. For the scenarios with anisotropic superconducting pairing, there is a deviation between the two treatments, especially for the effective $p_x$-wave order parameter near the Dirac cones which features zero-energy states at the interfaces. This indicates that a numerical, self-consistent approach becomes necessary when treating anisotropic superconducting pairing in graphene., Comment: 15 pages, 12 figures

We use a tight-binding Bogoliubov-de Gennes (BdG) formalism to self-consistently calculate the proximity effect, Josephson current, and local density of states in ballistic graphene SNS Josephson junctions. Both short and long junctions, with respect to the superconducting coherence length, are considered, as well as different doping levels of the graphene. We show that self-consistency does not notably change the current-phase relationship derived earlier for short junctions using the non-selfconsistent Dirac-BdG formalism but predict a significantly increased critical current with a stronger junction length dependence. In addition, we show that in junctions with no Fermi level mismatch between the N and S regions superconductivity persists even in the longest junctions we can investigate, indicating a diverging Ginzburg-Landau superconducting coherence length in the normal region., 8 pages, 6 figures

Riboswitches are complex folded RNA domains found in noncoding regions of mRNA that regulate gene expression upon small molecule binding. Recently, Breaker and coworkers reported a tandem aptamer riboswitch (VCI-II) that binds glycine cooperatively. Here, we use hydroxyl radical footprinting and small-angle X-ray scattering (SAXS) to study the conformations of this tandem aptamer as a function of Mg(2+) and glycine concentration. We fit a simple three-state thermodynamic model that describes the energetic coupling between magnesium-induced folding and glycine binding. Furthermore, we characterize the structural conformations of each of the three states: In low salt with no magnesium present, the VCI-II construct has an extended overall conformation, presumably representing unfolded structures. Addition of millimolar concentrations of Mg(2+) in the absence of glycine leads to a significant compaction and partial folding as judged by hydroxyl radical protections. In the presence of millimolar Mg(2+) concentrations, the tandem aptamer binds glycine cooperatively. The glycine binding transition involves a further compaction, additional tertiary packing interactions and further uptake of magnesium ions relative to the state in high Mg(2+) but no glycine. Employing density reconstruction algorithms, we obtain low resolution 3-D structures for all three states from the SAXS measurements. These data provide a first glimpse into the structural conformations of the VCI-II aptamer, establish rigorous constraints for further modeling, and provide a framework for future mechanistic studies.

We propose that the pseudogap state observed in the transition metal oxides can be explained by a three-dimensional flux state, which exhibits spontaneously generated currents in its ground state due to electron-electron correlations. We compare the energy of the flux state to other classes of mean field states, and find that it is stabilized over a wide range of $t$ and $\delta$. The signature of the state will be peaks in the neutron diffraction spectra, the location and intensity of which are presented. The dependence of the pseudogap in the optical conductivity is calculated based on the parameters in the model., Comment: submitted to Phys. Rev. B on January 8, 2002

We show that the flux line lattice in a model layered HTSC becomes unstable above a critical magnetic field with respect to a plastic deformation via penetration of pairs of point-like disclination defects. The instability is characterized by the competition between the elastic and the pinning energies and is essentially assisted by softening of the lattice induced by a dimensional crossover of the fluctuations as field increases. We confirm through a computer simulation that this indeed may lead to a phase transition from crystalline order at low fields to a topologically disordered phase at higher fields. We propose that this mechanism provides a model of the low temperature field--driven disordering transition observed in neutron diffraction experiments on ${\rm Bi_2Sr_2CaCu_2O_8\, }$ single crystals., 11 pages, 4 figures available upon request via snail mail from ryu@pacific.mps.ohio-state.edu

A relatively unknown protein structure motif forms stable isolated single alpha-helices, termed ER/K alpha-helices, in a wide variety of proteins and has been shown to be essential for the function of some molecular motors. The flexibility of the ER/K alpha-helix determines whether it behaves as a force transducer, rigid spacer, or flexible linker in proteins. In this study, we quantify this flexibility in terms of persistence length, namely the length scale over which it is rigid. We use single-molecule optical trapping and small-angle x-ray scattering, combined with Monte Carlo simulations to demonstrate that the Kelch ER/K alpha-helix behaves as a wormlike chain with a persistence length of approximately 15 nm or approximately 28 turns of alpha-helix. The ER/K alpha-helix length in proteins varies from 3 to 60 nm, with a median length of approximately 5 nm. Knowledge of its persistence length enables us to define its function as a rigid spacer in a translation initiation factor, as a force transducer in the mechanoenzyme myosin VI, and as a flexible spacer in the Kelch-motif-containing protein.

One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy, and small angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein’s hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

Functional RNAs fold into compact, well-defined tertiary structures despite strong electrostatic repulsion both within and between helices. To achieve these compact structures, many RNAs employ structural divalent cations, typically Mg2+. The simplest tertiary contact in nucleic acids is two helices, joined by some non-helical contact. To explore the fundamental characteristics of tertiary contact formation in nucleic acids we studied a system of two DNA helices tethered via a short PEG linker by both computational and experimental methods. Computationally, we predict the electrostatic repulsion between these helices as a function of Mg2+ and Na+ concentration. Experimentally, we linked the distal termini of these helices via a disulfide linker. Using small-angle x-ray scattering, we measured the fraction of intact disulfide bonds as a function of reducing strength of the buffer at a range of Mg2+ and Na+ concentration. Using these data we can extrapolate the magnitude of the strain on the disulfide bond, and thus the repulsion between the helices. Previous results show that the conformational ensemble is narrowly distributed around an extended, co-linear conformation at low salt and becomes more relaxed at high salt, but is unable to isolate a conformation in which the helices are stacked. Furthermore, previous results also suggest that specific interactions between Mg2+ and the phosphate backbone strongly are more important than simple ionic strength in determining the magnitude of the repulsive potential between the helices. In this project, we hope to experimentally demonstrate the energy of helix stacking and the different role of ionic strength and ionic specific interactions.