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Sample records for single protein revealed

  1. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs

    PubMed Central

    Smits, Arne H.; Lindeboom, Rik G.H.; Perino, Matteo; van Heeringen, Simon J.; Veenstra, Gert Jan C.; Vermeulen, Michiel

    2014-01-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs. PMID:25056316

  2. Revealing Two-State Protein-Protein Interaction of Calmodulin by Single-Molecule Spectroscopy

    SciTech Connect

    Liu, Ruchuan; Hu, Dehong; Tan, Xin; Lu, H PETER.

    2006-08-09

    We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of 28 amino-acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM-C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (F1AsH) that enables our unambiguously probing the CaM N-terminal target-binding domain motions at a millisecond timescale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between F1AsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal slow (at sub-second time scale) binding-unbinding motions of the N-terminal domain of the CaM in CaM-C28W complexes, which is a strong evidence of a two-state binding interaction of CaM-mediated cell signaling.

  3. Revealing Two-State Protein-Protein Interactions of Calmodulin by Single-Molecule Spectroscopy

    SciTech Connect

    Liu, Ruchuan; Hu, Dehong; Tan, Xin; Lu, H. Peter

    2006-08-01

    We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of a 28 amino acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM/C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (FlAsH) that enables our unambiguous probing of the CaM N-terminal target-binding domain motions on a millisecond time scale without convolution of the probe-dye random motions. Finally, by analyzing the distribution of FRET efficiency between FlAsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal binding-unbinding motions of the N-terminal domain of the CaM in CaM/C28W complexes, which is strong evidence of a two-state binding interaction of CaM-mediated cell signaling.

  4. Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

    PubMed Central

    Gallardo, Ignacio F.; Zhou, Yi; Gong, Fade; Yang, Soo-Hyun; Wold, Marc S.; Miller, Kyle M.; Paull, Tanya T.

    2016-01-01

    Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1—a recently identified human SSB that promotes DNA resection during homologous recombination—supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells. PMID:26884156

  5. Slowing down single-molecule trafficking through a protein nanopore reveals intermediates for peptide translocation

    NASA Astrophysics Data System (ADS)

    Mereuta, Loredana; Roy, Mahua; Asandei, Alina; Lee, Jong Kook; Park, Yoonkyung; Andricioaei, Ioan; Luchian, Tudor

    2014-01-01

    The microscopic details of how peptides translocate one at a time through nanopores are crucial determinants for transport through membrane pores and important in developing nano-technologies. To date, the translocation process has been too fast relative to the resolution of the single molecule techniques that sought to detect its milestones. Using pH-tuned single-molecule electrophysiology and molecular dynamics simulations, we demonstrate how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide. Molecular dynamics simulations of peptide translocation reveal the time- dependent ordering of intermediate structures of the translocating peptide inside the pore at atomic resolution. Calculations of the expected current ratios of the different pore-blocking microstates and their time sequencing are in accord with the recorded current traces.

  6. Quantum dot single molecule tracking reveals a wide range of diffusive motions of membrane transport proteins

    NASA Astrophysics Data System (ADS)

    Crane, Jonathan M.; Haggie, Peter M.; Verkman, A. S.

    2009-02-01

    Single particle tracking (SPT) provides information about the microscopic motions of individual particles in live cells. We applied SPT to study the diffusion of membrane transport proteins in cell plasma membranes in which individual proteins are labeled with quantum dots at engineered extracellular epitopes. Software was created to deduce particle diffusive modes from quantum dot trajectories. SPT of aquaporin (AQP) water channels and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels revealed several types of diffusion. AQP1 was freely mobile in cell membranes, showing rapid, Brownian-type diffusion. The full-length (M1) isoform of AQP4 also diffused rapidly, though the diffusion of a shorter (M23) isoform of AQP4 was highly restricted due to its supermolecular assembly in raft-like orthogonal arrays. CFTR mobility was also highly restricted, in a spring-like potential, due to its tethering to the actin cytoskeleton through PDZ-domain C-terminus interactions. The biological significance of regulated diffusion of membrane transport proteins is a subject of active investigation.

  7. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    NASA Astrophysics Data System (ADS)

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-09-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

  8. Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

    PubMed Central

    Tsytlonok, Maksym; Ibrahim, Shehu M.; Rowling, Pamela J.E.; Xu, Wenshu; Ruedas-Rama, Maria J.; Orte, Angel; Klenerman, David; Itzhaki, Laura S.

    2015-01-01

    Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated. PMID:25565106

  9. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane.

    PubMed

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  10. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    PubMed Central

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  11. QUANTUM DOT SINGLE MOLECULE TRACKING REVEALS A WIDE RANGE OF DIFFUSIVE MOTIONS OF MEMBRANE TRANSPORT PROTEINS.

    PubMed

    Crane, Jonathan M; Haggie, Peter M; Verkman, A S

    2009-03-01

    Single particle tracking (SPT) provides information about the microscopic motions of individual particles in live cells. We applied SPT to study the diffusion of membrane transport proteins in cell plasma membranes in which individual proteins are labeled with quantum dots at engineered extracellular epitopes. Software was created to deduce particle diffusive modes from quantum dot trajectories. SPT of aquaporin (AQP) water channels and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels revealed several types of diffusion. AQP1 was freely mobile in cell membranes, showing rapid, Brownian-type diffusion. The full-length (M1) isoform of AQP4 also diffused rapidly, though the diffusion of a shorter (M23) isoform of AQP4 was highly restricted due to its supermolecular assembly in raft-like orthogonal arrays. CFTR mobility was also highly restricted, in a spring-like potential, due to its tethering to the actin cytoskeleton through PDZ-domain C-terminus interactions. The biological significance of regulated diffusion of membrane transport proteins is a subject of active investigation.

  12. Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells.

    PubMed

    Godin, Antoine G; Rappaz, Benjamin; Potvin-Trottier, Laurent; Kennedy, Timothy E; De Koninck, Yves; Wiseman, Paul W

    2015-08-18

    Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique-spatial intensity distribution analysis (SpIDA)-that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and

  13. Nonlinearly Additive Forces in Multivalent Ligand Binding to a Single Protein Revealed with Force Spectroscopy

    SciTech Connect

    Ratto, T V; Rudd, R E; Langry, K C; Balhorn, R L; McElfresh, M W

    2005-07-15

    We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between ConcanavalinA (ConA) and {alpha}-D-mannose, but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 66, and 85 pN, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and non-specific binding. We analyze the binding configuration (i.e. serial versus parallel connections) through fitting the polymer stretching data with modified Worm-Like Chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.

  14. Conformational Memory of a Protein Revealed by Single-Molecule Spectroscopy.

    PubMed

    Schörner, Mario; Beyer, Sebastian Reinhardt; Southall, June; Cogdell, Richard J; Köhler, Jürgen

    2015-11-01

    Proteins are supramolecular machines that carry out a wide range of different functions, many of which require flexibility. Up until now spontaneous conformational fluctuations of proteins have always been assumed to reflect a stochastic random process. However, if changing between different conformational states was random, then it would be difficult to understand how conformational control of protein function could have evolved. Here we demonstrate that a single protein can show conformational memory. This is exactly the process that can facilitate the evolution of control of switching between two conformational states that can then be used to regulate protein function.

  15. Protein Configuration Landscape Fluctuations Revealed by Exciton Transition Polarizations in Single Light Harvesting Complexes.

    PubMed

    Tubasum, Sumera; Torbjörnsson, Magne; Yadav, Dheerendra; Camacho, Rafael; Söderlind, Gustaf; Scheblykin, Ivan G; Pullerits, Tõnu

    2016-02-01

    Protein is a flexible material with broad distribution of conformations forming an energy landscape of quasi-stationary states. Disentangling the system dynamics along this landscape is the key for understanding the functioning of the protein. Here we studied a photosynthetic antenna pigment-protein complex LH2 with single molecule two-dimensional polarization imaging. Modeling based on the Redfield relaxation theory well describes the observed polarization properties of LH2 fluorescence and fluorescence excitation, strongly suggesting that at 77 K the conformational subspace of the LH2 is limited to about three configurations with relatively frequent switching among each other. At room temperature the next level of fluctuations determines the conformational dynamics. The results support the multitier model of the energy landscape of proteins and demonstrate the potential of the method for the studies of structural dynamics in proteins.

  16. Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways

    PubMed Central

    Suksombat, Sukrit; Khafizov, Rustem; Kozlov, Alexander G; Lohman, Timothy M; Chemla, Yann R

    2015-01-01

    Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA. DOI: http://dx.doi.org/10.7554/eLife.08193.001 PMID:26305498

  17. Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay

    PubMed Central

    De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M.

    2011-01-01

    Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin. PMID:22006542

  18. Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility

    PubMed Central

    McCauley, Micah J.; Rueter, Emily M.; Rouzina, Ioulia; Maher, L. James; Williams, Mark C.

    2013-01-01

    Eukaryotic High-Mobility Group B (HMGB) proteins alter DNA elasticity while facilitating transcription, replication and DNA repair. We developed a new single-molecule method to probe non-specific DNA interactions for two HMGB homologs: the human HMGB2 box A domain and yeast Nhp6Ap, along with chimeric mutants replacing neutral N-terminal residues of the HMGB2 protein with cationic sequences from Nhp6Ap. Surprisingly, HMGB proteins constrain DNA winding, and this torsional constraint is released over short timescales. These measurements reveal the microscopic dissociation rates of HMGB from DNA. Separate microscopic and macroscopic (or local and non-local) unbinding rates have been previously proposed, but never independently observed. Microscopic dissociation rates for the chimeric mutants (∼10 s−1) are higher than those observed for wild-type proteins (∼0.1–1.0 s−1), reflecting their reduced ability to bend DNA through short-range interactions, despite their increased DNA-binding affinity. Therefore, transient local HMGB–DNA contacts dominate the DNA-bending mechanism used by these important architectural proteins to increase DNA flexibility. PMID:23143110

  19. Single molecule analysis of functionally asymmetric G protein-coupled receptor (GPCR) oligomers reveals diverse spatial and structural assemblies.

    PubMed

    Jonas, Kim C; Fanelli, Francesca; Huhtaniemi, Ilpo T; Hanyaloglu, Aylin C

    2015-02-13

    Formation of G protein-coupled receptors (GPCRs) into dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. We present a super-resolution imaging approach, resolving single GPCR molecules to ∼ 8 nm resolution in functional asymmetric dimers and oligomers using dual-color photoactivatable dyes and localization microscopy (PD-PALM). PD-PALM of two functionally defined mutant luteinizing hormone receptors (LHRs), a ligand-binding deficient receptor (LHR(B-)) and a signaling-deficient (LHR(S-)) receptor, which only function via intermolecular cooperation, favored oligomeric over dimeric formation. PD-PALM imaging of trimers and tetramers revealed specific spatial organizations of individual protomers in complexes where the ratiometric composition of LHR(B-) to LHR(S-) modulated ligand-induced signal sensitivity. Structural modeling of asymmetric LHR oligomers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Our findings reveal that diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.

  20. Direct observation of single stationary-phase bacteria reveals a surprisingly long period of constant protein production activity

    PubMed Central

    Gefen, Orit; Fridman, Ofer; Ronin, Irine; Balaban, Nathalie Q.

    2014-01-01

    Exponentially growing bacteria are rarely found in the wild, as microorganisms tend to spend most of their lifetime at stationary phase. Despite this general prevalence of stationary-phase bacteria, they are as yet poorly characterized. Our goal was to quantitatively study this phase by direct observation of single bacteria as they enter into stationary phase and by monitoring their activity over several days during growth arrest. For this purpose, we devised an experimental procedure for starving single Escherichia coli bacteria in microfluidic devices and measured their activity by monitoring the production rate of fluorescent proteins. When amino acids were the sole carbon source, the production rate decreased by an order of magnitude upon entry into stationary phase. We found that, even while growth-arrested, bacteria continued to produce proteins at a surprisingly constant rate over several days. Our identification of this newly observed period of constant activity in nongrowing cells, designated as constant activity stationary phase, makes possible the conduction of assays that require constant protein expression over time, and are therefore difficult to perform under exponential growth conditions. Moreover, we show that exogenous protein expression bears no fitness cost on the regrowth of the population when starvation ends. Further characterization of constant activity stationary phase—a phase where nongrowing bacteria can be quantitatively studied over several days in a reproducible manner—should contribute to a better understanding of this ubiquitous but overlooked physiological state of bacteria in nature. PMID:24344288

  1. Confined Diffusion Without Fences of a G-Protein-Coupled Receptor as Revealed by Single Particle Tracking

    PubMed Central

    Daumas, Frédéric; Destainville, Nicolas; Millot, Claire; Lopez, André; Dean, David; Salomé, Laurence

    2003-01-01

    Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the μ-opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients. PMID:12524289

  2. Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1

    PubMed Central

    Milenkovic, Ljiljana; Weiss, Lucien E.; Yoon, Joshua; Roth, Theodore L.; Su, YouRong S.; Sahl, Steffen J.; Scott, Matthew P.; Moerner, W. E.

    2015-01-01

    Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation. PMID:26100903

  3. Single-molecule Force Spectroscopy Reveals the Calcium Dependence of the Alternative Conformations in the Native State of a βγ-Crystallin Protein.

    PubMed

    Scholl, Zackary N; Li, Qing; Yang, Weitao; Marszalek, Piotr E

    2016-08-26

    Although multidomain proteins predominate the proteome of all organisms and are expected to display complex folding behaviors and significantly greater structural dynamics as compared with single-domain proteins, their conformational heterogeneity and its impact on their interaction with ligands are poorly understood due to a lack of experimental techniques. The multidomain calcium-binding βγ-crystallin proteins are particularly important because their deterioration and misfolding/aggregation are associated with melanoma tumors and cataracts. Here we investigate the mechanical stability and conformational dynamics of a model calcium-binding βγ-crystallin protein, Protein S, and elaborate on its interactions with calcium. We ask whether domain interactions and calcium binding affect Protein S folding and potential structural heterogeneity. Our results from single-molecule force spectroscopy show that the N-terminal (but not the C-terminal) domain is in equilibrium with an alternative conformation in the absence of Ca(2+), which is mechanically stable in contrast to other proteins that were observed to sample a molten globule under similar conditions. Mutagenesis experiments and computer simulations reveal that the alternative conformation of the N-terminal domain is caused by structural instability produced by the high charge density of a calcium binding site. We find that this alternative conformation in the N-terminal domain is diminished in the presence of calcium and can also be partially eliminated with a hitherto unrecognized compensatory mechanism that uses the interaction of the C-terminal domain to neutralize the electronegative site. We find that up to 1% of all identified multidomain calcium-binding proteins contain a similarly highly charged site and therefore may exploit a similar compensatory mechanism to prevent structural instability in the absence of ligand. PMID:27378818

  4. Novel circular single-stranded DNA viruses identified in marine invertebrates reveal high sequence diversity and consistent predicted intrinsic disorder patterns within putative structural proteins

    PubMed Central

    Rosario, Karyna; Schenck, Ryan O.; Harbeitner, Rachel C.; Lawler, Stephanie N.; Breitbart, Mya

    2015-01-01

    Viral metagenomics has recently revealed the ubiquitous and diverse nature of single-stranded DNA (ssDNA) viruses that encode a conserved replication initiator protein (Rep) in the marine environment. Although eukaryotic circular Rep-encoding ssDNA (CRESS-DNA) viruses were originally thought to only infect plants and vertebrates, recent studies have identified these viruses in a number of invertebrates. To further explore CRESS-DNA viruses in the marine environment, this study surveyed CRESS-DNA viruses in various marine invertebrate species. A total of 27 novel CRESS-DNA genomes, with Reps that share less than 60.1% identity with previously reported viruses, were recovered from 21 invertebrate species, mainly crustaceans. Phylogenetic analysis based on the Rep revealed a novel clade of CRESS-DNA viruses that included approximately one third of the marine invertebrate associated viruses identified here and whose members may represent a novel family. Investigation of putative capsid proteins (Cap) encoded within the eukaryotic CRESS-DNA viral genomes from this study and those in GenBank demonstrated conserved patterns of predicted intrinsically disordered regions (IDRs), which can be used to complement similarity-based searches to identify divergent structural proteins within novel genomes. Overall, this study expands our knowledge of CRESS-DNA viruses associated with invertebrates and explores a new tool to evaluate divergent structural proteins encoded by these viruses. PMID:26217327

  5. Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

    PubMed Central

    Gorzelnik, Karl V.; Cui, Zhicheng; Reed, Catrina A.; Jakana, Joanita; Young, Ry; Zhang, Junjie

    2016-01-01

    Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qβ, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A2, binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qβ with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A2 and the gRNA. A2 contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the surrounding coat protein shell to potentially facilitate RNA release during infection. We observe a fixed pattern of gRNA organization among all viral particles, with the major and minor grooves of RNA helices clearly visible. A single layer of RNA directly contacts every copy of the coat protein, with one-third of the interactions occurring at operator-like RNA hairpins. These RNA–coat interactions stabilize the tertiary structure of gRNA within the virion, which could further provide a roadmap for capsid assembly. PMID:27671640

  6. Single-Nucleotide Mutations in FMR1 Reveal Novel Functions and Regulatory Mechanisms of the Fragile X Syndrome Protein FMRP

    PubMed Central

    Suhl, Joshua A.; Warren, Stephen T.

    2015-01-01

    Fragile X syndrome is a monogenic disorder and a common cause of intellectual disability. Despite nearly 25 years of research on FMR1, the gene underlying the syndrome, very few pathological mutations other than the typical CGG-repeat expansion have been reported. This is in contrast to other X-linked, monogenic, intellectual disability disorders, such as Rett syndrome, where many point mutations have been validated as causative of the disorder. As technology has improved and significantly driven down the cost of sequencing, allowing for whole genes to be sequenced with relative ease, in-depth sequencing studies on FMR1 have recently been performed. These studies have led to the identification of novel variants in FMR1, where some of which have been functionally evaluated and are likely pathogenic. In this review, we discuss recently identified FMR1 variants, the ways these novel variants cause dysfunction, and how they reveal new regulatory mechanisms and functionalities of the gene. PMID:26819560

  7. Secreted protein gene derived-single nucleotide polymorphisms (SP-SNPs) reveal population diversity and differentiation of Puccinia striiformis f. sp. tritici in the United States.

    PubMed

    Xia, Chongjing; Wan, Anmin; Wang, Meinan; Jiwan, Derick A; See, Deven R; Chen, Xianming

    2016-05-01

    Single nucleotide polymorphism (SNP) is a powerful molecular marker technique that has been widely used in population genetics and molecular mapping studies for various organisms. However, the technique has not been used for studying Puccinia striiformis f. sp. tritici (Pst), the wheat stripe rust pathogen. In this study, we developed over a hundred secreted protein gene-derived SNP (SP-SNP) markers and used 92 markers to study the population structure of Pst. From 352 isolates collected in the United States, we identified 242 multi-locus genotypes. The SP-SNP genotypes had a moderate, but significant correlation with the virulence phenotype data. Clustering of the multi-locus genotypes was consistent by various analyses, revealing distinct genetic groups. Analysis of molecular variance detected significant differences between the eastern and western US Pst populations. High heterozygosity was found in the US population with significant differences identified among epidemiological regions. Analysis of population differentiation revealed that populations between the eastern and western US were highly differentiated while moderate differentiation was found in populations within the western or eastern US. Isolates from the western US were more diverse than isolates from the eastern US. The information is useful for guiding the disease management in different epidemiological regions. PMID:27109369

  8. Mutational Analysis of the T4 Gp59 Helicase Loader Reveals Its Sites for Interaction with Helicase, Single-stranded Binding Protein, and DNA*

    PubMed Central

    Dolezal, Darin; Jones, Charles E.; Lai, Xiaoqin; Brister, J. Rodney; Mueser, Timothy C.; Nossal, Nancy G.; Hinton, Deborah M.

    2012-01-01

    Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB. PMID:22427673

  9. Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

    PubMed Central

    Ghodke, Harshad; Wang, Hong; Hsieh, Ching L.; Woldemeskel, Selamawit; Watkins, Simon C.; Rapić-Otrin, Vesna; Van Houten, Bennett

    2014-01-01

    How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3–0.8, 8.1, and 113–126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism. PMID:24760829

  10. 2D DIGE Does Not Reveal all: A Scotopic Report Suggests Differential Expression of a Single “Calponin Family Member” Protein for Tetany of Sphincters!

    PubMed Central

    Chaudhury, Arun

    2015-01-01

    Using 2D differential gel electrophoresis (DIGE) and mass spectrometry (MS), a recent report by Rattan and Ali (2015) compared proteome expression between tonically contracted sphincteric smooth muscles of the internal anal sphincter (IAS), in comparison to the adjacent rectum [rectal smooth muscles (RSM)] that contracts in a phasic fashion. The study showed the differential expression of a single 23 kDa protein SM22, which was 1.87 fold, overexpressed in RSM in comparison to IAS. Earlier studies have shown differences in expression of different proteins like Rho-associated protein kinase II, myosin light chain kinase, myosin phosphatase, and protein kinase C between IAS and RSM. The currently employed methods, despite its high-throughput potential, failed to identify these well-characterized differences between phasic and tonic muscles. This calls into question the fidelity and validatory potential of the otherwise powerful technology of 2D DIGE/MS. These discrepancies, when redressed in future studies, will evolve this recent report as an important baseline study of “sphincter proteome.” Proteomics techniques are currently underutilized in examining pathophysiology of hypertensive/hypotensive disorders involving gastrointestinal sphincters, including achalasia, gastroesophageal reflux disease (GERD), spastic pylorus, seen during diabetes or chronic chemotherapy, intestinal pseudo-obstruction, and recto-anal incontinence. Global proteome mapping may provide instant snapshot of the complete repertoire of differential proteins, thus expediting to identify the molecular pathology of gastrointestinal motility disorders currently labeled “idiopathic” and facilitating practice of precision medicine. PMID:26151053

  11. A Selection for Assembly Reveals That a Single Amino Acid Mutant of the Bacteriophage MS2 Coat Protein Forms a Smaller Virus-like Particle.

    PubMed

    Asensio, Michael A; Morella, Norma M; Jakobson, Christopher M; Hartman, Emily C; Glasgow, Jeff E; Sankaran, Banumathi; Zwart, Peter H; Tullman-Ercek, Danielle

    2016-09-14

    Virus-like particles are used to encapsulate drugs, imaging agents, enzymes, and other biologically active molecules in order to enhance their function. However, the size of most virus-like particles is inflexible, precluding the design of appropriately sized containers for different applications. Here, we describe a chromatographic selection for virus-like particle assembly. Using this selection, we identified a single amino acid substitution to the coat protein of bacteriophage MS2 that mediates a uniform switch in particle geometry from T = 3 to T = 1 icosahedral symmetry. The resulting smaller particle retains the ability to be disassembled and reassembled in vitro and to be chemically modified to load cargo into its interior cavity. The pair of 27 and 17 nm MS2 particles will allow direct examination of the effect of size on function in established applications of virus-like particles, including drug delivery and imaging. PMID:27549001

  12. Single quantum dot tracking reveals that an individual multivalent HIV-1 Tat protein transduction domain can activate machinery for lateral transport and endocytosis.

    PubMed

    Suzuki, Yasuhiro; Roy, Chandra Nath; Promjunyakul, Warunya; Hatakeyama, Hiroyasu; Gonda, Kohsuke; Imamura, Junji; Vasudevanpillai, Biju; Ohuchi, Noriaki; Kanzaki, Makoto; Higuchi, Hideo; Kaku, Mitsuo

    2013-08-01

    The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines. PMID:23732912

  13. Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

    PubMed Central

    Roy, Chandra Nath; Promjunyakul, Warunya; Hatakeyama, Hiroyasu; Gonda, Kohsuke; Imamura, Junji; Vasudevanpillai, Biju; Ohuchi, Noriaki; Kanzaki, Makoto; Higuchi, Hideo; Kaku, Mitsuo

    2013-01-01

    The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines. PMID:23732912

  14. Single-molecule studies reveal that DEAD box protein DDX1 promotes oligomerization of HIV-1 Rev on the Rev response element.

    PubMed

    Robertson-Anderson, Rae M; Wang, Jun; Edgcomb, Stephen P; Carmel, Andrew B; Williamson, James R; Millar, David P

    2011-07-29

    Oligomeric assembly of Rev on the Rev response element (RRE) is essential for the nuclear export of unspliced and singly spliced human immunodeficiency virus type 1 viral mRNA transcripts. Several host factors, including the human DEAD box protein DDX1, are also known to be required for efficient Rev function. In this study, spontaneous assembly and dissociation of individual Rev-RRE complexes in the presence or absence of DDX1 were observed in real time via single-molecule total internal reflection fluorescence microscopy. Binding of up to eight fluorescently labeled Rev monomers to a single RRE molecule was visualized, and the event frequencies and corresponding binding and dissociation rates for the different Rev-RRE stoichiometries were determined. The presence of DDX1 eliminated a second kinetic phase present during the initial Rev binding step, attributed to nonproductive nucleation events, resulting in increased occurrence of higher-order Rev-RRE stoichiometries. This effect was further enhanced upon the addition of a non-hydrolyzable ATP analog (adenylyl-imidophosphate), whereas ADP had no effect beyond that of DDX1 alone. Notably, the first three Rev monomer binding events were accelerated in the presence of DDX1 and adenylyl-imidophosphate, while the dissociation rates remained unchanged. Measurements performed across a range of DDX1 concentrations suggest that DDX1 targets Rev rather than the RRE to promote oligomeric assembly. Moreover, DDX1 is able to restore the oligomerization activity of a Rev mutant that is otherwise unable to assemble on the RRE beyond a monomeric complex. Taken together, these results suggest that DDX1 acts as a cellular cofactor by promoting oligomerization of Rev on the RRE. PMID:21763499

  15. Watching Single Proteins Using Engineered Nanopores

    PubMed Central

    Movileanu, Liviu

    2014-01-01

    Recent studies in the area of single-molecule detection of proteins with nanopores show a great promise in fundamental science, bionanotechnology and proteomics. In this mini-review, I discuss a comprehensive array of examinations of protein detection and characterization using protein and solid-state nanopores. These investigations demonstrate the power of the single-molecule nanopore measurements to reveal a broad range of functional, structural, biochemical and biophysical features of proteins, such as their backbone flexibility, enzymatic activity, binding affinity as well as their concentration, size and folding state. Engineered nanopores in organic materials and in inorganic membranes coupled with surface modification and protein engineering might provide a new generation of sensing devices for molecular biomedical diagnosis. PMID:24370252

  16. Revealing ligand binding sites and quantifying subunit variants of noncovalent protein complexes in a single native top-down FTICR MS experiment.

    PubMed

    Li, Huilin; Wongkongkathep, Piriya; Van Orden, Steve L; Ogorzalek Loo, Rachel R; Loo, Joseph A

    2014-12-01

    "Native" mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.

  17. Revealing Ligand Binding Sites and Quantifying Subunit Variants of Non-Covalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment

    PubMed Central

    Li, Huilin; Wongkongkathep, Piriya; Van Orden, Steve L.; Loo, Rachel R. Ogorzalek; Loo, Joseph A.

    2015-01-01

    “Native” mass spectrometry (MS) has been proven increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD MS hADH dimer shows that each subunit (E and S chain) binds not only to two zinc atoms, but also the NAD+/NADH ligand, with a higher NAD+/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains. PMID:24912433

  18. Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF-RecO-RecR complex in DNA repair and recombination.

    PubMed

    Hegde, S P; Qin, M H; Li, X H; Atkinson, M A; Clark, A J; Rajagopalan, M; Madiraju, M V

    1996-12-10

    The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF-RecO-RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF-RecO-Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF-RecO-RecR complex functions as an anti-Ssb factor. PMID:8962075

  19. Optical tweezers reveal how proteins alter replication

    NASA Astrophysics Data System (ADS)

    Chaurasiya, Kathy

    Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

  20. Protein Vivisection Reveals Elusive Intermediates in Folding

    SciTech Connect

    Zheng, Zhongzhou; Sosnick, Tobin R.

    2010-05-25

    Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here, we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu {yields} Glu{sup -}) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the {beta}5-strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.

  1. Protein vivisection reveals elusive intermediates in folding.

    PubMed

    Zheng, Zhongzhou; Sosnick, Tobin R

    2010-04-01

    Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here, we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu-->Glu(-)) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the beta5-strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.

  2. Atomic force microscopy reveals the mechanical design of a modular protein

    NASA Astrophysics Data System (ADS)

    Li, Hongbin; Oberhauser, Andres F.; Fowler, Susan B.; Clarke, Jane; Fernandez, Julio M.

    2000-06-01

    Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering.

  3. Single molecule studies reveal new mechanisms for microtubule severing

    NASA Astrophysics Data System (ADS)

    Ross, Jennifer; Diaz-Valencia, Juan Daniel; Morelli, Margaret; Zhang, Dong; Sharp, David

    2011-03-01

    Microtubule-severing enzymes are hexameric complexes made from monomeric enzyme subunits that remove tubulin dimers from the microtubule lattice. Severing proteins are known to remodel the cytoskeleton during interphase and mitosis, and are required in proper axon morphology and mammalian bone and cartilage development. We have performed the first single molecule imaging to determine where and how severing enzymes act to cut microtubules. We have focused on the original member of the group, katanin, and the newest member, fidgetin to compare their biophysical activities in vitro. We find that, as expected, severing proteins localize to areas of activity. Interestingly, the association is very brief: they do not stay bound nor do they bind cooperatively at active sites. The association duration changes with the nucleotide content, implying that the state in the catalytic cycle dictates binding affinity with the microtubule. We also discovered that, at lower concentrations, both katanin and fidgetin can depolymerize taxol-stabilized microtubules by removing terminal dimers. These studies reveal the physical regulation schemes to control severing activity in cells, and ultimately regulate cytoskeletal architecture. This work is supported by the March of Dimes Grant #5-FY09-46.

  4. Protein mechanics: from single molecules to functional biomaterials.

    PubMed

    Li, Hongbin; Cao, Yi

    2010-10-19

    Elastomeric proteins act as the essential functional units in a wide variety of biomechanical machinery and serve as the basic building blocks for biological materials that exhibit superb mechanical properties. These proteins provide the desired elasticity, mechanical strength, resilience, and toughness within these materials. Understanding the mechanical properties of elastomeric protein-based biomaterials is a multiscale problem spanning from the atomistic/molecular level to the macroscopic level. Uncovering the design principles of individual elastomeric building blocks is critical both for the scientific understanding of multiscale mechanics of biomaterials and for the rational engineering of novel biomaterials with desirable mechanical properties. The development of single-molecule force spectroscopy techniques has provided methods for characterizing mechanical properties of elastomeric proteins one molecule at a time. Single-molecule atomic force microscopy (AFM) is uniquely suited to this purpose. Molecular dynamic simulations, protein engineering techniques, and single-molecule AFM study have collectively revealed tremendous insights into the molecular design of single elastomeric proteins, which can guide the design and engineering of elastomeric proteins with tailored mechanical properties. Researchers are focusing experimental efforts toward engineering artificial elastomeric proteins with mechanical properties that mimic or even surpass those of natural elastomeric proteins. In this Account, we summarize our recent experimental efforts to engineer novel artificial elastomeric proteins and develop general and rational methodologies to tune the nanomechanical properties of elastomeric proteins at the single-molecule level. We focus on general design principles used for enhancing the mechanical stability of proteins. These principles include the development of metal-chelation-based general methodology, strategies to control the unfolding hierarchy of

  5. Protein single crystal growth under microgravity

    NASA Astrophysics Data System (ADS)

    Littke, Walter; John, Christina

    1986-08-01

    Crystal growth conditions for proteins under microgravity were investigated with two model compounds (β-galactosidase and lysozyme). The single crystals obtained have been found to be significantly larger than those prepared in the same environment on earth.

  6. Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions.

    PubMed

    Lee, Hong-Won; Ryu, Ji Young; Yoo, Janghyun; Choi, Byungsan; Kim, Kipom; Yoon, Tae-Young

    2013-10-01

    Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time, single-molecule fluorescence imaging as its detection scheme. Bait proteins are pulled down onto the imaging plane of a total internal reflection (TIR) microscope. With unpurified cells or tissue extracts kept in reaction chambers, we observe single protein-protein interactions between the surface-immobilized bait and the fluorescent protein-labeled prey proteins in real time. Such direct recording provides an improvement of five orders of magnitude in the time resolution of co-IP analysis. With the single-molecule sensitivity and millisecond time resolution, which distinguish our method from other methods for measuring weak protein-protein interactions, it is possible to quantify the interaction kinetics and active fraction of native, unlabeled bait proteins. Real-time single-molecule co-IP analysis, which takes ∼4 h to complete from lysate preparation to kinetic analysis, provides a general avenue for revealing the rich kinetic picture of target protein-protein interactions, and it can be used, for example, to investigate the molecular lesions that drive individual cancers at the level of protein-protein interactions.

  7. Protein Structures Revealed at Record Pace

    SciTech Connect

    Hura, Greg

    2009-01-01

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  8. Protein Structures Revealed at Record Pace

    SciTech Connect

    Greg Hura

    2009-07-09

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  9. Protein Structures Revealed at Record Pace

    ScienceCinema

    Hura, Greg

    2013-05-29

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  10. Protein Structures Revealed at Record Pace

    ScienceCinema

    Greg Hura

    2016-07-12

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  11. Growth of shaped single crystals of proteins

    NASA Astrophysics Data System (ADS)

    Moreno, Abel; Rondón, Deyanira; García-Ruiz, Juan Ma.

    1996-09-01

    We present a procedure for obtaining protein single crystals that fill the capillary tubes in which they grow. The implementation was typical of the gel acupuncture method and the four different proteins are used as examples: lysozyme (HEW), thaumatin I, ferritin and insulin. Rod- and prismatic-shaped protein single crystals of these four proteins were grown inside capillary tubes of 0.2, 0.3, 0.5 mm in diameter and, for the case of lysozyme, up to 1.2 mm in diameter. The maximum length measured along the long axes of the rod crystals was 1.6 mm again for lysozyme crystals. It was observed that, once the capillary tube was filled, the crystal continues to grow by diffusion of the precipitating agent throughout the porous network formed by the protein crystal structure. We also discuss the possibility of growing these cylinders of crystalline proteins by the addition of protein solution to the mother liquor through the upper end of the glass capillary while the precipitating agent diffuses through the protein crystal itself. X-ray diffraction patterns confirm the single crystal character of the protein rods.

  12. Microsecond protein dynamics observed at the single-molecule level

    PubMed Central

    Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

    2015-01-01

    How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape. PMID:26151767

  13. Microsecond protein dynamics observed at the single-molecule level

    NASA Astrophysics Data System (ADS)

    Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

    2015-07-01

    How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape.

  14. Single Filaments to Reveal the Multiple Flavors of Actin.

    PubMed

    Jégou, Antoine; Romet-Lemonne, Guillaume

    2016-05-24

    A number of key cell processes rely on specific assemblies of actin filaments, which are all constructed from nearly identical building blocks: the abundant and extremely conserved actin protein. A central question in the field is to understand how different filament networks can coexist and be regulated. Discoveries in science are often related to technical advances. Here, we focus on the ongoing single filament revolution and discuss how these techniques have greatly contributed to our understanding of actin assembly. In particular, we highlight how they have refined our understanding of the many protein-based regulatory mechanisms that modulate actin assembly. It is now becoming apparent that other factors give filaments a specific identity that determines which proteins will bind to them. We argue that single filament techniques will play an essential role in the coming years as we try to understand the many ways actin filaments can take different flavors and unveil how these flavors modulate the action of regulatory proteins. We discuss different factors known to make actin filaments distinguishable by regulatory proteins and speculate on their possible consequences.

  15. Nanoscience of single polymer chains revealed by nanofishing.

    PubMed

    Nakajima, Ken; Nishi, Toshio

    2006-01-01

    The invention of atomic force microscopy (AFM) enabled us to study the statistical properties of single polymer chains by a method called "nanofishing," which stretches a single polymer chain adsorbed on a substrate with its one end by picking it at the other end. A force-extension curve obtained for a single polystyrene chain in a Theta solvent (cyclohexane) shows good agreement with a worm-like chain model and, therefore, gives microscopic information about entropic elasticity. Furthermore, the nanofishing technique can be used for dynamic viscoelastic measurement of single polymer chains. An AFM cantilever is mechanically oscillated at its resonant frequency during the stretching process. This technique enables the estimation of quantitative and simultaneous elongation-dependent changes of stiffness and viscosity of a single chain with the use of a phenomenological model. In this study, the effect of solvent on viscosity in low extension regions reveals that the viscosity is attributed to monomer-solvent friction. Thus, static and dynamic nanofishing techniques are shown to give powerful experimental proofs for several basic questions in polymer physics. The techniques are expected to reveal hidden properties of polymer chains or polymer solutions by any types of macroscopic measurements in the future. PMID:17099889

  16. Single-particle study of protein assembly

    NASA Astrophysics Data System (ADS)

    Kiang, Ching-Hwa

    2001-10-01

    A study of protein assembly in solution with single-particle imaging and reconstruction techniques using cryoelectron microscopy is reported. The human glutamine synthetase enzyme, important in brain metabolism, and previously assumed to be assembled into a homogeneous quaternary structure, is found to be heterogeneous, with three oligomeric states that co-exist at room temperature. This result corrects an old structural and kinetic model determined by ensemble averaging techniques that assumed a homogeneous system. Unexpectedly fast protein dissociation kinetics results from a stabilized transition state.

  17. Synthetic protein interactions reveal a functional map of the cell

    PubMed Central

    Berry, Lisa K; Ólafsson, Guðjón; Ledesma-Fernández, Elena; Thorpe, Peter H

    2016-01-01

    To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001 PMID:27098839

  18. Photocurrent of a single photosynthetic protein

    NASA Astrophysics Data System (ADS)

    Gerster, Daniel; Reichert, Joachim; Bi, Hai; Barth, Johannes V.; Kaniber, Simone M.; Holleitner, Alexander W.; Visoly-Fisher, Iris; Sergani, Shlomi; Carmeli, Itai

    2012-10-01

    Photosynthesis is used by plants, algae and bacteria to convert solar energy into stable chemical energy. The initial stages of this process--where light is absorbed and energy and electrons are transferred--are mediated by reaction centres composed of chlorophyll and carotenoid complexes. It has been previously shown that single small molecules can be used as functional components in electric and optoelectronic circuits, but it has proved difficult to control and probe individual molecules for photovoltaic and photoelectrochemical applications. Here, we show that the photocurrent generated by a single photosynthetic protein--photosystem I--can be measured using a scanning near-field optical microscope set-up. One side of the protein is anchored to a gold surface that acts as an electrode, and the other is contacted by a gold-covered glass tip. The tip functions as both counter electrode and light source. A photocurrent of ~10 pA is recorded from the covalently bound single-protein junctions, which is in agreement with the internal electron transfer times of photosystem I.

  19. Single-cell chromatin accessibility reveals principles of regulatory variation

    PubMed Central

    Buenrostro, Jason D.; Wu, Beijing; Litzenburger, Ulrike M.; Ruff, Dave; Gonzales, Michael L.; Snyder, Michael P.; Chang, Howard Y.; Greenleaf, William J.

    2015-01-01

    Cell-to-cell variation is a universal feature of life that impacts a wide range of biological phenomena, from developmental plasticity1,2 to tumor heterogeneity3. While recent advances have improved our ability to document cellular phenotypic variation4–8 the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of cellular DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells via assay for transposase-accessible chromatin using sequencing (ATAC-seq). Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single-cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provides insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type specific accessibility variance across 8 cell types. Targeted perturbations of cell cycle or transcription factor signaling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome topological domains9 de novo, linking single-cell accessibility variation to three-dimensional genome organization. All together, single-cell analysis of DNA accessibility provides new insight into cellular variation of the “regulome.” PMID:26083756

  20. Single-cell chromatin accessibility reveals principles of regulatory variation.

    PubMed

    Buenrostro, Jason D; Wu, Beijing; Litzenburger, Ulrike M; Ruff, Dave; Gonzales, Michael L; Snyder, Michael P; Chang, Howard Y; Greenleaf, William J

    2015-07-23

    Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'.

  1. Molecular force modulation spectroscopy revealing the dynamic response of single bacteriorhodopsins.

    PubMed

    Janovjak, Harald; Müller, Daniel J; Humphris, Andrew D L

    2005-02-01

    Recent advances in atomic force microscopy allowed globular and membrane proteins to be mechanically unfolded on a single-molecule level. Presented is an extension to the existing force spectroscopy experiments. While unfolding single bacteriorhodopsins from native purple membranes, small oscillation amplitudes (6-9 nm) were supplied to the vertical displacement of the cantilever at a frequency of 3 kHz. The phase and amplitude response of the cantilever-protein system was converted to reveal the elastic (conservative) and viscous (dissipative) contributions to the unfolding process. The elastic response (stiffness) of the extended parts of the protein were in the range of a few tens pN/nm and could be well described by the derivative of the wormlike chain model. Discrete events in the viscous response coincided with the unfolding of single secondary structure elements and were in the range of 1 microNs/m. In addition, these force modulation spectroscopy experiments revealed novel mechanical unfolding intermediates of bacteriorhodopsin. We found that kinks result in a loss of unfolding cooperativity in transmembrane helices. Reconstructing force-distance spectra by the integration of amplitude-distance spectra verified their position, offering a novel approach to detect intermediates during the forced unfolding of single proteins. PMID:15574708

  2. Exploring one-state downhill protein folding in single molecules

    PubMed Central

    Liu, Jianwei; Campos, Luis A.; Cerminara, Michele; Wang, Xiang; Ramanathan, Ravishankar; English, Douglas S.; Muñoz, Victor

    2012-01-01

    A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (ca. 106 s-1) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out free-diffusion single-molecule FRET experiments with 50-μs resolution and maximal photoprotection using a recently developed Trolox-cysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRET efficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the one-state downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics. PMID:22184219

  3. Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states

    PubMed Central

    Schlau-Cohen, Gabriela S.; Wang, Quan; Southall, June; Cogdell, Richard J.; Moerner, W. E.

    2013-01-01

    Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities. PMID:23776245

  4. Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states.

    PubMed

    Schlau-Cohen, Gabriela S; Wang, Quan; Southall, June; Cogdell, Richard J; Moerner, W E

    2013-07-01

    Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.

  5. Single-cell protein from waste cellulose

    NASA Technical Reports Server (NTRS)

    Dunlap, C. E.; Callihan, C. D.

    1973-01-01

    The recycle, reuse, or reclamation of single cell protein from liquid and solid agricultural waste fibers by a fermentation process is reported. It is shown that cellulose comprises the bulk of the fibers at 50% to 55% of the dry weight of the refuse and that its biodegradability is of prime importance in the choice of a substrate. The application of sodium hydroxide followed by heat and pressure serves to de-polymerize and disrupt lignin structure while swelling the cellulose to increase water uptake and pore volume. Some of the lignin, hemi-celluloses, ash, and cellulose of the material is hydrolized and solubilized. Introduction of microorganisms to the substrate fibers mixed with nutrients produces continuous fermentation of cellulose for further protein extraction and purification.

  6. Experimental study of single protein mechanics and protein rates of unfolding

    NASA Astrophysics Data System (ADS)

    Hermans, Rodolfo I.

    This dissertation is a multidisciplinary approach to the understanding of the mechanical properties of proteins and their kinetics of unfolding, through the use of an atomic force microscope in force-clamp mode to perform single-molecule force spectroscopy. It begins with the development of a new experimental framework, which includes the design and construction of a new faster atomic force microscopy instrumentation, the definition of new bias-free experimental protocols, and the application of modern statistical tools for data analysis, all of these optimized for the use in force spectroscopy at the single molecule level. Through this new framework new insight was gained on the laws governing the mechanics of a protein in solution. The kinetics of unfolding of the protein ubiquitin were experimentally observed to feature a broad power-law distribution of unfolding rates, quality characteristic of glassy kinetics, which challenges the commonly accepted simplest single rate two-state model models for protein unfolding. Also, the transition between two highly stretched structural configurations of a protein was directly observed to be rate-limited, revealing the magnitude of the intrinsic friction limiting the speed of the first stage of protein folding. This document is presented with detailed technical and mathematical considerations with the intention to serve as handbook for the experimentalist interested in the study of polymers using force-clamp single molecule force spectroscopy.

  7. Kinetics of single DNA molecule denaturation by T4 Gene 32 protein

    NASA Astrophysics Data System (ADS)

    Pant, Kiran; Karpel, Richard L.; Williams, Mark C.

    2003-03-01

    Bacteriophage T4 gene 32 protein (32 protein) specifically binds single-stranded DNA, a property essential for its role in DNA replication, recombination, and repair. Although on a thermodynamic basis, single-stranded DNA binding proteins should lower the thermal melting temperature of double-stranded DNA (dsDNA), 32 protein does not. Using single molecule force spectroscopy, we show for the first time that 32 protein is capable of slowly destabilizing natural dsDNA. Direct measurements of single DNA molecule denaturation and renaturation kinetics in the presence of 32 protein and its proteolytic fragments reveal three types of kinetic behavior, attributable to specific protein structural domains, which regulate 32 protein's helix-destabilizing capabilities. This regulation is potentially biologically significant because uncontrolled helix-destabilization would be lethal to the cell. We also obtain equilibrium measurements of the DNA helix-coil transition free energy in the presence of these proteins for the first time.

  8. Temperature-cycle microscopy reveals single-molecule conformational heterogeneity.

    PubMed

    Yuan, Haifeng; Gaiduk, Alexander; Siekierzycka, Joanna R; Fujiyoshi, Satoru; Matsushita, Michio; Nettels, Daniel; Schuler, Benjamin; Seidel, Claus A M; Orrit, Michel

    2015-03-01

    Our previous temperature-cycle study reported FRET transitions between different states on FRET-labeled polyprolines [Yuan et al., PCCP, 2011, 13, 1762]. The conformational origin of such transitions, however, was left open. In this work, we apply temperature-cycle microscopy of single FRET-labeled polyproline and dsDNA molecules and compare their responses to resolve the conformational origin of different FRET states. We observe different steady-state FRET distributions and different temperature-cycle responses in the two samples. Our temperature-cycle results on single molecules resemble the results in steady-state measurements but reveal a dark state which could not be observed otherwise. By comparing the timescales and probabilities of different FRET states in temperature-cycle traces, we assign the conformational heterogeneity reflected by different FRET states to linker dynamics, dye-chain and dye-dye interactions. The dark state and low-FRET state are likely due to dye-dye interactions at short separations.

  9. Network analysis reveals common host protein/s modulating pathogenesis of neurotropic viruses

    PubMed Central

    Ghosh, Sourish; Mukherjee, Sriparna; Sengupta, Nabonita; Roy, Arunava; Dey, Dhritiman; Chakraborty, Surajit; Chattopadhyay, Dhrubajyoti; Banerjee, Arpan; Basu, Anirban

    2016-01-01

    Network analysis through graph theory provides a quantitative approach to characterize specific proteins and their constituent assemblies that underlie host-pathogen interactions. In the present study, graph theory was used to analyze the interactome designed out of 50 differentially expressing proteins from proteomic analysis of Chandipura Virus (CHPV, Family: Rhabdoviridae) infected mouse brain tissue to identify the primary candidates for intervention. Using the measure of degree centrality, that quantifies the connectedness of a single protein within a milieu of several other interacting proteins, DJ-1 was selected for further molecular validation. To elucidate the generality of DJ-1’s role in propagating infection its role was also monitored in another RNA virus, Japanese Encephalitis Virus (JEV, Family: Flaviviridae) infection. Concurrently, DJ-1 got over-expressed in response to reactive oxygen species (ROS) generation following viral infection which in the early phase of infection migrated to mitochondria to remove dysfunctional mitochondria through the process of mitophagy. DJ-1 was also observed to modulate the viral replication and interferon responses along with low-density lipoprotein (LDL) receptor expression in neurons. Collectively these evidences reveal a comprehensive role for DJ-1 in neurotropic virus infection in the brain. PMID:27581498

  10. Network analysis reveals common host protein/s modulating pathogenesis of neurotropic viruses.

    PubMed

    Ghosh, Sourish; Mukherjee, Sriparna; Sengupta, Nabonita; Roy, Arunava; Dey, Dhritiman; Chakraborty, Surajit; Chattopadhyay, Dhrubajyoti; Banerjee, Arpan; Basu, Anirban

    2016-01-01

    Network analysis through graph theory provides a quantitative approach to characterize specific proteins and their constituent assemblies that underlie host-pathogen interactions. In the present study, graph theory was used to analyze the interactome designed out of 50 differentially expressing proteins from proteomic analysis of Chandipura Virus (CHPV, Family: Rhabdoviridae) infected mouse brain tissue to identify the primary candidates for intervention. Using the measure of degree centrality, that quantifies the connectedness of a single protein within a milieu of several other interacting proteins, DJ-1 was selected for further molecular validation. To elucidate the generality of DJ-1's role in propagating infection its role was also monitored in another RNA virus, Japanese Encephalitis Virus (JEV, Family: Flaviviridae) infection. Concurrently, DJ-1 got over-expressed in response to reactive oxygen species (ROS) generation following viral infection which in the early phase of infection migrated to mitochondria to remove dysfunctional mitochondria through the process of mitophagy. DJ-1 was also observed to modulate the viral replication and interferon responses along with low-density lipoprotein (LDL) receptor expression in neurons. Collectively these evidences reveal a comprehensive role for DJ-1 in neurotropic virus infection in the brain. PMID:27581498

  11. Network Clustering Revealed the Systemic Alterations of Mitochondrial Protein Expression

    PubMed Central

    Koo, Hyun-Jung; Park, Wook-Ha; Yang, Jae-Seong; Yu, Myeong-Hee; Kim, Sanguk; Pak, Youngmi Kim

    2011-01-01

    The mitochondrial protein repertoire varies depending on the cellular state. Protein component modifications caused by mitochondrial DNA (mtDNA) depletion are related to a wide range of human diseases; however, little is known about how nuclear-encoded mitochondrial proteins (mt proteome) changes under such dysfunctional states. In this study, we investigated the systemic alterations of mtDNA-depleted (ρ0) mitochondria by using network analysis of gene expression data. By modularizing the quantified proteomics data into protein functional networks, systemic properties of mitochondrial dysfunction were analyzed. We discovered that up-regulated and down-regulated proteins were organized into two predominant subnetworks that exhibited distinct biological processes. The down-regulated network modules are involved in typical mitochondrial functions, while up-regulated proteins are responsible for mtDNA repair and regulation of mt protein expression and transport. Furthermore, comparisons of proteome and transcriptome data revealed that ρ0 cells attempted to compensate for mtDNA depletion by modulating the coordinated expression/transport of mt proteins. Our results demonstrate that mt protein composition changed to remodel the functional organization of mitochondrial protein networks in response to dysfunctional cellular states. Human mt protein functional networks provide a framework for understanding how cells respond to mitochondrial dysfunctions. PMID:21738461

  12. Single cell protein as an occupational hazard.

    PubMed Central

    Ekenvall, L; Dölling, B; Göthe, C J; Ebbinghaus, L; von Stedingk, L V; Wasserman, J

    1983-01-01

    Single cell protein (SCP) intended for animal feed purposes was produced in a pilot plant. The SCP consisted of Methylomonas methanolica, a pseudomonas species which is an obligate methanol user. The SCP was cultured in fermenters and later dewatered and dried in a spray-drier. Seven of eight research workers had febrile reactions 6-12 hours after exposure to SCP dust. All workers had high titres of IgG and IgM antibodies against the pseudomonas species as measured with indirect ELISA and passive haemagglutination techniques. The mechanism behind the febrile reaction is judged to be a non-immunological reaction caused by endotoxins. By increasing the particle size of the SCP through using different drying procedures, a product which generated less dust was obtained. PMID:6830720

  13. Single-molecule mechanics of protein-labelled DNA handles.

    PubMed

    Jadhav, Vivek S; Brüggemann, Dorothea; Wruck, Florian; Hegner, Martin

    2016-01-01

    DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA-protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot-streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein-DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time

  14. Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis.

    PubMed

    Shin, Jaehoon; Berg, Daniel A; Zhu, Yunhua; Shin, Joseph Y; Song, Juan; Bonaguidi, Michael A; Enikolopov, Grigori; Nauen, David W; Christian, Kimberly M; Ming, Guo-li; Song, Hongjun

    2015-09-01

    Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes.

  15. Single-molecule imaging reveals a collapsed conformational state for DNA-bound cohesin

    PubMed Central

    Stigler, Johannes; Çamdere, Gamze Ö.; Koshland, Douglas E.; Greene, Eric C.

    2016-01-01

    Cohesin is essential for the hierarchical organization of the eukaryotic genome and plays key roles in many aspects of chromosome biology. The conformation of cohesin bound to DNA remains poorly defined, leaving crucial gaps in our understanding of how cohesin fulfills its biological functions. Here we use single molecule microscopy to directly observe the dynamic and functional characteristics of cohesin bound to DNA. We show that cohesin can undergo rapid one-dimensional (1D) diffusion along DNA, but individual nucleosomes, nucleosome arrays, and other protein obstacles significantly restrict its mobility. We further demonstrate that DNA motor proteins can readily push cohesin along DNA, but they cannot pass through the interior of the cohesin ring. Together, our results reveal that DNA-bound cohesin has a central pore that is substantially smaller than anticipated. These findings have direct implications for understanding how cohesin and other SMC proteins interact with and distribute along chromatin. PMID:27117417

  16. Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging

    PubMed Central

    Yaginuma, Hideyuki; Kawai, Shinnosuke; Tabata, Kazuhito V.; Tomiyama, Keisuke; Kakizuka, Akira; Komatsuzaki, Tamiki; Noji, Hiroyuki; Imamura, Hiromi

    2014-01-01

    Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions. Quantitative measurement of the absolute ATP concentration in individual cells has not been achieved because of the lack of reliable methods. In this study, we developed a new genetically-encoded ratiometric fluorescent ATP indicator “QUEEN”, which is composed of a single circularly-permuted fluorescent protein and a bacterial ATP binding protein. Unlike previous FRET-based indicators, QUEEN was apparently insensitive to bacteria growth rate changes. Importantly, intracellular ATP concentrations of numbers of bacterial cells calculated from QUEEN fluorescence were almost equal to those from firefly luciferase assay. Thus, QUEEN is suitable for quantifying the absolute ATP concentration inside bacteria cells. Finally, we found that, even for a genetically-identical Escherichia coli cell population, absolute concentrations of intracellular ATP were significantly diverse between individual cells from the same culture, by imaging QUEEN signals from single cells. PMID:25283467

  17. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

    PubMed Central

    Shankaran, Mahalakshmi; King, Chelsea L.; Angel, Thomas E.; Holmes, William E.; Li, Kelvin W.; Colangelo, Marc; Price, John C.; Turner, Scott M.; Bell, Christopher; Hamilton, Karyn L.; Miller, Benjamin F.; Hellerstein, Marc K.

    2015-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  18. Single-molecule mechanics of protein-labelled DNA handles

    PubMed Central

    Wruck, Florian

    2016-01-01

    Summary DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition

  19. Probing Protein Channel Dynamics At The Single Molecule Level.

    NASA Astrophysics Data System (ADS)

    Lee, M. Ann; Dunn, Robert C.

    1997-03-01

    It would be difficult to overstate the importance played by protein ion channels in cellular function. These macromolecular pores allow the passage of ions across the cellular membrane and play indispensable roles in all aspects of neurophysiology. While the patch-clamp technique continues to provide elegant descriptions of the kinetic processes involved in ion channel gating, the associated conformational changes remain a mystery. We are using the spectroscopic capabilities and single molecule fluorescence sensitivity of near-field scanning optical microscopy (NSOM) to probe these dynamics at the single channel level. Using a newly developed cantilevered NSOM probe capable of probing soft biological samples with single molecule fluorescence sensitivity, we have begun mapping the location of single NMDA receptors in intact rat cortical neurons with <100 nm spatial resolution. We will also present recent results exploring the conformational changes accompanying activation of nuclear pore channels located in the nuclear membrane of Xenopus oocytes. Our recent NSOM and AFM measurements on single nuclear pore complexes reveal large conformational changes taking place upon activation, providing rich, new molecular level details of channel function.

  20. Proteomics Reveals Novel Drosophila Seminal Fluid Proteins Transferred at Mating

    PubMed Central

    Findlay, Geoffrey D; Yi, Xianhua; MacCoss, Michael J; Swanson, Willie J

    2008-01-01

    Across diverse taxa, seminal fluid proteins (Sfps) transferred at mating affect the reproductive success of both sexes. Such reproductive proteins often evolve under positive selection between species; because of this rapid divergence, Sfps are hypothesized to play a role in speciation by contributing to reproductive isolation between populations. In Drosophila, individual Sfps have been characterized and are known to alter male sperm competitive ability and female post-mating behavior, but a proteomic-scale view of the transferred Sfps has been missing. Here we describe a novel proteomic method that uses whole-organism isotopic labeling to detect transferred Sfps in mated female D. melanogaster. We identified 63 proteins, which were previously unknown to function in reproduction, and confirmed the transfer of dozens of predicted Sfps. Relative quantification of protein abundance revealed that several of these novel Sfps are abundant in seminal fluid. Positive selection and tandem gene duplication are the prevailing forces of Sfp evolution, and comparative proteomics with additional species revealed lineage-specific changes in seminal fluid content. We also report a proteomic-based gene discovery method that uncovered 19 previously unannotated genes in D. melanogaster. Our results demonstrate an experimental method to identify transferred proteins in any system that is amenable to isotopic labeling, and they underscore the power of combining proteomic and evolutionary analyses to shed light on the complex process of Drosophila reproduction. PMID:18666829

  1. Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content

    PubMed Central

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit; Saari, Heikki; Ibañez, Elisa Lazaro; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2015-01-01

    Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level. PMID:26649679

  2. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins*

    PubMed Central

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina; Cruz-Zaragoza, Luis Daniel; Garcia, Esther; Clausen, Mathias P.; Schliebs, Wolfgang; Erdmann, Ralf; Eggeling, Christian

    2016-01-01

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future. PMID:27311714

  3. Silk protein aggregation kinetics revealed by Rheo-IR.

    PubMed

    Boulet-Audet, Maxime; Terry, Ann E; Vollrath, Fritz; Holland, Chris

    2014-02-01

    The remarkable mechanical properties of silk fibres stem from a multi-scale hierarchical structure created when an aqueous protein "melt" is converted to an insoluble solid via flow. To directly relate a silk protein's structure and function in response to flow, we present the first application of a Rheo-IR platform, which couples cone and plate rheology with attenuated total reflectance infrared spectroscopy. This technique provides a new window into silk processing by linking shear thinning to an increase in molecular alignment, with shear thickening affecting changes in the silk protein's secondary structure. Additionally, compared to other static characterization methods for silk, Rheo-IR proved particularly useful at revealing the intrinsic difference between natural (native) and reconstituted silk feedstocks. Hence Rheo-IR offers important novel insights into natural silk processing. This has intrinsic academic merit, but it might also be useful when designing reconstituted silk analogues alongside other polymeric systems, whether natural or synthetic.

  4. Single Molecule and Collective Dynamics of Motor Protein Coupled with Mechano-Sensitive Chemical Reaction

    NASA Astrophysics Data System (ADS)

    Iwaki, Mitsuhiro; Marcucci, Lorenzo; Togashi, Yuichi; Yanagida, Toshio

    2013-12-01

    Motor proteins such as myosin and kinesin hydrolyze ATP into ADP and Pi to convert chemical energy into mechanical work. This resultsin various motile processes like muscle contraction, vesicle transport and cell division. Recent single molecule experiments have revealed that external load applied to these motor proteins perturb not only the mechanical motion, but the ATP hydrolysis cycle as well, making these molecules mechano-enzymes. Here, we describe our single molecule detection techniques to reveal the mechano-enzymatic properties of myosin and introduce recent progress from both experimental and theoretical approaches at the single- and multiple-molecule level.

  5. Membrane protein properties revealed through data-rich electrostatics calculations

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.; Grabe, Michael

    2015-01-01

    SUMMARY The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem including: full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane induced pKa shifts, calculation of non-polar energies, and command-line scripting for large scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane potentially revealing interesting functional information. PMID:26118532

  6. Single-molecule studies of kinesin family motor proteins

    NASA Astrophysics Data System (ADS)

    Fordyce, Polly

    Kinesin family motor proteins drive many essential cellular processes, including cargo transport and mitotic spindle assembly and regulation. They accomplish these tasks by converting the chemical energy released from the hydrolysis of adenosine triphosphate (ATP) directly into mechanical motion along microtubules in cells. Optical traps allow us to track and apply force to individual motor proteins, and have already revealed many details of the movement of conventional kinesin, although the precise mechanism by which chemical energy is converted into mechanical motion is unclear. Other kinesin family members remain largely uncharacterized. This dissertation details the use of a novel optical-trapping assay to study Eg5, a Kinesin-5 family member involved in both spindle assembly and pole separation during mitosis. We demonstrate that individual Eg5 dimers are relatively slow and force-insensitive motors that take about 8 steps, on average, before detaching from the microtubule. Key differences in processivity and force-response between Eg5 and conventional kinesin suggest ways in which the two motors might have evolved to perform very different tasks in cells. This dissertation also details efforts to unravel how chemical energy is converted into mechanical motion by simultaneously measuring mechanical transitions (with an optical trap) and nucleotide binding and release (with single-molecule fluorescence) for individual conventional kinesin motors. We constructed a combined instrument, demonstrated its capabilities by unzipping fluorescently-labeled DNA duplexes, and used this instrument to record the motion of individual conventional kinesin motors powered by the hydrolysis of fluorescent nucleotides. Preliminary data reveal the challenges inherent in such measurements and guide proposals for future experimental approaches. Finally, this dissertation includes several chapters intended to serve as practical guides to understanding, constructing, and maintaining

  7. Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae

    PubMed Central

    Krishnamoorthy, Archana

    2015-01-01

    Luciferase is a useful, noninvasive reporter of gene regulation that can be continuously monitored over long periods of time; however, its use is problematic in fast-growing microbes like bacteria and yeast because rapidly changing cell numbers and metabolic states also influence bioluminescence, thereby confounding the reporter's signal. Here we show that these problems can be overcome in the budding yeast Saccharomyces cerevisiae by simultaneously monitoring bioluminescence from two different colors of beetle luciferase, where one color (green) reports activity of a gene of interest, while a second color (red) is stably expressed and used to continuously normalize green bioluminescence for fluctuations in signal intensity that are unrelated to gene regulation. We use this dual-luciferase strategy in conjunction with a light-inducible promoter system to test whether different phases of yeast respiratory oscillations are more suitable for heterologous protein production than others. By using pulses of light to activate production of a green luciferase while normalizing signal variation to a red luciferase, we show that the early reductive phase of the yeast metabolic cycle produces more luciferase than other phases. PMID:26162874

  8. RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation.

    PubMed

    Yu, DongMei; Zhang, Chao; Qin, PeiWu; Cornish, Peter V; Xu, Dong

    2014-11-01

    Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.

  9. Profiling of urinary proteins in Karan Fries cows reveals more than 1550 proteins.

    PubMed

    Bathla, Shveta; Rawat, Preeti; Baithalu, Rubina; Yadav, Munna Lal; Naru, Jasmine; Tiwari, Anurag; Kumar, Sudarshan; Balhara, Ashok K; Singh, Surender; Chaudhary, Suman; Kumar, Rajesh; Lotfan, Masoud; Behare, Pradip; Phulia, Sushil K; Mohanty, Tushar K; Kaushik, Jai K; Nallapeta, Shivramaiah; Singh, Inderjeet; Ambatipudi, Srinivas K; Mohanty, Ashok K

    2015-09-01

    Urine is a non-invasive source of biological fluid, which reflects the physiological status of the mammals. We have profiled the cow urinary proteome and analyzed its functional significance. The urine collected from three healthy cows was concentrated by diafiltration (DF) followed by protein extraction using three methods, namely methanol, acetone, and ammonium sulphate (AS) precipitation and Proteo Spin urine concentration kit (PS). The quality of the protein was assessed by two-dimensional gel electrophoresis (2DE). In-gel digestion method revealed more proteins (1191) in comparison to in-solution digestion method (541). Collectively, 938, 606 and 444 proteins were identified in LC-MS/MS after in-gel and in-solution tryptic digestion of proteins prepared by AS, PS and DF methods, respectively resulting in identification of a total of 1564 proteins. Gene ontology (GO) using Panther7.0 grouped the majority of the proteins into cytoplasmic (location), catalytic activity (function), and metabolism (biological processes), while Cytoscape grouped proteins into complement and coagulation cascades; protease inhibitor activity and wound healing. Functional significance of few selected proteins seems to play important role in their physiology. Comparative analysis with human urine revealed 315 overlapping proteins. This study reports for the first time evidence of more than 1550 proteins in urine of healthy cow donors. This article is part of a Special Issue entitled: Proteomics in India.

  10. Single-cell transcriptomics reveals receptor transformations during olfactory neurogenesis.

    PubMed

    Hanchate, Naresh K; Kondoh, Kunio; Lu, Zhonghua; Kuang, Donghui; Ye, Xiaolan; Qiu, Xiaojie; Pachter, Lior; Trapnell, Cole; Buck, Linda B

    2015-12-01

    The sense of smell allows chemicals to be perceived as diverse scents. We used single-neuron RNA sequencing to explore the developmental mechanisms that shape this ability as nasal olfactory neurons mature in mice. Most mature neurons expressed only one of the ~1000 odorant receptor genes (Olfrs) available, and at a high level. However, many immature neurons expressed low levels of multiple Olfrs. Coexpressed Olfrs localized to overlapping zones of the nasal epithelium, suggesting regional biases, but not to single genomic loci. A single immature neuron could express Olfrs from up to seven different chromosomes. The mature state in which expression of Olfr genes is restricted to one per neuron emerges over a developmental progression that appears to be independent of neuronal activity involving sensory transduction molecules.

  11. Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells.

    PubMed

    Pichon, Xavier; Bastide, Amandine; Safieddine, Adham; Chouaib, Racha; Samacoits, Aubin; Basyuk, Eugenia; Peter, Marion; Mueller, Florian; Bertrand, Edouard

    2016-09-12

    Translation is an essential step in gene expression. In this study, we used an improved SunTag system to label nascent proteins and image translation of single messenger ribonucleoproteins (mRNPs) in human cells. Using a dedicated reporter RNA, we observe that translation of single mRNPs stochastically turns on and off while they diffuse through the cytoplasm. We further measure a ribosome density of 1.3 per kilobase and an elongation rate of 13-18 amino acids per second. Tagging the endogenous POLR2A gene revealed similar elongation rates and ribosomal densities and that nearly all messenger RNAs (mRNAs) are engaged in translation. Remarkably, tagging of the heavy chain of dynein 1 (DYNC1H1) shows this mRNA accumulates in foci containing three to seven RNA molecules. These foci are translation sites and thus represent specialized translation factories. We also observe that DYNC1H1 polysomes are actively transported by motors, which may deliver the mature protein at appropriate cellular locations. The SunTag should be broadly applicable to study translational regulation in live single cells. PMID:27597760

  12. Single muscle fiber proteomics reveals unexpected mitochondrial specialization

    PubMed Central

    Murgia, Marta; Nagaraj, Nagarjuna; Deshmukh, Atul S; Zeiler, Marlis; Cancellara, Pasqua; Moretti, Irene; Reggiani, Carlo; Schiaffino, Stefano; Mann, Matthias

    2015-01-01

    Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity. PMID:25643707

  13. Metal-enhanced fluorescence of single green fluorescent protein (GFP)

    SciTech Connect

    Fu Yi; Zhang Jian; Lakowicz, Joseph R.

    2008-11-28

    The green fluorescent protein (GFP) has emerged as a powerful reporter molecule for monitoring gene expression, protein localization, and protein-protein interaction. However, the detection of low concentrations of GFPs is limited by the weakness of the fluorescent signal and the low photostability. In this report, we observed the proximity of single GFPs to metallic silver nanoparticles increases its fluorescence intensity approximately 6-fold and decreases the decay time. Single protein molecules on the silvered surfaces emitted 10-fold more photons as compared to glass prior to photobleaching. The photostability of single GFP has increased to some extent. Accordingly, we observed longer duration time and suppressed blinking. The single-molecule lifetime histograms indicate the relatively heterogeneous distributions of protein mutants inside the structure.

  14. Structure of mega-hemocyanin reveals protein origami in snails.

    PubMed

    Gatsogiannis, Christos; Hofnagel, Oliver; Markl, Jürgen; Raunser, Stefan

    2015-01-01

    Mega-hemocyanin is a 13.5 MDa oxygen transporter found in the hemolymph of some snails. Similar to typical gastropod hemocyanins, it is composed of 400 kDa building blocks but has additional 550 kDa subunits. Together, they form a large, completely filled cylinder. The structural basis for this highly complex protein packing is not known so far. Here, we report the electron cryomicroscopy (cryo-EM) structure of mega-hemocyanin complexes from two different snail species. The structures reveal that mega-hemocyanin is composed of flexible building blocks that differ in their conformation, but not in their primary structure. Like a protein origami, these flexible blocks are optimally packed, implementing different local symmetries and pseudosymmetries. A comparison between the two structures suggests a surprisingly simple evolutionary mechanism leading to these large oxygen transporters. PMID:25482543

  15. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    SciTech Connect

    Feng, Yingang; Song, Xiaxia; Lin, Jinzhong; Xuan, Jinsong; Cui, Qiu; Wang, Jinfeng

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  16. Semisynthetic protein nanoreactor for single-molecule chemistry

    PubMed Central

    Lee, Joongoo; Bayley, Hagan

    2015-01-01

    The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach. PMID:26504203

  17. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics.

    PubMed

    Tasic, Bosiljka; Menon, Vilas; Nguyen, Thuc Nghi; Kim, Tae Kyung; Jarsky, Tim; Yao, Zizhen; Levi, Boaz; Gray, Lucas T; Sorensen, Staci A; Dolbeare, Tim; Bertagnolli, Darren; Goldy, Jeff; Shapovalova, Nadiya; Parry, Sheana; Lee, Changkyu; Smith, Kimberly; Bernard, Amy; Madisen, Linda; Sunkin, Susan M; Hawrylycz, Michael; Koch, Christof; Zeng, Hongkui

    2016-02-01

    Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. We constructed a cellular taxonomy of one cortical region, primary visual cortex, in adult mice on the basis of single-cell RNA sequencing. We identified 49 transcriptomic cell types, including 23 GABAergic, 19 glutamatergic and 7 non-neuronal types. We also analyzed cell type-specific mRNA processing and characterized genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we found that some of our transcriptomic cell types displayed specific and differential electrophysiological and axon projection properties, thereby confirming that the single-cell transcriptomic signatures can be associated with specific cellular properties.

  18. Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

    PubMed

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-03-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  19. Revealing different aggregation pathways of amyloidogenic proteins by ultrasound velocimetry.

    PubMed

    Smirnovas, Vytautas; Winter, Roland

    2008-04-15

    In this work, we performed a detailed thermodynamic study, including ultrasound velocimetry, densimetry, calorimetry, and FTIR spectroscopy, of an aggregation-prone protein (insulin) under different salt-screening conditions to gain a deeper insight into the scenario of physicochemical events during its temperature-induced unfolding and aggregation reactions. Differences in aggregation and fibrillization pathways are reflected in changes of the partial molar volume, the coefficients of thermal expansion and compressibility, and the infrared spectral properties of the protein. Combining all experimental data allows setting up a scheme for the temperature-dependent insulin aggregation reaction in the presence and absence of NaCl. As revealed by complementary atomic force microscopy studies, under charge-screening conditions, a process involving structural reorganization, ripening, and formation of more compact nuclei from amorphous oligomers is involved in the formation of mature fibrillar morphologies. In this work, our focus was to put forward a comprehensive discussion of the use of ultrasound velocimetry in disentangling different aggregation pathways. In fact, ultrasound velocimetry proved to be very sensitive to changes in aggregation pathway, highlighting the importance of density and compressibility changes in the different aggregation and fibrillization reactions of the protein.

  20. Protein Laboratories in Single Location | Poster

    Cancer.gov

    By Andrew Stephen, Timothy Veenstra, and Gordon Whiteley, Guest Writers, and Ken Michaels, Staff Writer The Laboratory of Proteomics and Analytical Technologies (LPAT), Antibody Characterization Laboratory (ACL), and Protein Chemistry Laboratory (PCL), previously located on different floors or in different buildings, are now together on the first floor of C wing in the ATRF.

  1. Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis.

    PubMed

    Shin, Jaehoon; Berg, Daniel A; Zhu, Yunhua; Shin, Joseph Y; Song, Juan; Bonaguidi, Michael A; Enikolopov, Grigori; Nauen, David W; Christian, Kimberly M; Ming, Guo-li; Song, Hongjun

    2015-09-01

    Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes. PMID:26299571

  2. Conformational dynamics of nonsynonymous variants at protein interfaces reveals disease association.

    PubMed

    Butler, Brandon M; Gerek, Z Nevin; Kumar, Sudhir; Ozkan, S Banu

    2015-03-01

    Recent studies have shown that the protein interface sites between individual monomeric units in biological assemblies are enriched in disease-associated non-synonymous single nucleotide variants (nsSNVs). To elucidate the mechanistic underpinning of this observation, we investigated the conformational dynamic properties of protein interface sites through a site-specific structural dynamic flexibility metric (dfi) for 333 multimeric protein assemblies. dfi measures the dynamic resilience of a single residue to perturbations that occurred in the rest of the protein structure and identifies sites contributing the most to functionally critical dynamics. Analysis of dfi profiles of over a thousand positions harboring variation revealed that amino acid residues at interfaces have lower average dfi (31%) than those present at non-interfaces (50%), which means that protein interfaces have less dynamic flexibility. Interestingly, interface sites with disease-associated nsSNVs have significantly lower average dfi (23%) as compared to those of neutral nsSNVs (42%), which directly relates structural dynamics to functional importance. We found that less conserved interface positions show much lower dfi for disease nsSNVs as compared to neutral nsSNVs. In this case, dfi is better as compared to the accessible surface area metric, which is based on the static protein structure. Overall, our proteome-wide conformational dynamic analysis indicates that certain interface sites play a critical role in functionally related dynamics (i.e., those with low dfi values), therefore mutations at those sites are more likely to be associated with disease.

  3. Single-cell dynamics reveals sustained growth during diauxic shifts.

    PubMed

    Boulineau, Sarah; Tostevin, Filipe; Kiviet, Daniel J; ten Wolde, Pieter Rein; Nghe, Philippe; Tans, Sander J

    2013-01-01

    Stochasticity in gene regulation has been characterized extensively, but how it affects cellular growth and fitness is less clear. We study the growth of E. coli cells as they shift from glucose to lactose metabolism, which is characterized by an obligatory growth arrest in bulk experiments that is termed the lag phase. Here, we follow the growth dynamics of individual cells at minute-resolution using a single-cell assay in a microfluidic device during this shift, while also monitoring lac expression. Mirroring the bulk results, the majority of cells displays a growth arrest upon glucose exhaustion, and resume when triggered by stochastic lac expression events. However, a significant fraction of cells maintains a high rate of elongation and displays no detectable growth lag during the shift. This ability to suppress the growth lag should provide important selective advantages when nutrients are scarce. Trajectories of individual cells display a highly non-linear relation between lac expression and growth, with only a fraction of fully induced levels being sufficient for achieving near maximal growth. A stochastic molecular model together with measured dependencies between nutrient concentration, lac expression level, and growth accurately reproduces the observed switching distributions. The results show that a growth arrest is not obligatory in the classic diauxic shift, and underscore that regulatory stochasticity ought to be considered in terms of its impact on growth and survival. PMID:23637881

  4. Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors

    PubMed Central

    Midde, Krishna K.; Aznar, Nicolas; Laederich, Melanie B.; Ma, Gary S.; Kunkel, Maya T.; Newton, Alexandra C.; Ghosh, Pradipta

    2015-01-01

    Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK–GIV–Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states. PMID:25713130

  5. Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors.

    PubMed

    Midde, Krishna K; Aznar, Nicolas; Laederich, Melanie B; Ma, Gary S; Kunkel, Maya T; Newton, Alexandra C; Ghosh, Pradipta

    2015-03-01

    Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK-GIV-Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states.

  6. Single-cell genomics reveals organismal interactions in uncultivated marine protists.

    PubMed

    Yoon, Hwan Su; Price, Dana C; Stepanauskas, Ramunas; Rajah, Veeran D; Sieracki, Michael E; Wilson, William H; Yang, Eun Chan; Duffy, Siobain; Bhattacharya, Debashish

    2011-05-01

    Whole-genome shotgun sequence data from three individual cells isolated from seawater, followed by analysis of ribosomal DNA, indicated that the cells represented three divergent clades of picobiliphytes. In contrast with the recent description of this phylum, we found no evidence of plastid DNA nor of nuclear-encoded plastid-targeted proteins, which suggests that these picobiliphytes are heterotrophs. Genome data from one cell were dominated by sequences from a widespread single-stranded DNA virus. This virus was absent from the other two cells, both of which contained non-eukaryote DNA derived from marine Bacteroidetes and large DNA viruses. By using shotgun sequencing of uncultured marine picobiliphytes, we revealed the distinct interactions of individual cells.

  7. Analysis of protein transport in the Brassica oleracea vasculature reveals protein-specific destinations.

    PubMed

    Niu, Chenxing; Anstead, James; Verchot, Jeanmarie

    2012-03-01

    We investigated the vascular transport properties of exogenously applied proteins to Brassica oleracea plants and compared their delivery to various aerial parts of the plant with carboxy fluorescein (CF) dye. We identified unique properties for each protein. Alexafluor-tagged bovine serum albumin (Alexa-BSA) and Alexafluor-tagged Histone H1 (Alexa-Histone) moved slower than CF dye throughout the plant. Interestingly, Alexa-Histone was retained in the phloem and phloem parenchyma while Alexa-BSA moved into the apoplast. One possibility is that Alexa-Histone sufficiently resembles plant endogenous proteins and is retained in the vascular stream, while Alexa-BSA is exported from the cell as a foreign protein. Both proteins diffuse from the leaf veins into the leaf lamina. Alexa-BSA accumulated in the leaf epidermis while Alexa-Histone accumulated mainly in the mesophyll layers. Fluorescein-tagged hepatitis C virus core protein (fluorescein-HCV) was also delivered to B. oleracea plants and is larger than Alexa-BSA. This protein moves more rapidly than BSA through the plant and was restricted to the leaf veins. Fluorescein-HCV failed to unload to the leaf lamina. These combined data suggest that there is not a single default pathway for the vascular transfer of exogenous proteins in B. oleracea plants. Specific protein properties appear to determine their destination and transport properties within the phloem. PMID:22476467

  8. RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome.

    PubMed

    Silverman, Ian M; Li, Fan; Alexander, Anissa; Goff, Loyal; Trapnell, Cole; Rinn, John L; Gregory, Brian D

    2014-01-07

    Although numerous approaches have been developed to map RNA-binding sites of individual RNA-binding proteins (RBPs), few methods exist that allow assessment of global RBP-RNA interactions. Here, we describe PIP-seq, a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply PIP-seq to the HeLa transcriptome and compare binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP-binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites.

  9. Homogeneous vs heterogeneous polymerization catalysis revealed by single-particle fluorescence microscopy.

    PubMed

    Esfandiari, N Melody; Blum, Suzanne A

    2011-11-16

    A high-sensitivity and high-resolution single-particle fluorescence microscopy technique differentiated between homogeneous and heterogeneous metathesis polymerization catalysis by imaging the location of the early stages of polymerization. By imaging single polymers and single crystals of Grubbs II, polymerization catalysis was revealed to be solely homogeneous rather than heterogeneous or both.

  10. Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation

    PubMed Central

    2016-01-01

    The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo. PMID:26751094

  11. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    PubMed

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

  12. Membrane protein structures without crystals, by single particle electron cryomicroscopy

    PubMed Central

    Vinothkumar, Kutti R

    2015-01-01

    It is an exciting period in membrane protein structural biology with a number of medically important protein structures determined at a rapid pace. However, two major hurdles still remain in the structural biology of membrane proteins. One is the inability to obtain large amounts of protein for crystallization and the other is the failure to get well-diffracting crystals. With single particle electron cryomicroscopy, both these problems can be overcome and high-resolution structures of membrane proteins and other labile protein complexes can be obtained with very little protein and without the need for crystals. In this review, I highlight recent advances in electron microscopy, detectors and software, which have allowed determination of medium to high-resolution structures of membrane proteins and complexes that have been difficult to study by other structural biological techniques. PMID:26435463

  13. Single-Molecule Nanocatalysis Reveals Catalytic Activation Energy of Single Nanocatalysts.

    PubMed

    Chen, Tao; Zhang, Yuwei; Xu, Weilin

    2016-09-28

    By monitoring the temperature-dependent catalytic activity of single Au nanocatalysts for a fluorogenic reaction, we derive the activation energies via multiple methods for two sequential catalytic steps (product formation and dissociation) on single nanocatalysts. The wide distributions of activation energies across multiple individual nanocatalysts indicate a huge static heterogeneity among the individual nanocatalysts. The compensation effect and isokinetic relationship of catalytic reactions are observed at the single particle level. This study exemplifies another function of single-molecule nanocatalysis and improves our understanding of heterogeneous catalysis.

  14. Probing Protein Fluctuations, Folding and Misfolding at Single-molecule Resolution

    NASA Astrophysics Data System (ADS)

    Deniz, Ashok

    2010-03-01

    The conformational fluctuations and folding of proteins are key for their function in cells and organisms. Conversely, misfolding and aggregation can cause disease, although amyloids with functional significance are also being identified. To better understand these aspects of protein biophysics, we utilize single-molecule fluorescence and complementary methods to directly study complex protein dynamics, structural distributions, and conformational transitions. In one example, we used these methods to investigate disorder and disorder-to-order transitions in intrinsically disordered proteins (IDPs). IDPs are an interesting class of proteins which are relatively unstructured in isolation, but can often fold by interacting with binding partners. These complex systems are increasingly found to play major roles in biology and disease. In one case, we used a combination of single-molecule FRET (smFRET), coincidence and correlation analyses to probe the native structural features of a yeast protein Sup35, whose amyloid state is believed to be used in a beneficial context in yeast. We find that the monomeric protein populates a compact and rapidly fluctuating ensemble of conformations. In another case, we studied the binding-coupled folding of the IDP alpha-synuclein, whose misfolding and aggregation have been linked to Parkinson's disease. Single-molecule measurements directly revealed a complex multi-state folding landscape for this protein. Observations of a transient folding intermediate using microfluidic mixing, and links to misfolding and aggregation will also be discussed. Our results highlight single-molecule methodology that is broadly applicable to map protein folding and misfolding landscapes.

  15. Tracking single Kv2.1 channels in live cells reveals anomalous subdiffusion and ergodicity breaking

    NASA Astrophysics Data System (ADS)

    Weigel, Aubrey; Simon, Blair; Tamkun, Michael; Krapf, Diego

    2011-03-01

    The dynamic organization of the plasma membrane is responsible for essential cellular processes, such as receptor trafficking and signaling. By studying the dynamics of transmembrane proteins a greater understanding of these processes as a whole can be achieved. It is broadly observed that the diffusion pattern of membrane protein displays anomalous subdiffusion. However, the mechanisms responsible for this behavior are not yet established. We explore the dynamics of the voltage gated potassium channel Kv2.1 by using single-particle tracking. We analyze Kv2.1 channel trajectories in terms of the time and ensemble distributions of square displacements. Our results reveal that all Kv2.1 channels experience anomalous subdiffusion and we observe that the Kv2.1 diffusion pattern is non-ergodic. We further investigated the role of the actin cytoskeleton in these channel dynamics by applying actin depolymerizing drugs. It is seen that with the breakdown of the actin cytoskeleton the Kv2.1 channel trajectories recover ergodicity.

  16. Mechanical disassembly of single virus particles reveals kinetic intermediates predicted by theory.

    PubMed

    Castellanos, Milagros; Pérez, Rebeca; Carrillo, Pablo J P; de Pablo, Pedro J; Mateu, Mauricio G

    2012-06-01

    New experimental approaches are required to detect the elusive transient intermediates predicted by simulations of virus assembly or disassembly. Here, an atomic force microscope (AFM) was used to mechanically induce partial disassembly of single icosahedral T=1 capsids and virions of the minute virus of mice. The kinetic intermediates formed were imaged by AFM. The results revealed that induced disassembly of single minute-virus-of-mice particles is frequently initiated by loss of one of the 20 equivalent capsomers (trimers of capsid protein subunits) leading to a stable, nearly complete particle that does not readily lose further capsomers. With lower frequency, a fairly stable, three-fourths-complete capsid lacking one pentamer of capsomers and a free, stable pentamer were obtained. The intermediates most frequently identified (capsids missing one capsomer, capsids missing one pentamer of capsomers, and free pentamers of capsomers) had been predicted in theoretical studies of reversible capsid assembly based on thermodynamic-kinetic models, molecular dynamics, or oligomerization energies. We conclude that mechanical manipulation and imaging of simple virus particles by AFM can be used to experimentally identify kinetic intermediates predicted by simulations of assembly or disassembly.

  17. The protein composition of reconstituted 30S ribosomal subunits: the effects of single protein omission.

    PubMed

    Buck, M A; Olah, T V; Perrault, A R; Cooperman, B S

    1991-06-01

    Using reverse phase HPLC, we have been able to quantify the protein compositions of reconstituted 30S ribosomal subunits, formed either with the full complement of 30S proteins in the reconstitution mix or with a single protein omitted. We denote particles formed in the latter case as SPORE (single protein omission reconstitution) particles. An important goal in 30S reconstitution studies is the formation of reconstituted subunits having uniform protein composition, preferably corresponding to one copy of each protein per reconstituted particle. Here we describe procedures involving variation of the protein:rRNA ratio that approach this goal. In SPORE particles the omission of one protein often results in the partial loss in uptake of other proteins. We also describe procedures to increase the uptake of such proteins into SPORE particles, thus enhancing the utility of the SPORE approach in defining the role of specific proteins in 30S structure and function. The losses of proteins other than the omitted protein provide a measure of protein:protein interaction within the 30S subunit. Most of these losses are predictable on the basis of other such measures. However, we do find evidence for several long-range protein:protein interactions (S6:S3, S6:S12, S10:S16, and S6:S4) that have not been described previously.

  18. Single-molecule imaging studies of protein dynamics

    NASA Astrophysics Data System (ADS)

    Zareh, Shannon Kian G.

    2011-12-01

    Single-molecule fluorescence imaging is a powerful method for studying biological events. The work of this thesis primarily focuses on single molecule studies of the dynamics of Green Fluorescent Protein (GFP) and other fluorescent-labeled proteins by utilizing Total Internal Reflection Fluorescence (TIRF) microscopy and imaging. The single molecule experiments of this thesis covered three broad topics. First, the adsorption mechanisms of proteins onto hydrophobic and hydrophilic fused silica surfaces were imaged and reversible and irreversible adsorption mechanisms were observed. The second topic covered a new technique for measuring the diffusion coefficient of Brownian diffusing proteins, in particular GFP, in solution via a single image. The corresponding experiments showed a relationship between the intensity profile width and the diffusion coefficient of the diffusing molecules. The third topic covered an in vivo experiment involving imaging and quantifying prokaryotic cell metabolism protein dynamics inside the Bacillus subtilis bacteria, in which a helical diffusion pattern for the protein was observed. These topics are presented in the chronological order of the experiments conducted.

  19. Protein painting reveals solvent-excluded drug targets hidden within native protein–protein interfaces

    PubMed Central

    Luchini, Alessandra; Espina, Virginia; Liotta, Lance A.

    2014-01-01

    Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein–protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets. PMID:25048602

  20. Dynamic Coupling among Protein Binding, Sliding, and DNA Bending Revealed by Molecular Dynamics.

    PubMed

    Tan, Cheng; Terakawa, Tsuyoshi; Takada, Shoji

    2016-07-13

    Protein binding to DNA changes the DNA's structure, and altered DNA structure can, in turn, modulate the dynamics of protein binding. This mutual dependency is poorly understood. Here we investigated dynamic couplings among protein binding to DNA, protein sliding on DNA, and DNA bending by applying a coarse-grained simulation method to the bacterial architectural protein HU and 14 other DNA-binding proteins. First, we verified our method by showing that the simulated HU exhibits a weak preference for A/T-rich regions of DNA and a much higher affinity for gapped and nicked DNA, consistent with biochemical experiments. The high affinity was attributed to a local DNA bend, but not the specific chemical moiety of the gap/nick. The long-time dynamic analysis revealed that HU sliding is associated with the movement of the local DNA bending site. Deciphering single sliding steps, we found the coupling between HU sliding and DNA bending is akin to neither induced-fit nor population-shift; instead they moved concomitantly. This is reminiscent of a cation transfer on DNA and can be viewed as a protein version of polaron-like sliding. Interestingly, on shorter time scales, HU paused when the DNA was highly bent at the bound position and escaped from pauses once the DNA spontaneously returned to a less bent structure. The HU sliding is largely regulated by DNA bending dynamics. With 14 other proteins, we explored the generality and versatility of the dynamic coupling and found that 6 of the 15 assayed proteins exhibit the polaron-like sliding. PMID:27309278

  1. Watching Individual Proteins Acting on Single Molecules of DNA

    PubMed Central

    Amitani, Ichiro; Liu, Bian; Dombrowski, Christopher C.; Baskin, Ronald J.; Kowalczykowski, Stephen C.

    2011-01-01

    In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual proteins or assemblies of proteins acting on single molecules of DNA. The general experimental design involves attaching a single DNA molecule to a polystyrene microsphere that is then used as a microscopic handle to manipulate individual DNA molecules with a laser trap. Visualization is achieved by fluorescently labeling either the DNA or the protein of interest, followed by direct imaging using high-sensitivity fluorescence microscopy. We describe the sample preparation and instrumentation used to visualize the interaction of individual proteins with single molecules of DNA. As examples, we describe the application of these methods to the study of proteins involved in recombination-mediated DNA repair, a process essential for the maintenance of genomic integrity. PMID:20580968

  2. Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands

    PubMed Central

    Lu, Yao; Xue, Qiong; Eisele, Markus R.; Sulistijo, Endah S.; Brower, Kara; Han, Lin; Amir, El-ad David; Pe’er, Dana; Miller-Jensen, Kathryn; Fan, Rong

    2015-01-01

    Despite recent advances in single-cell genomic, transcriptional, and mass-cytometric profiling, it remains a challenge to collect highly multiplexed measurements of secreted proteins from single cells for comprehensive analysis of functional states. Herein, we combine spatial and spectral encoding with polydimethylsiloxane (PDMS) microchambers for codetection of 42 immune effector proteins secreted from single cells, representing the highest multiplexing recorded to date for a single-cell secretion assay. Using this platform to profile differentiated macrophages stimulated with lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4), reveals previously unobserved deep functional heterogeneity and varying levels of pathogenic activation. Uniquely protein profiling on the same single cells before and after LPS stimulation identified a role for macrophage inhibitory factor (MIF) to potentiate the activation of LPS-induced cytokine production. Advanced clustering analysis identified functional subsets including quiescent, polyfunctional fully activated, partially activated populations with different cytokine profiles. This population architecture is conserved throughout the cell activation process and prevails as it is extended to other TLR ligands and to primary macrophages derived from a healthy donor. This work demonstrates that the phenotypically similar cell population still exhibits a large degree of intrinsic heterogeneity at the functional and cell behavior level. This technology enables full-spectrum dissection of immune functional states in response to pathogenic or environmental stimulation, and opens opportunities to quantify deep functional heterogeneity for more comprehensive and accurate immune monitoring. PMID:25646488

  3. Plasmon-enhanced emission from single fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Donehue, Jessica E.; Haas, Beth L.; Wertz, Esther; Talicska, Courtney N.; Biteen, Julie S.

    2013-02-01

    In this work, we use evaporated gold nanoparticle films (GNPFs) as substrates for plasmon-enhanced imaging of two fluorescent proteins (FPs): mCherry and YFP. Through single-molecule epifluorescence microscopy, we show enhancement of single FP emission in the presence of GNPFs. The gold-coupled FPs demonstrate emission up to four times brighter and seven times longer lived, yielding order-of-magnitude enhancements in total photons detected. Ultimately, this results in increased localization accuracies for single-molecule imaging. Furthermore, we introduce preliminary results for enhancement of mCherry-labeled TcpP membrane proteins inside live Vibrio cholerae cells coupled to GNPFs. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

  4. Analysis of single nucleic acid molecules with protein nanopores

    PubMed Central

    Maglia, Giovanni; Heron, Andrew J.; Stoddart, David; Japrung, Deanpen; Bayley, Hagan

    2011-01-01

    We describe the methods used in our laboratory for the analysis of single nucleic acid molecules with protein nanopores. The technical section is preceded by a review of the variety of experiments that can be done with protein nanopores. The end goal of much of this work is single-molecule DNA sequencing, although sequencing is not discussed explicitly here. The technical section covers the equipment required for nucleic acid analysis, the preparation and storage of the necessary materials, and aspects of signal processing and data analysis. PMID:20627172

  5. Network based approaches reveal clustering in protein point patterns

    NASA Astrophysics Data System (ADS)

    Parker, Joshua; Barr, Valarie; Aldridge, Joshua; Samelson, Lawrence E.; Losert, Wolfgang

    2014-03-01

    Recent advances in super-resolution imaging have allowed for the sub-diffraction measurement of the spatial location of proteins on the surfaces of T-cells. The challenge is to connect these complex point patterns to the internal processes and interactions, both protein-protein and protein-membrane. We begin analyzing these patterns by forming a geometric network amongst the proteins and looking at network measures, such the degree distribution. This allows us to compare experimentally observed patterns to models. Specifically, we find that the experimental patterns differ from heterogeneous Poisson processes, highlighting an internal clustering structure. Further work will be to compare our results to simulated protein-protein interactions to determine clustering mechanisms.

  6. Single molecule surface enhanced resonance Raman scattering (SERRS) of the enhanced green fluorescent protein (EGFP)

    NASA Astrophysics Data System (ADS)

    Hofkens, Johan; De Schryver, Frans C.; Cotlet, Mircea; Habuchi, Satoshi

    2004-06-01

    One of the most intriguing findings in single molecule spectroscopy (SMS) is the observation of Raman spectra of individual molecules, despite the small cross section of the transitions involved. The observation of the spectra can be explained by the surface enhanced Raman scattering (SERRS) effect. At the single-molecule level, the SERRS-spectra recorded as a function of time reveal inhomogeneous behaviour such as on/off blinking, spectral diffusion, intensity fluctuations of vibrational line, and even splitting of some lines within the spectrum of one molecule. Single-molecule SERRS (SM-SERRS) spectroscopy opens up exciting opportunities in the field of biophysics and biomedical spectroscopy. The first example of single protein SERRS was performed on hemoglobin. However, the possibility of extracting the heme group by silver sols can not be excluded. Here we report on SM-SERRS spectra of enhanced green fluorescent protein (EGFP) in which the chromophore is kept in the protein. The time series of SM-SERRS spectra suggest the conversion of the EGFP chromophore between the deprotonated and the protonated form. Autocorrelation analysis of SM-SERRS trajectory reveals the presence of fast dynamics taking place in the protein. Our findings show the potential of the technique to study structural dynamics of protein molecules.

  7. Imaging multiple intermediates of single-virus membrane fusion mediated by distinct fusion proteins.

    PubMed

    Joo, Kye-Il; Tai, April; Lee, Chi-Lin; Wong, Clement; Wang, Pin

    2010-09-01

    Membrane fusion plays an essential role in the entry of enveloped viruses into target cells. The merging of viral and target cell membranes is catalyzed by viral fusion proteins, which involves multiple sequential steps in the fusion process. However, the fusion mechanisms mediated by different fusion proteins involve multiple transient intermediates that have not been well characterized. Here, we report a synthetic virus platform that allows us to better understand the different fusion mechanisms driven by the diverse types fusion proteins. The platform consists of lentiviral particles coenveloped with a surface antibody, which serves as the binding protein, along with a fusion protein derived from either influenza virus (HAmu) or Sindbis virus (SINmu). By using a single virus tracking technique, we demonstrated that both HAmu- and SINmu-bearing viruses enter cells through clathrin-dependent endocytosis, but they required different endosomal trafficking routes to initiate viral fusion. Direct observation of single viral fusion events clearly showed that hemifusion mediated by SINmu upon exposure to low pH occurs faster than that mediated by HAmu. Monitoring sequential fusion processes by dual labeling the outer and inner leaflets of viral membranes also revealed that the SINmu-mediated hemifusion intermediate is relatively long-lived as compared with that mediated by HAmu. Taken together, we have demonstrated that the combination of this versatile viral platform with the techniques of single virus tracking can be a powerful tool for revealing molecular details of fusion mediated by various fusion proteins.

  8. Reveal protein dynamics by combining computer simulation and neutron scattering

    NASA Astrophysics Data System (ADS)

    Hong, Liang; Smith, Jeremy; CenterMolecular Biophysics Team

    2014-03-01

    Protein carries out most functions in living things on the earth through characteristic modulation of its three-dimensional structure over time. Understanding the microscopic nature of the protein internal motion and its connection to the function and structure of the biomolecule is a central topic in biophysics, and of great practical importance for drug design, study of diseases, and the development of renewable energy, etc. Under physiological conditions, protein exhibits a complex dynamics landscape, i.e., a variety of diffusive and conformational motions occur on similar time and length scales. This variety renders difficult the derivation of a simplified description of protein internal motions in terms of a small number of distinct, additive components. This difficulty is overcome by our work using a combined approach of Molecular Dynamics (MD) simulations and the Neutron Scattering experiments. Our approach enables distinct protein motions to be characterized separately, furnishing an in-depth understanding of the connection between protein structure, dynamics and function.

  9. Single-molecule protein sequencing through fingerprinting: computational assessment

    NASA Astrophysics Data System (ADS)

    Yao, Yao; Docter, Margreet; van Ginkel, Jetty; de Ridder, Dick; Joo, Chirlmin

    2015-10-01

    Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.

  10. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. PMID:26659058

  11. Revealing Higher Order Protein Structure Using Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Chait, Brian T.; Cadene, Martine; Olinares, Paul Dominic; Rout, Michael P.; Shi, Yi

    2016-06-01

    The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope.

  12. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering.

  13. Single Nucleus Genome Sequencing Reveals High Similarity among Nuclei of an Endomycorrhizal Fungus

    PubMed Central

    Zhang, Zhonghua; Ivanov, Sergey; Saunders, Diane G. O.; Mu, Desheng; Pang, Erli; Cao, Huifen; Cha, Hwangho; Lin, Tao; Zhou, Qian; Shang, Yi; Li, Ying; Sharma, Trupti; van Velzen, Robin; de Ruijter, Norbert; Aanen, Duur K.; Win, Joe; Kamoun, Sophien; Bisseling, Ton; Geurts, René; Huang, Sanwen

    2014-01-01

    Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya. PMID:24415955

  14. Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

    PubMed Central

    Hoskins, Aaron A; Rodgers, Margaret L; Friedman, Larry J; Gelles, Jeff; Moore, Melissa J

    2016-01-01

    The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation. DOI: http://dx.doi.org/10.7554/eLife.14166.001 PMID:27244240

  15. The Single-Molecule Approach to Membrane Protein Stoichiometry.

    PubMed

    Nichols, Michael G; Hallworth, Richard

    2016-01-01

    The advent of techniques for imaging solitary fluorescent molecules has made possible many new kinds of biological experiments. Here, we describe the application of single-molecule imaging to the problem of subunit stoichiometry in membrane proteins. A membrane protein of unknown stoichiometry, prestin, is coupled to the fluorescent enhanced green fluorescent protein (eGFP) and synthesized in the human embryonic kidney (HEK) cell line. We prepare adherent membrane fragments containing prestin-eGFP by osmotic lysis. The molecules are then exposed to continuous low-level excitation until their fluorescence reaches background levels. Their fluorescence decreases in discrete equal-amplitude steps, consistent with the photobleaching of single fluorophores. We count the number of steps required to photobleach each molecule. The molecular stoichiometry is then deduced using a binomial model.

  16. Ribosomal History Reveals Origins of Modern Protein Synthesis

    PubMed Central

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  17. Ribosomal history reveals origins of modern protein synthesis.

    PubMed

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  18. Common fluorescent proteins for single-molecule localization microscopy

    NASA Astrophysics Data System (ADS)

    Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

    2015-07-01

    Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

  19. Surface passivation for single-molecule protein studies.

    PubMed

    Chandradoss, Stanley D; Haagsma, Anna C; Lee, Young Kwang; Hwang, Jae-Ho; Nam, Jwa-Min; Joo, Chirlmin

    2014-04-24

    Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

  20. Protein adhesion force dynamics and single adhesion events.

    PubMed Central

    Sagvolden, G

    1999-01-01

    Using the manipulation force microscope, a novel atomic force microscope, the adhesion forces of bovine serum albumin, myoglobin, ferritin, and lysozyme proteins to glass and polystyrene substrates were characterized by following the force necessary to displace an adsorbed protein-covered microsphere over several orders of magnitude in time. This force was consistent with a power law with exponent a = 0.37 +/- 0.03 on polystyrene, indicating that there is no typical time scale for adhesion on this substrate. On glass, the rate of adhesion depended strongly on protein charge. Forces corresponding to single protein adhesion events were identified. The typical rupture force of a single lysozyme, ferritin, bovine serum albumin, and myoglobin protein adhering to glass was estimated to be 90 +/- 10 pN, 115 +/- 13 pN, 277 +/- 44 pN, and 277 +/- 44 pN, respectively, using a model of the experimental system. These forces, as well as the force amplitudes on hydrophobic polystyrene, correlate with protein stiffness. PMID:10388777

  1. Near-membrane protein dynamics revealed by evanescent field microscopy

    NASA Astrophysics Data System (ADS)

    Bezzerides, Vassilios J.; Clapham, David E.

    2004-05-01

    Evanescent Field (EF) microscopy is used to investigate the spatial and temporal dynamics of proteins in living cells. A genetically engineered ion channel fused to a fluorescent tag is expressed in cells and imaged with an objective-based EF microscope. Images are obtained from a CCD and analyzed to determine fluorescence and velocity of individual protein containing vesicles. An inverse correlation between fluorescent intensity and average motility provides a method for determination of membrane localization. Stimulation and subsequent decrease in ion channel activity is correlated with loss of protein from membrane as shown by EF microscopy and patch-clamp electrophysiology.

  2. Spectroscopy reveals that ethyl esters interact with proteins in wine.

    PubMed

    Di Gaspero, Mattia; Ruzza, Paolo; Hussain, Rohanah; Vincenzi, Simone; Biondi, Barbara; Gazzola, Diana; Siligardi, Giuliano; Curioni, Andrea

    2017-02-15

    Impairment of wine aroma after vinification is frequently associated to bentonite treatments and this can be the result of protein removal, as recently demonstrated for ethyl esters. To evaluate the existence of an interaction between wine proteins and ethyl esters, the effects induced by these fermentative aroma compounds on the secondary structure and stability of VVTL1, a Thaumatin-like protein purified from wine, was analyzed by Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The secondary structure of wine VVTL1 was not strongly affected by the presence of selected ethyl esters. In contrast, VVTL1 stability was slightly increased by the addition of ethyl-octanoate, -decanoate and -dodecanoate, but decreased by ethyl-hexanoate. This indicates the existence of an interaction between VVTL1 and at least some aroma compounds produced during fermentation. The data suggest that proteins removal from wine by bentonite can result in indirect removal of at least some aroma compounds associated with them. PMID:27664648

  3. Single-file diffusion of protein drugs through cylindrical nanochannels.

    PubMed

    Yang, Seung Yun; Yang, Jeong-A; Kim, Eung-Sam; Jeon, Gumhye; Oh, Eun Ju; Choi, Kwan Yong; Hahn, Sei Kwang; Kim, Jin Kon

    2010-07-27

    A new drug delivery device using cylindrical block copolymer nanochannels was successfully developed for controlled protein drug delivery applications. Depending on the hydrodynamic diameter of the protein drugs, the pore size in cylindrical nanochannels could be controlled precisely down to 6 nm by Au deposition. Zero-order release of bovine serum albumin (BSA) and human growth hormone (hGH) by single-file diffusion, which has been observed for gas diffusion through zeolite pores, was realized up to 2 months without protein denaturation. Furthermore, a nearly constant in vivo release of hGH from the drug delivery nanodevice implanted to Sprague-Dawley (SD) rats was continued up to 3 weeks, demonstrating the feasibility for long-term controlled delivery of therapeutic protein drugs.

  4. Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles.

    PubMed

    Serna, Naroa; Céspedes, María Virtudes; Saccardo, Paolo; Xu, Zhikun; Unzueta, Ugutz; Álamo, Patricia; Pesarrodona, Mireia; Sánchez-Chardi, Alejandro; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio; Ferrer-Miralles, Neus

    2016-07-01

    A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles. PMID:26949165

  5. Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles.

    PubMed

    Serna, Naroa; Céspedes, María Virtudes; Saccardo, Paolo; Xu, Zhikun; Unzueta, Ugutz; Álamo, Patricia; Pesarrodona, Mireia; Sánchez-Chardi, Alejandro; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio; Ferrer-Miralles, Neus

    2016-07-01

    A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles.

  6. Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction

    PubMed Central

    Hu, Jianfei; Neiswinger, Johnathan; Zhang, Jin; Zhu, Heng; Qian, Jiang

    2015-01-01

    Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process. PMID:26393507

  7. Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction.

    PubMed

    Hu, Jianfei; Neiswinger, Johnathan; Zhang, Jin; Zhu, Heng; Qian, Jiang

    2015-01-01

    Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process.

  8. Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx

    PubMed Central

    Dynes, Joseph L.; Amcheslavsky, Anna; Cahalan, Michael D.

    2016-01-01

    Orai1 comprises the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel. When bound and activated by stromal interacting molecule 1 (STIM1), an endoplasmic reticulum (ER)-resident calcium sensor, Orai1 channels possess high selectivity for calcium but extremely small conductance that has precluded direct recording of single-channel currents. We have developed an approach to visualize Orai1 activity by fusing Orai1 to fluorescent, genetically encoded calcium indicators (GECIs). The GECI–Orai1 probes reveal local Ca2+ influx at STIM1–Orai1 puncta. By whole cell recording, these fusions are fully functional as CRAC channels. When GECI–Orai1 and the CRAC-activating domain (CAD) of STIM1 were coexpressed at low levels and imaged using a total internal reflectance fluorescence microscope, cells exhibited sporadic fluorescence transients the size of diffraction-limited spots and the brightness of a few activated GECI proteins. Transients typically rose rapidly and fell into two classes according to duration: briefer “flickers” lasting only a few hundred milliseconds, and longer “pulses” lasting one to several seconds. The size, intensity, trace shape, frequency, distribution, physiological characteristics, and association with CAD binding together demonstrate that GECI–Orai1 fluorescence transients correspond to single-channel Orai1 responses. Single Orai1 channels gated by CAD, and small Orai1 puncta gated by STIM1, exhibit repetitive fluctuations in single-channel output. CAD binding supports a role in open state maintenance and reveals a second phase of CAD/STIM1 binding after channel opening. These first recordings of single-channel Orai1 currents reveal unexpected dynamics, and when paired with CAD association, support multiple single-channel states. PMID:26712003

  9. Single-molecule studies reveal the function of a third polymerase in the replisome

    PubMed Central

    Georgescu, Roxana E; Kurth, Isabel; O'Donnell, Mike E

    2013-01-01

    The Escherichia coli replisome contains three polymerases, one more than necessary to duplicate the two parental strands. Using single-molecule studies, we reveal two advantages conferred by the third polymerase. First, dipolymerase replisomes are inefficient at synthesizing lagging strands, leaving single-strand gaps, whereas tripolymerase replisomes fill strands almost to completion. Second, tripolymerase replisomes are much more processive than dipolymerase replisomes. These features account for the unexpected three-polymerase-structure of bacterial replisomes. PMID:22157955

  10. Maleimide photolithography for single-molecule protein-protein interaction analysis in micropatterns.

    PubMed

    Waichman, Sharon; You, Changjiang; Beutel, Oliver; Bhagawati, Maniraj; Piehler, Jacob

    2011-01-15

    Spatial organization of proteins into microscopic structures has important applications in fundamental and applied research. Preserving the function of proteins in such microstructures requires generic methods for site-specific capturing through affinity handles. Here, we present a versatile bottom-up surface micropatterning approach based on surface functionalization with maleimides, which selectively react with organic thiols. Upon UV irradiation through a photomask, the functionality of illuminated maleimide groups was efficiently destroyed. Remaining maleimides in nonilluminated regions were further reacted with different thiol-functionalized groups for site-specific protein immobilization under physiological conditions. Highly selective immobilization of His-tagged proteins into tris(nitrilotriacetic acid) functionalized microstructures with very high contrast was possible even by direct capturing of proteins from crude cell lysates. Moreover, we employed phosphopantetheinyl transfer from surface-immobilized coenzyme A to ybbR-tagged proteins in order to implement site-specific, covalent protein immobilization into microstructures. The functional integrity of the immobilized protein was confirmed by monitoring protein-protein interactions in real time. Moreover, we demonstrate quantitative single-molecule analysis of protein-protein interactions with proteins selectively captured into these high-contrast micropatterns.

  11. Single-molecule atomic force microscopy reveals clustering of the yeast plasma-membrane sensor Wsc1.

    PubMed

    Heinisch, Jürgen J; Dupres, Vincent; Wilk, Sabrina; Jendretzki, Arne; Dufrêne, Yves F

    2010-06-14

    Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains ("rafts") is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of approximately 200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches.

  12. Quantification of protein copy number in single mitochondria: The Bcl-2 family proteins.

    PubMed

    Chen, Chaoxiang; Zhang, Xiang; Zhang, Shuyue; Zhu, Shaobin; Xu, Jingyi; Zheng, Yan; Han, Jinyan; Zeng, Jin-Zhang; Yan, Xiaomei

    2015-12-15

    Bcl-2 family proteins, represented by antiapoptotic protein Bcl-2 and proapoptotic protein Bax, are key regulators of mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome c release, it is essential to know the number of proteins in individual mitochondria. Here, we report an effective method to quantify the copy number and distribution of proteins in single mitochondria via immunofluorescent labeling and sensitive detection by a laboratory-built high sensitivity flow cytometer (HSFCM). Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488 (AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to the number of proteins per mitochondrion. Under the normal condition, the measured mean copy numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively. A significant variation in protein copy number was identified, which ranged from 130 to 6000 (2.5-97.5%) for Bcl-2 and from 65 to 700 (2.5-97.5%) for Bax, respectively. We observed an approximately 4.4 fold increase of Bax copy number per mitochondrion upon 9h of apoptosis stimulation while the abundance of Bcl-2 remained almost unchanged. To the best of our knowledge, this is the first report of Bcl-2 family protein copy number and variance in single mitochondria. Collectively, we demonstrate that the HSFCM-based immunoassay provides a rapid and sensitive method for determining protein copy number distribution in single mitochondria.

  13. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    SciTech Connect

    Timmins, P.A.; Pebay-Peyroula, E.

    1994-12-31

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H{sub 2}O/D{sub 2}O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

  14. Fully Quantified Spectral Imaging Reveals in Vivo Membrane Protein Interactions

    PubMed Central

    King, Christopher; Stoneman, Michael; Raicu, Valerica; Hristova, Kalina

    2016-01-01

    Here we introduce the Fully Quantified Spectral Imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. We use the FSI method to study the lateral interactions of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes, in vivo. The knowledge gained through the use of the new method challenges the current understanding of VEGFR2 signaling. PMID:26787445

  15. Coupled motion in proteins revealed by pressure perturbation

    PubMed Central

    Fu, Yinan; Kasinath, Vignesh; Moorman, Veronica R.; Nucci, Nathaniel V.; Hilser, Vincent J.; Wand, A. Joshua

    2012-01-01

    The cooperative nature of protein substructure and internal motion is a critical aspect of their functional competence about which little is known experimentally. NMR relaxation is used here to monitor the effects of high-pressure on fast internal motion in the protein ubiquitin. In contrast to the main chain, the motions of the methyl-bearing side chains have a large and variable pressure dependence. Within the core, this pressure sensitivity correlates with the magnitude of motion at ambient pressure. Spatial clustering of the dynamic response to applied hydrostatic pressure is also seen indicating localized cooperativity of motion on the sub-nanosecond time scale and suggesting regions of variable compressibility. These and other features indicate that the native ensemble contains a significant fraction of members with characteristics ascribed to the recently postulated “dry molten globule.” The accompanying variable side chain conformational entropy helps complete our view of the thermodynamic architecture underlying protein stability, folding and function. PMID:22452540

  16. The dark energy of proteins comes to light: conformational entropy and its role in protein function revealed by NMR relaxation.

    PubMed

    Wand, A Joshua

    2013-02-01

    Historically it has been virtually impossible to experimentally determine the contribution of residual protein entropy to fundamental protein activities such as the binding of ligands. Recent progress has illuminated the possibility of employing NMR relaxation methods to quantitatively determine the role of changes in conformational entropy in molecular recognition by proteins. The method rests on using fast internal protein dynamics as a proxy. Initial results reveal a large and variable role for conformational entropy in the binding of ligands by proteins. Such a role for conformational entropy in molecular recognition has significant implications for enzymology, signal transduction, allosteric regulation and the development of protein-directed pharmaceuticals. PMID:23246280

  17. Electrochemical atomic force microscopy imaging of redox-immunomarked proteins on native potyviruses: from subparticle to single-protein resolution.

    PubMed

    Nault, Laurent; Taofifenua, Cécilia; Anne, Agnès; Chovin, Arnaud; Demaille, Christophe; Besong-Ndika, Jane; Cardinale, Daniela; Carette, Noëlle; Michon, Thierry; Walter, Jocelyne

    2015-05-26

    We show herein that electrochemical atomic force microscopy (AFM-SECM), operated in molecule touching (Mt) mode and combined with redox immunomarking, enables the in situ mapping of the distribution of proteins on individual virus particles and makes localization of individual viral proteins possible. Acquisition of a topography image allows isolated virus particles to be identified and structurally characterized, while simultaneous acquisition of a current image allows the sought after protein, marked by redox antibodies, to be selectively located. We concomitantly show that Mt/AFM-SECM, due to its single-particle resolution, can also uniquely reveal the way redox functionalization endowed to viral particles is distributed both statistically among the viruses and spatially over individual virus particles. This possibility makes Mt/AFM-SECM a unique tool for viral nanotechnology. PMID:25905663

  18. Electrochemical atomic force microscopy imaging of redox-immunomarked proteins on native potyviruses: from subparticle to single-protein resolution.

    PubMed

    Nault, Laurent; Taofifenua, Cécilia; Anne, Agnès; Chovin, Arnaud; Demaille, Christophe; Besong-Ndika, Jane; Cardinale, Daniela; Carette, Noëlle; Michon, Thierry; Walter, Jocelyne

    2015-05-26

    We show herein that electrochemical atomic force microscopy (AFM-SECM), operated in molecule touching (Mt) mode and combined with redox immunomarking, enables the in situ mapping of the distribution of proteins on individual virus particles and makes localization of individual viral proteins possible. Acquisition of a topography image allows isolated virus particles to be identified and structurally characterized, while simultaneous acquisition of a current image allows the sought after protein, marked by redox antibodies, to be selectively located. We concomitantly show that Mt/AFM-SECM, due to its single-particle resolution, can also uniquely reveal the way redox functionalization endowed to viral particles is distributed both statistically among the viruses and spatially over individual virus particles. This possibility makes Mt/AFM-SECM a unique tool for viral nanotechnology.

  19. Single-cell genomics reveal low recombination frequencies in freshwater bacteria of the SAR11 clade

    PubMed Central

    2013-01-01

    Background The SAR11 group of Alphaproteobacteria is highly abundant in the oceans. It contains a recently diverged freshwater clade, which offers the opportunity to compare adaptations to salt- and freshwaters in a monophyletic bacterial group. However, there are no cultivated members of the freshwater SAR11 group and no genomes have been sequenced yet. Results We isolated ten single SAR11 cells from three freshwater lakes and sequenced and assembled their genomes. A phylogeny based on 57 proteins indicates that the cells are organized into distinct microclusters. We show that the freshwater genomes have evolved primarily by the accumulation of nucleotide substitutions and that they have among the lowest ratio of recombination to mutation estimated for bacteria. In contrast, members of the marine SAR11 clade have one of the highest ratios. Additional metagenome reads from six lakes confirm low recombination frequencies for the genome overall and reveal lake-specific variations in microcluster abundances. We identify hypervariable regions with gene contents broadly similar to those in the hypervariable regions of the marine isolates, containing genes putatively coding for cell surface molecules. Conclusions We conclude that recombination rates differ dramatically in phylogenetic sister groups of the SAR11 clade adapted to freshwater and marine ecosystems. The results suggest that the transition from marine to freshwater systems has purged diversity and resulted in reduced opportunities for recombination with divergent members of the clade. The low recombination frequencies of the LD12 clade resemble the low genetic divergence of host-restricted pathogens that have recently shifted to a new host. PMID:24286338

  20. Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1 alpha reveals a new post-translational protein modification.

    PubMed

    Rosenberry, T L; Krall, J A; Dever, T E; Haas, R; Louvard, D; Merrick, W C

    1989-05-01

    Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988) J. Biol. Chem. 263, 8244-8252). In particular, the protein was enriched in cytosolic and microsomal fractions relative to plasma membrane and thus did not appear to correspond to the class of proteins with glycoinositol phospholipid anchors, the only post-translational protein modification involving ethanolamine that had been described previously. Two-dimensional polyacrylamide gel analysis involving isoelectric focusing followed by electrophoresis in sodium dodecyl sulfate indicated that the protein was very basic, and nitrocellulose blots of one- and two-dimensional gels subjected to 3H autoradiography and immunostaining with antisera to purified rabbit elongation factor (EF) 1 alpha revealed that the protein was EF-1 alpha. Copurification of rabbit EF-1 alpha and the [3H]ethanolamine-labeled protein from K562 cells further supported this identification. Analysis of tryptic fragments produced from the copurified proteins by reverse-phase high pressure liquid chromatography showed two radiolabeled peptides. Amino acid analysis demonstrated 1 residue of ethanolamine in each peptide, and peptide sequencing revealed that the ethanolamine-containing component(s) was attached to Glu301 and Glu374 in the EF-1 alpha protein sequence deduced from a human EF-1 alpha cDNA. These data confirm a new class of post-translational protein modifications involving ethanolamine. PMID:2708357

  1. Charged single alpha-helices in proteomes revealed by a consensus prediction approach.

    PubMed

    Gáspári, Zoltán; Süveges, Dániel; Perczel, András; Nyitray, László; Tóth, Gábor

    2012-04-01

    Charged single α-helices (CSAHs) constitute a recently recognized protein structural motif. Its presence and role is characterized in only a few proteins. To explore its general features, a comprehensive study is necessary. We have set up a consensus prediction method available as a web service (at http://csahserver.chem.elte.hu) and downloadable scripts capable of predicting CSAHs from protein sequences. Using our method, we have performed a comprehensive search on the UniProt database. We found that the motif is very rare but seems abundant in proteins involved in symbiosis and RNA binding/processing. Although there are related proteins with CSAH segments, the motif shows no deep conservation in protein families. We conclude that CSAH-containing proteins, although rare, are involved in many key biological processes. Their conservation pattern and prevalence in symbiosis-associated proteins suggest that they might be subjects of relatively rapid molecular evolution and thus can contribute to the emergence of novel functions.

  2. Protein energy malnutrition revealing an esophageal foreign body.

    PubMed

    Kouassi, Yao Mathurin; Vroh, Bi Tah Sylvain; Buraima, Fataho; Toure, Abdoulaye; Tanon-Anoh, Marie-Josée

    2010-12-01

    It is not unusual for a foreign body to be swallowed and be lodged in the esophagus. It is however, very unusual for such a foreign body to remain lodged for a period of 8 months. This particular case, a 15-month-old male infant, is under focus because of the time length the foreign body remained in the esophagus without local complications, what is unusual is a protein energy malnutrition complication. The neck and chest X-ray permitted the foreign body identification. The management of esophageal foreign body requires a multidisciplinary approach among otorhinolaryngologist, radiologist and pediatrician.

  3. Transcriptional abundance is not the single force driving the evolution of bacterial proteins

    PubMed Central

    2013-01-01

    Background Despite rapid progress in understanding the mechanisms that shape the evolution of proteins, the relative importance of various factors remain to be elucidated. In this study, we have assessed the effects of 16 different biological features on the evolutionary rates (ERs) of protein-coding sequences in bacterial genomes. Results Our analysis of 18 bacterial species revealed new correlations between ERs and constraining factors. Previous studies have suggested that transcriptional abundance overwhelmingly constrains the evolution of yeast protein sequences. This transcriptional abundance leads to selection against misfolding or misinteractions. In this study we found that there was no single factor in determining the evolution of bacterial proteins. Not only transcriptional abundance (codon adaptation index and expression level), but also protein-protein associations (PPAs), essentiality (ESS), subcellular localization of cytoplasmic membrane (SLM), transmembrane helices (TMH) and hydropathicity score (HS) independently and significantly affected the ERs of bacterial proteins. In some species, PPA and ESS demonstrate higher correlations with ER than transcriptional abundance. Conclusions Different forces drive the evolution of protein sequences in yeast and bacteria. In bacteria, the constraints are involved in avoiding a build-up of toxic molecules caused by misfolding/misinteraction (transcriptional abundance), while retaining important functions (ESS, PPA) and maintaining the cell membrane (SLM, TMH and HS). Each of these independently contributes to the variation in protein evolution. PMID:23914835

  4. From protein structure to function via single crystal optical spectroscopy

    PubMed Central

    Ronda, Luca; Bruno, Stefano; Bettati, Stefano; Storici, Paola; Mozzarelli, Andrea

    2015-01-01

    The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic “artifacts,” including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density maps, thus limiting the relevance of structure determinations. Moreover, for most of these structures, no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in infereing protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman, and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5′-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms. PMID:25988179

  5. Single-point single-molecule FRAP distinguishes inner and outer nuclear membrane protein distribution

    PubMed Central

    Mudumbi, Krishna C; Schirmer, Eric C; Yang, Weidong

    2016-01-01

    The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. NETs are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM). Quantitative determination of the distribution of NETs on the ONM and INM is limited in available approaches, which moreover provide no information about translocation rates in the two membranes. Here we demonstrate a single-point single-molecule FRAP microscopy technique that enables determination of distribution and translocation rates for NETs in vivo. PMID:27558844

  6. Single-point single-molecule FRAP distinguishes inner and outer nuclear membrane protein distribution.

    PubMed

    Mudumbi, Krishna C; Schirmer, Eric C; Yang, Weidong

    2016-01-01

    The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. NETs are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM). Quantitative determination of the distribution of NETs on the ONM and INM is limited in available approaches, which moreover provide no information about translocation rates in the two membranes. Here we demonstrate a single-point single-molecule FRAP microscopy technique that enables determination of distribution and translocation rates for NETs in vivo. PMID:27558844

  7. Replication protein A and more: single-stranded DNA-binding proteins in eukaryotic cells.

    PubMed

    Liu, Ting; Huang, Jun

    2016-07-01

    Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombinational repair, and maintenance of genome stability. In human, the major SSB, replication protein A (RPA), is a stable heterotrimer composed of subunits of RPA1, RPA2, and RPA3, each of which is conserved not only in mammals but also in all other eukaryotic species. In addition to RPA, other SSBs have also been identified in the human genome, including sensor of single-stranded DNA complexes 1 and 2 (SOSS1/2). In this review, we summarize our current understanding of how these SSBs contribute to the maintenance of genome stability.

  8. Metabolomics of a single vacuole reveals metabolic dynamism in an alga Chara australis.

    PubMed

    Oikawa, Akira; Matsuda, Fumio; Kikuyama, Munehiro; Mimura, Tetsuro; Saito, Kazuki

    2011-10-01

    Metabolomics is the most reliable analytical method for understanding metabolic diversity in single organelles derived from single cells. Although metabolites such as phosphate compounds are believed to be localized in different organelles in a highly specific manner, the process of metabolite compartmentalization in the cell is not thoroughly understood. The analysis of metabolites in single organelles has consequently presented a significant challenge. In this study, we used a metabolomic method to elucidate the localization and dynamics of 125 known metabolites isolated from the vacuole and cytoplasm of a single cell of the alga Chara australis. The amount of metabolites in the vacuole and the cytoplasm fluctuated asynchronously under various stress conditions, suggesting that metabolites are spatially regulated within the cell. Metabolite transport across the vacuolar membrane can be directly detected using the microinjection technique, which may reveal a previously unknown function of the vacuole.

  9. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state

    PubMed Central

    Rotem, Assaf; Ram, Oren; Shoresh, Noam; Sperling, Ralph A.; Goren, Alon; Weitz, David A.; Bernstein, Bradley E.

    2015-01-01

    Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells, and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell is sparse, comprising on the order of 1000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of sub-populations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone. PMID:26458175

  10. Shrimp single WAP domain (SWD)-containing protein exhibits proteinase inhibitory and antimicrobial activities.

    PubMed

    Amparyup, Piti; Donpudsa, Suchao; Tassanakajon, Anchalee

    2008-01-01

    Single WAP domain (SWD)-containing proteins are small proteins with a C-terminal region containing a single whey acidic protein (WAP) domain. In the present study, the cDNAs representing three isoforms of SWD proteins (SWDPm1, SWDPm2 and SWDPm3) were identified from hemocytes of the black tiger shrimp, Penaeus monodon. The deduced peptides revealed that they contain a putative signal peptide of 24 amino acids and encode for a mature peptide of 69, 68 and 56 amino acids, respectively, which contain typical characters similar to those of the shrimp SWD proteins (type III crustin) with a Pro-Arg region and a WAP domain towards the C-terminus. Tissue distribution analysis by RT-PCR showed that all three SWDPm transcripts were primarily found in hemocytes. Transcript expression of SWDPm1 was down-regulated upon injection with Staphylococcus aureus whilst there was no change of SWDPm2 and SWDPm3 expression. In contrast, white spot syndrome virus (WSSV) injection resulted in a biphasic response with up-regulation of SWDPm1 and SWDPm2 transcripts at 6h followed by significant down-regulation by 24h after infection. Genomic organization of the SWDPm2 gene revealed the presence of three exons interrupted by two introns. To characterize the biological functions of the SWD protein, the mature SWDPm2 protein encoding cDNA was cloned and expressed in Escherichia coli. Purified recombinant (r)SWDPm2 exhibits antibacterial activity against several Gram-positive, but not Gram-negative, bacteria and is a competitive inhibitor of subtilisin A with an inhibition constant (Ki) of 1.98nM. Thus, rSWDPm2 may contribute to the inhibitory regulation of subtilisin A from bacterial infection and P. monodon SWD protein likely function as immune effectors in defense against invasion of shrimp pathogens. PMID:18602420

  11. Production and feeding of single-cell protein

    SciTech Connect

    Ferranti, M.P.; Fiechter, A.

    1983-01-01

    This book addresses the technical and economic factors which must be considered when evaluating plans for the production of animal feed proteins from agricultural and forestry wastes. The work is divided into three parts, the first focusing on pretreatment and hydrolysis of lignocellulostic materials, the second on upgrading of whey and the third on nutrition and toxicology. The presentation concludes with a Round Table discussion including evaluations and recommendations for submission to the Commission of the European Communities. CONTENTS: Special Section: Pretreatment and Degradation of Lignocellulosic Materials. Subject Area 1: Production of SCP Enriched Substrate from Cellulosic Materials. Lignin and Lignocellulose. Process Development. Carbohydrates. Subject Area 2: Single Cell Protein from Whey. Subject Area 3: Nutrition and Toxicology. Round Table Discussion: Evaluation and Recommendations. List of Participants. Index of Authors.

  12. Interaction of DNA and Proteins with Single Nanopores

    NASA Astrophysics Data System (ADS)

    Kasianowicz, J. J.

    2006-03-01

    The bacterial toxins Staphylococcus aureus alpha-hemolysin and Bacillus anthracis protective antigen kill cells in part by forming ion channels in target membranes. We are using electrophysiology, molecular biology/protein biochemistry and computer modeling to study how biopolymers (e.g., single-stranded DNA and proteins) bind to and transport through these nanometer-scale pores. The results provide insight into the mechanism by which these toxins work and are the basis for several potential nanobiotechnology applications including ultra-rapid DNA sequencing, the sensitive and selective detection of a wide range of analytes and high throughput screening of therapeutic agents against several anthrax toxins. In collaboration with V.M. Stanford, M. Misakian, B. Nablo, S.E. Henrickson, NIST, EEEL, Gaithersburg, MD; T. Nguyen, R. Gussio, NCI, Ft. Detrick, MD; and K.M. Halverson, S. Bavari, R.G. Panchal, USAMRIID, Ft. Detrick, MD.

  13. Probing protein-DNA interactions by unzipping a single DNA double helix.

    PubMed Central

    Koch, Steven J; Shundrovsky, Alla; Jantzen, Benjamin C; Wang, Michelle D

    2002-01-01

    We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes (BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of approximately 25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (K(A)) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K(A) for EcoRI binding. The measured values are in good agreement with previous measurements of K(A) over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions. PMID:12124289

  14. Sequence similarity network reveals common ancestry of multidomain proteins.

    PubMed

    Song, Nan; Joseph, Jacob M; Davis, George B; Durand, Dannie

    2008-04-01

    We address the problem of homology identification in complex multidomain families with varied domain architectures. The challenge is to distinguish sequence pairs that share common ancestry from pairs that share an inserted domain but are otherwise unrelated. This distinction is essential for accuracy in gene annotation, function prediction, and comparative genomics. There are two major obstacles to multidomain homology identification: lack of a formal definition and lack of curated benchmarks for evaluating the performance of new methods. We offer preliminary solutions to both problems: 1) an extension of the traditional model of homology to include domain insertions; and 2) a manually curated benchmark of well-studied families in mouse and human. We further present Neighborhood Correlation, a novel method that exploits the local structure of the sequence similarity network to identify homologs with great accuracy based on the observation that gene duplication and domain shuffling leave distinct patterns in the sequence similarity network. In a rigorous, empirical comparison using our curated data, Neighborhood Correlation outperforms sequence similarity, alignment length, and domain architecture comparison. Neighborhood Correlation is well suited for automated, genome-scale analyses. It is easy to compute, does not require explicit knowledge of domain architecture, and classifies both single and multidomain homologs with high accuracy. Homolog predictions obtained with our method, as well as our manually curated benchmark and a web-based visualization tool for exploratory analysis of the network neighborhood structure, are available at http://www.neighborhoodcorrelation.org. Our work represents a departure from the prevailing view that the concept of homology cannot be applied to genes that have undergone domain shuffling. In contrast to current approaches that either focus on the homology of individual domains or consider only families with identical domain

  15. Conformational dynamics of single protein molecules studied by direct mechanical manipulation.

    PubMed

    Heidarsson, Pétur O; Naqvi, Mohsin M; Sonar, Punam; Valpapuram, Immanuel; Cecconi, Ciro

    2013-01-01

    Advances in single-molecule manipulation techniques have recently enabled researchers to study a growing array of biological processes in unprecedented detail. Individual molecules can now be manipulated with subnanometer precision along a simple and well-defined reaction coordinate, the molecular end-to-end distance, and their conformational changes can be monitored in real time with ever-improving time resolution. The behavior of biomolecules under tension continues to unravel at an accelerated pace and often in combination with computational studies that reveal the atomistic details of the process under investigation. In this chapter, we explain the basic principles of force spectroscopy techniques, with a focus on optical tweezers, and describe some of the theoretical models used to analyze and interpret single-molecule manipulation data. We then highlight some recent and exciting results that have emerged from this research field on protein folding and protein-ligand interactions.

  16. Single pentagon in a hexagonal carbon lattice revealed by scanning tunneling microscopy

    SciTech Connect

    An, B.; Fukuyama, S.; Yokogawa, K.; Yoshimura, M.; Egashira, M.; Korai, Y.; Mochida, I.

    2001-06-04

    The electronic structure of a single pentagon in a hexagonal carbon lattice has been revealed on an atomic scale by scanning tunneling microscopy. The pentagon is located at the apex of the conical protuberance of the graphitic particle. The enhanced charge density localized at each carbon atom in the pentagon is identified, and the ringlike pattern of the ({radical}3{times}{radical}3)R30{degree} superstructure of graphite is clearly observed around the pentagon. {copyright} 2001 American Institute of Physics.

  17. The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing.

    PubMed

    Björklund, Åsa K; Forkel, Marianne; Picelli, Simone; Konya, Viktoria; Theorell, Jakob; Friberg, Danielle; Sandberg, Rickard; Mjösberg, Jenny

    2016-04-01

    Innate lymphoid cells (ILCs) are increasingly appreciated as important participants in homeostasis and inflammation. Substantial plasticity and heterogeneity among ILC populations have been reported. Here we have delineated the heterogeneity of human ILCs through single-cell RNA sequencing of several hundreds of individual tonsil CD127(+) ILCs and natural killer (NK) cells. Unbiased transcriptional clustering revealed four distinct populations, corresponding to ILC1 cells, ILC2 cells, ILC3 cells and NK cells, with their respective transcriptomes recapitulating known as well as unknown transcriptional profiles. The single-cell resolution additionally divulged three transcriptionally and functionally diverse subpopulations of ILC3 cells. Our systematic comparison of single-cell transcriptional variation within and between ILC populations provides new insight into ILC biology during homeostasis, with additional implications for dysregulation of the immune system.

  18. Pheromone binding proteins of Epiphyas postvittana (Lepidoptera: Tortricidae) are encoded at a single locus.

    PubMed

    Newcomb, R D; Sirey, T M; Rassam, M; Greenwood, D R

    2002-11-01

    The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in

  19. Single-molecule chemical reaction reveals molecular reaction kinetics and dynamics.

    PubMed

    Zhang, Yuwei; Song, Ping; Fu, Qiang; Ruan, Mingbo; Xu, Weilin

    2014-06-25

    Understanding the microscopic elementary process of chemical reactions, especially in condensed phase, is highly desirable for improvement of efficiencies in industrial chemical processes. Here we show an approach to gaining new insights into elementary reactions in condensed phase by combining quantum chemical calculations with a single-molecule analysis. Elementary chemical reactions in liquid-phase, revealed from quantum chemical calculations, are studied by tracking the fluorescence of single dye molecules undergoing a reversible redox process. Statistical analyses of single-molecule trajectories reveal molecular reaction kinetics and dynamics of elementary reactions. The reactivity dynamic fluctuations of single molecules are evidenced and probably arise from either or both of the low-frequency approach of the molecule to the internal surface of the SiO2 nanosphere or the molecule diffusion-induced memory effect. This new approach could be applied to other chemical reactions in liquid phase to gain more insight into their molecular reaction kinetics and the dynamics of elementary steps.

  20. Network Analysis of Circular Permutations in Multidomain Proteins Reveals Functional Linkages for Uncharacterized Proteins

    PubMed Central

    Adjeroh, Donald; Jiang, Yue; Jiang, Bing-Hua; Lin, Jie

    2014-01-01

    Various studies have implicated different multidomain proteins in cancer. However, there has been little or no detailed study on the role of circular multidomain proteins in the general problem of cancer or on specific cancer types. This work represents an initial attempt at investigating the potential for predicting linkages between known cancer-associated proteins with uncharacterized or hypothetical multidomain proteins, based primarily on circular permutation (CP) relationships. First, we propose an efficient algorithm for rapid identification of both exact and approximate CPs in multidomain proteins. Using the circular relations identified, we construct networks between multidomain proteins, based on which we perform functional annotation of multidomain proteins. We then extend the method to construct subnetworks for selected cancer subtypes, and performed prediction of potential link-ages between uncharacterized multidomain proteins and the selected cancer types. We include practical results showing the performance of the proposed methods. PMID:25741177

  1. Molecular shape and binding force of Mycoplasma mobile's leg protein Gli349 revealed by an AFM study

    SciTech Connect

    Lesoil, Charles; Nonaka, Takahiro; Sekiguchi, Hiroshi; Osada, Toshiya; Miyata, Makoto; Afrin, Rehana; Ikai, Atsushi

    2010-01-15

    Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349 kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the 'leg' of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70 pN. These findings strongly support the idea that Gli349 is the 'leg' protein of M. mobile, responsible for binding and also most probably force generation during gliding motility.

  2. Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block.

    PubMed

    Li, Hong; Chen, Xiao Yan; Kong, Qing You; Liu, Jia

    2002-06-01

    The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section. The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a defined cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.

  3. Insight into the Assembly of Viruses with Vertical Single β-barrel Major Capsid Proteins.

    PubMed

    Gil-Carton, David; Jaakkola, Salla T; Charro, Diego; Peralta, Bibiana; Castaño-Díez, Daniel; Oksanen, Hanna M; Bamford, Dennis H; Abrescia, Nicola G A

    2015-10-01

    Archaeal viruses constitute the least explored niche within the virosphere. Structure-based approaches have revealed close relationships between viruses infecting organisms from different domains of life. Here, using biochemical and cryo-electron microscopy techniques, we solved the structure of euryarchaeal, halophilic, internal membrane-containing Haloarcula hispanica icosahedral virus 2 (HHIV-2). We show that the density of the two major capsid proteins (MCPs) recapitulates vertical single β-barrel proteins and that disulfide bridges stabilize the capsid. Below, ordered density is visible close to the membrane and at the five-fold vertices underneath the host-interacting vertex complex underpinning membrane-protein interactions. The HHIV-2 structure exemplifies the division of conserved architectural elements of a virion, such as the capsid, from those that evolve rapidly due to selective environmental pressure such as host-recognizing structures. We propose that in viruses with two vertical single β-barrel MCPs the vesicle is indispensable, and membrane-protein interactions serve as protein-railings for guiding the assembly. PMID:26320579

  4. Analysis and Interpretation of Single Molecule Protein Unfolding Kinetics

    NASA Astrophysics Data System (ADS)

    Lannon, Herbert; Brujic, Jasna

    2012-02-01

    The kinetics of protein unfolding under a stretching force has been extensively studied by atomic force microscopy (AFM) over the past decade [1]. Experimental artifacts at the single molecule level introduce uncertainties in the data analysis that have led to several competing physical models for the unfolding process. For example, the unfolding dynamics of the protein ubiquitin under constant force has been described by probability distributions as diverse as exponential [2,3], a sum of exponentials, log-normal [4], and more recently a function describing static disorder in the Arrhenius model [5]. A new method for data analysis is presented that utilizes maximum likelihood estimation (MLE) combined with other traditional statistical tests to unambiguously rank the consistency of these and other models with the experimental data. These techniques applied to the ubiquitin unfolding data shows that the probability of unfolding is best fit with a stretched exponential distribution, with important implications on the complexity of the mechanism of protein unfolding. [4pt] [1] Carrion-Vazquez, et. al. Springer Series in Biophys. 2006 [0pt] [2] Fernandez et. al. Science 2004 [0pt] [3] Brujic et. al. Nat. Phys 2006 [0pt] [4] Garcia-Manyes et. al. Biophys. J. 2007 [0pt] [5] Kuo et. al. PNAS 2010

  5. Single-molecule studies of unconventional motor protein myosin VI

    NASA Astrophysics Data System (ADS)

    Kim, HyeongJun

    Myosin VI is one of the myosin superfamily members that are actin-based molecular motors. It has received special attention due to its distinct features as compared to other myosins, such as its opposite directionality and a much larger step size than expected given the length of its "leg". This dissertation presents the author.s graduate work of several single-molecule studies on myosin VI. Special attention was paid to some of myosin VI.s tail domains that consist of proximal tail (PT), medial tail (MT), distal tail (DT) domains and cargo-binding domain (CBD). The functional form of myosin VI in cells is still under debate. Although full length myosin VI proteins in cytosolic extracts of cells were monomers from earlier studies, there are several reasons why it is now believed that myosin VI could exist as a dimer. If this is true and dimerization occurs, the next logical question would be which parts of myosin VI are dimerization regions? One model claimed that the CBD is the sole dimerization region. A competing model claimed that there must be another region that could be involved in dimerization, based on their observation that a construct without the CBD could still dimerize. Our single-molecule experiment with progressively truncated myosin VI constructs showed that the MT domain is a dimerization region, supporting the latter model. Additional single-molecule experiments and molecular dynamics (MD) simulation done with our collaborators suggest that electrostatic salt bridges formed between positive and negative amino acid residues are mainly responsible for the MT domain dimerization. After resolving this, we are left with another important question which is how myosin VI can take such a large step. Recent crystal structure showed that one of the tail domains preceding the MT domain, called the PT domain, is a three-helix bundle. The most easily conceivable way might be an unfolding of the three-helix bundle upon dimerization, allowing the protein to

  6. A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting.

    PubMed

    Herbert, Kristina M; Sarkar, Susanta K; Mills, Maria; Delgado De la Herran, Hilda C; Neuman, Keir C; Steitz, Joan A

    2016-02-01

    During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem-loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.

  7. Cellular localization and trafficking of vascular adhesion protein-1 as revealed by an N-terminal GFP fusion protein.

    PubMed

    Weston, Chris J; Shepherd, Emma L; Adams, David H

    2013-06-01

    Recent studies of vascular adhesion protein-1 (VAP-1) have greatly advanced our understanding of the important role this protein plays in the establishment and progression of inflammatory disease. To facilitate more detailed studies on the function of VAP-1, we developed a GFP-fusion protein that enabled us to monitor the trafficking of the protein in three selected cell types: hepatic sinusoidal endothelial cells, liver myofibroblasts and an hepatic stellate cell line (LX-2). The fusion protein was detected as punctate cytoplasmic GFP staining, but was present only at low levels at the cell surface in all cell types studied. The subcellular distribution of the protein was not altered in a catalytically inactive mutant form of the protein (Tyr471Phe) or in the presence of exogenous VAP-1 substrate (methylamine) or inhibitor (semicarbazide). The GFP-VAP-1 protein was localized to the Golgi apparatus (GM-130), endoplasmic reticulum (GRP94) and early endosomes (EEA-1). Additional staining for VAP-1 revealed that the overexpressed protein was also present in vesicles that were negative for GFP fluorescent signal and did not express EEA-1. We propose that these vesicles are responsible for recycling the fusion protein and that the fluorescence of the GFP moiety is quenched at the low pH within these vesicles. This feature of the protein makes it well suited for live cell imaging studies where we wish to track protein that is being actively trafficked within the cell in preference to that which is being recycled.

  8. Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering.

    PubMed

    Carrion-Vazquez, M; Oberhauser, A F; Fisher, T E; Marszalek, P E; Li, H; Fernandez, J M

    2000-01-01

    Mechanical unfolding and refolding may regulate the molecular elasticity of modular proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein engineering techniques allow the construction of homomeric polyproteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp). PMID:11106807

  9. Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity

    PubMed Central

    Horn, Abigail E.; Kugel, Jennifer F.; Goodrich, James A.

    2016-01-01

    Transcription by RNA polymerase II (Pol II) is a complex process that requires general transcription factors and Pol II to assemble on DNA into preinitiation complexes that can begin RNA synthesis upon binding of NTPs (nucleoside triphosphate). The pathways by which preinitiation complexes form, and how this impacts transcriptional activity are not completely clear. To address these issues, we developed a single molecule system using TIRF (total internal reflection fluorescence) microscopy and purified human transcription factors, which allows us to visualize transcriptional activity at individual template molecules. We see that stable interactions between polymerase II (Pol II) and a heteroduplex DNA template do not depend on general transcription factors; however, transcriptional activity is highly dependent upon TATA-binding protein, TFIIB and TFIIF. We also found that subsets of general transcription factors and Pol II can form stable complexes that are precursors for functional transcription complexes upon addition of the remaining factors and DNA. Ultimately we found that Pol II, TATA-binding protein, TFIIB and TFIIF can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA. Single molecule studies can be used to learn how different modes of preinitiation complex assembly impact transcriptional activity. PMID:27112574

  10. Molecular Mechanics of Single Protein Molecules Measured with the Atomic Force Microscope

    NASA Astrophysics Data System (ADS)

    Hansma, Paul K.

    2000-03-01

    After a short history of AFM development in our lab, including recent developments with small cantilevers, this talk will focus on 1) pulling single protein molecules to explore the forces involved in unfolding and 2) watching single protein molecules in action to learn how they function mechanically. 1) Pulling experiments on proteins used as marine adhesives in abalone shells and other biological composite materials reveal modules bound together by sacrificial bonds that are weaker than the backbone bonds in the polypeptide chain.1 These self-healing modules provide effective energy absorption and appear to be a real key to understanding the impressive fracture resistance of biological composite materials. For example, the abalone shell is 3000 times more fracture resistant than a single crystal of calcium carbonate, despite the fact that 97% of the mass of the shell is crystalline calcium carbonate. 2) It is becoming possible, again with AFMs, to learn how some enzymes, nature's nanomachines, do their exquisite materials synthesis and processing. The talk will focus on the chaperonin system of GroEL and GroES that processes incorrectly folded proteins and assists them in refolding correctly. It is becoming possible not only to see single molecule events such as the association and disassociation of the GroEL-Gro-ES complex, but also to measure potential energy functions for the molecules in various conformational states. These new measurements, together with detailed structural measurements from other techniques, give new clues about how these proteins use the energy of ATP to do their work. Since the AFMs of today are very far from fundamental limits, this is only the beginning. 1. B. L. Smith, T. E. Schaffer, M. Viani, J. B. Thompson, N. A. Frederick, J. Kindt, A. Belcher, G. D. Stucky, D. E. Morse and P. K. Hansma, Nature 399, 761 (1999)

  11. Single-virus tracking approach to reveal the interaction of Dengue virus with autophagy during the early stage of infection

    NASA Astrophysics Data System (ADS)

    Chu, Li-Wei; Huang, Yi-Lung; Lee, Jin-Hui; Huang, Long-Ying; Chen, Wei-Jun; Lin, Ya-Hsuan; Chen, Jyun-Yu; Xiang, Rui; Lee, Chau-Hwang; Ping, Yueh-Hsin

    2014-01-01

    Dengue virus (DENV) is one of the major infectious pathogens worldwide. DENV infection is a highly dynamic process. Currently, no antiviral drug is available for treating DENV-induced diseases since little is known regarding how the virus interacts with host cells during infection. Advanced molecular imaging technologies are powerful tools to understand the dynamics of intracellular interactions and molecular trafficking. This study exploited a single-virus particle tracking technology to address whether DENV interacts with autophagy machinery during the early stage of infection. Using confocal microscopy and three-dimensional image analysis, we showed that DENV triggered the formation of green fluorescence protein-fused microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta, and DENV-induced autophagosomes engulfed DENV particles within 15-min postinfection. Moreover, single-virus particle tracking revealed that both DENV particles and autophagosomes traveled together during the viral infection. Finally, in the presence of autophagy suppressor 3-methyladenine, the replication of DENV was inhibited and the location of DENV particles spread in cytoplasma. In contrast, the numbers of newly synthesized DENV were elevated and the co-localization of DENV particles and autophagosomes was detected while the cells were treated with autophagy inducer rapamycin. Taken together, we propose that DENV particles interact with autophagosomes at the early stage of viral infection, which promotes the replication of DENV.

  12. Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

    PubMed Central

    Zhang, Zhengjian; English, Brian P.; Grimm, Jonathan B.; Kazane, Stephanie A.; Hu, Wenxin; Tsai, Albert; Inouye, Carla; You, Changjiang; Piehler, Jacob; Schultz, Peter G.; Lavis, Luke D.; Revyakin, Andrey; Tjian, Robert

    2016-01-01

    Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A “step-wise” preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions. PMID:27798851

  13. Nuclear RNA-seq of single neurons reveals molecular signatures of activation

    PubMed Central

    Lacar, Benjamin; Linker, Sara B.; Jaeger, Baptiste N.; Krishnaswami, Suguna; Barron, Jerika; Kelder, Martijn; Parylak, Sarah; Paquola, Apuã; Venepally, Pratap; Novotny, Mark; O'Connor, Carolyn; Fitzpatrick, Conor; Erwin, Jennifer; Hsu, Jonathan Y.; Husband, David; McConnell, Michael J.; Lasken, Roger; Gage, Fred H.

    2016-01-01

    Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo. PMID:27090946

  14. Mpath maps multi-branching single-cell trajectories revealing progenitor cell progression during development

    PubMed Central

    Chen, Jinmiao; Schlitzer, Andreas; Chakarov, Svetoslav; Ginhoux, Florent; Poidinger, Michael

    2016-01-01

    Single-cell RNA-sequencing offers unprecedented resolution of the continuum of state transition during cell differentiation and development. However, tools for constructing multi-branching cell lineages from single-cell data are limited. Here we present Mpath, an algorithm that derives multi-branching developmental trajectories using neighborhood-based cell state transitions. Applied to mouse conventional dendritic cell (cDC) progenitors, Mpath constructs multi-branching trajectories spanning from macrophage/DC progenitors through common DC progenitor to pre-dendritic cells (preDC). The Mpath-generated trajectories detect a branching event at the preDC stage revealing preDC subsets that are exclusively committed to cDC1 or cDC2 lineages. Reordering cells along cDC development reveals sequential waves of gene regulation and temporal coupling between cell cycle and cDC differentiation. Applied to human myoblasts, Mpath recapitulates the time course of myoblast differentiation and isolates a branch of non-muscle cells involved in the differentiation. Our study shows that Mpath is a useful tool for constructing cell lineages from single-cell data. PMID:27356503

  15. Nuclear RNA-seq of single neurons reveals molecular signatures of activation.

    PubMed

    Lacar, Benjamin; Linker, Sara B; Jaeger, Baptiste N; Krishnaswami, Suguna; Barron, Jerika; Kelder, Martijn; Parylak, Sarah; Paquola, Apuã; Venepally, Pratap; Novotny, Mark; O'Connor, Carolyn; Fitzpatrick, Conor; Erwin, Jennifer; Hsu, Jonathan Y; Husband, David; McConnell, Michael J; Lasken, Roger; Gage, Fred H

    2016-01-01

    Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo. PMID:27090946

  16. Electronic transport in single-helical protein molecules: Effects of multiple charge conduction pathways and helical symmetry

    NASA Astrophysics Data System (ADS)

    Kundu, Sourav; Karmakar, S. N.

    2016-07-01

    We propose a tight-binding model to investigate electronic transport properties of single helical protein molecules incorporating both the helical symmetry and the possibility of multiple charge transfer pathways. Our study reveals that due to existence of both the multiple charge transfer pathways and helical symmetry, the transport properties are quite rigid under influence of environmental fluctuations which indicates that these biomolecules can serve as better alternatives in nanoelectronic devices than its other biological counterparts e.g., single-stranded DNA.

  17. Interconvertible lac repressor-DNA loops revealed by single-molecule experiments.

    PubMed

    Wong, Oi Kwan; Guthold, Martin; Erie, Dorothy A; Gelles, Jeff

    2008-09-30

    At many promoters, transcription is regulated by simultaneous binding of a protein to multiple sites on DNA, but the structures and dynamics of such transcription factor-mediated DNA loops are poorly understood. We directly examined in vitro loop formation mediated by Escherichia coli lactose repressor using single-molecule structural and kinetics methods. Small ( approximately 150 bp) loops form quickly and stably, even with out-of-phase operator spacings. Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein-DNA complexes. The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic "V" conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain. The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions. PMID:18828671

  18. Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells

    PubMed Central

    Lee, Timothy K.; Meng, Kevin; Shi, Handuo; Huang, Kerwyn Casey

    2016-01-01

    The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis. PMID:27774981

  19. High-Throughput Sequencing Reveals Single Nucleotide Variants in Longer-Kernel Bread Wheat

    PubMed Central

    Chen, Feng; Zhu, Zibo; Zhou, Xiaobian; Yan, Yan; Dong, Zhongdong; Cui, Dangqun

    2016-01-01

    The transcriptomes of bread wheat Yunong 201 and its ethyl methanesulfonate derivative Yunong 3114 were obtained by next-sequencing technology. Single nucleotide variants (SNVs) in the wheat strains were explored and compared. A total of 5907 and 6287 non-synonymous SNVs were acquired for Yunong 201 and 3114, respectively. A total of 4021 genes with SNVs were obtained. The genes that underwent non-synonymous SNVs were significantly involved in ATP binding, protein phosphorylation, and cellular protein metabolic process. The heat map analysis also indicated that most of these mutant genes were significantly differentially expressed at different developmental stages. The SNVs in these genes possibly contribute to the longer kernel length of Yunong 3114. Our data provide useful information on wheat transcriptome for future studies on wheat functional genomics. This study could also help in illustrating the gene functions of the non-synonymous SNVs of Yunong 201 and 3114. PMID:27551288

  20. High-Throughput Sequencing Reveals Single Nucleotide Variants in Longer-Kernel Bread Wheat.

    PubMed

    Chen, Feng; Zhu, Zibo; Zhou, Xiaobian; Yan, Yan; Dong, Zhongdong; Cui, Dangqun

    2016-01-01

    The transcriptomes of bread wheat Yunong 201 and its ethyl methanesulfonate derivative Yunong 3114 were obtained by next-sequencing technology. Single nucleotide variants (SNVs) in the wheat strains were explored and compared. A total of 5907 and 6287 non-synonymous SNVs were acquired for Yunong 201 and 3114, respectively. A total of 4021 genes with SNVs were obtained. The genes that underwent non-synonymous SNVs were significantly involved in ATP binding, protein phosphorylation, and cellular protein metabolic process. The heat map analysis also indicated that most of these mutant genes were significantly differentially expressed at different developmental stages. The SNVs in these genes possibly contribute to the longer kernel length of Yunong 3114. Our data provide useful information on wheat transcriptome for future studies on wheat functional genomics. This study could also help in illustrating the gene functions of the non-synonymous SNVs of Yunong 201 and 3114. PMID:27551288

  1. A novel single-cell screening platform reveals proteome plasticity during yeast stress responses

    PubMed Central

    Breker, Michal; Gymrek, Melissa

    2013-01-01

    Uncovering the mechanisms underlying robust responses of cells to stress is crucial for our understanding of cellular physiology. Indeed, vast amounts of data have been collected on transcriptional responses in Saccharomyces cerevisiae. However, only a handful of pioneering studies describe the dynamics of proteins in response to external stimuli, despite the fact that regulation of protein levels and localization is an essential part of such responses. Here we characterized unprecedented proteome plasticity by systematically tracking the localization and abundance of 5,330 yeast proteins at single-cell resolution under three different stress conditions (DTT, H2O2, and nitrogen starvation) using the GFP-tagged yeast library. We uncovered a unique “fingerprint” of changes for each stress and elucidated a new response arsenal for adapting to radical environments. These include bet-hedging strategies, organelle rearrangement, and redistribution of protein localizations. All data are available for download through our online database, LOQATE (localization and quantitation atlas of yeast proteome). PMID:23509072

  2. Protein engineering: single or multiple site-directed mutagenesis.

    PubMed

    Hsieh, Pei-Chung; Vaisvila, Romualdas

    2013-01-01

    Site-directed mutagenesis techniques are invaluable tools in molecular biology to study the structural and functional properties of a protein. To expedite the time required and simplify methods for mutagenesis, we recommend two protocols in this chapter. The first method for single site-directed mutagenesis, which includes point mutations, insertions, or deletions, can be achieved by an inverse PCR strategy with mutagenic primers and the high-fidelity Phusion(®) DNA Polymerase to introduce a site-directed mutation with exceptional efficiency. The second method is for engineering multiple mutations into a gene of interest. This can be completed in one step by PCR with mutagenic primers and by assembling all mutagenized PCR products using the Gibson Assembly™ Master Mix. This method allows multiple nucleotides to be changed simultaneously, which not only saves time but also reagents compared to traditional methods of mutagenesis. PMID:23423897

  3. Structure and mechanics of proteins from single molecules to cells

    NASA Astrophysics Data System (ADS)

    Brown, Andre E.

    2009-07-01

    Physical factors drive evolution and play important roles in motility and attachment as well as in differentiation. As animal cells adhere to survive, they generate force and "feel" various mechanical features of their surroundings and respond to externally applied forces. This mechanosensitivity requires a substrate for cells to adhere to and a mechanism for cells to apply force, followed by a cellular response to the mechanical properties of the substrate. We have taken an outside-in approach to characterize several aspects of cellular mechanosensitivity. First, we used single molecule force spectroscopy to measure how fibrinogen, an extracellular matrix protein that forms the scaffold of blood clots, responds to applied force and found that it rapidly unfolds in 23 nm steps at forces around 100 pN. Second, we used tensile testing to measure the force-extension behavior of fibrin gels and found that they behave almost linearly to strains of over 100%, have extensibilities of 170 +/- 15%, and undergo a large volume decrease that corresponds to a large and negative peak in compressibility at low strain, which indicates a structural transition. Using electron microscopy and X-ray scattering we concluded that these properties are likely due to coiled-coil unfolding, as observed at the single molecule level in fibrinogen. Moving inside cells, we used total internal reflection fluorescence and atomic force microscopy to image self-assembled myosin filaments. These filaments of motor proteins that are responsible for cell and muscle contractility were found to be asymmetric, with an average of 32% more force generating heads on one half than the other. This could imply a force imbalance, so that rather than being simply contractile, myosin filaments may also be motile in cells.

  4. Single-molecule Study of Nucleocapsid Protein Chaperoned DNA Hairpin Structural Dynamics

    NASA Astrophysics Data System (ADS)

    Zeng, Yining; Cosa, Gonzalo; Liu, Hsiao-Wei; Landes, Christy; Makarov, Dmitrii; Barbara, Paul; Musier-Forsyth, Karin

    2006-03-01

    In HIV-1 reverse transcription, the nucleocapsid protein, NC, induces secondary structure fluctuations in the transactivation response (TAR) region of DNA and RNA hairpins. Time resolved single-molecule fluorescence resonance energy transfer was used to study NC chaperoned secondary fluctuations of DNA hairpins. The experiments reveal that the NC induced secondary fluctuations are limited to the terminal stems, and the mechanism for the fluctuations is complex. The dynamic processes occur over a wide time range, i.e. ˜5 to >250 milliseconds and involve long-lived intermediates. The dynamic role of DNA loop regions and NC binding/dissociation events are discussed.

  5. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

    PubMed

    Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

    2011-09-27

    The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

  6. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

    PubMed Central

    Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

    2011-01-01

    The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification. PMID:21952135

  7. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

    SciTech Connect

    Klopffleisch, Karsten; Phan, Nguyen; Chen, Jay; Panstruga, Ralph; Uhrig, Joachim; Jones, Alan M

    2011-01-01

    The heterotrimeric G-protein complex is minimally composed of G{alpha}, G{beta}, and G{gamma} subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

  8. Phylogenetic analyses in cornus substantiate ancestry of xylem supercooling freezing behavior and reveal lineage of desiccation related proteins.

    PubMed

    Karlson, Dale T; Xiang, Qiu-Yun; Stirm, Vicki E; Shirazi, A M; Ashworth, Edward N

    2004-07-01

    The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue- or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue- or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.

  9. Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain

    PubMed Central

    Lake, Blue B.; Ai, Rizi; Kaeser, Gwendolyn E.; Salathia, Neeraj S.; Yung, Yun C.; Liu, Rui; Wildberg, Andre; Gao, Derek; Fung, Ho-Lim; Chen, Song; Vijayaraghavan, Raakhee; Wong, Julian; Chen, Allison; Sheng, Xiaoyan; Kaper, Fiona; Shen, Richard; Ronaghi, Mostafa; Fan, Jian-Bing; Wang, Wei; Chun, Jerold; Zhang, Kun

    2016-01-01

    The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from post-mortem brain, generating 3,227 sets of single neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish novel and orthologous neuronal subtypes as well as regional identity within the human brain. PMID:27339989

  10. Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain.

    PubMed

    Lake, Blue B; Ai, Rizi; Kaeser, Gwendolyn E; Salathia, Neeraj S; Yung, Yun C; Liu, Rui; Wildberg, Andre; Gao, Derek; Fung, Ho-Lim; Chen, Song; Vijayaraghavan, Raakhee; Wong, Julian; Chen, Allison; Sheng, Xiaoyan; Kaper, Fiona; Shen, Richard; Ronaghi, Mostafa; Fan, Jian-Bing; Wang, Wei; Chun, Jerold; Zhang, Kun

    2016-06-24

    The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain. PMID:27339989

  11. Controlling Brownian motion of single protein molecules and single fluorophores in aqueous buffer.

    PubMed

    Cohen, Adam E; Moerner, W E

    2008-05-12

    We present an Anti-Brownian Electrokinetic trap (ABEL trap) capable of trapping individual fluorescently labeled protein molecules in aqueous buffer. The ABEL trap operates by tracking the Brownian motion of a single fluorescent particle in solution, and applying a time-dependent electric field designed to induce an electrokinetic drift that cancels the Brownian motion. The trapping strength of the ABEL trap is limited by the latency of the feedback loop. In previous versions of the trap, this latency was set by the finite frame rate of the camera used for video-tracking. In the present system, the motion of the particle is tracked entirely in hardware (without a camera or image-processing software) using a rapidly rotating laser focus and lock-in detection. The feedback latency is set by the finite rate of arrival of photons. We demonstrate trapping of individual molecules of the protein GroEL in buffer, and we show confinement of single fluorophores of the dye Cy3 in water.

  12. Vibrio cholerae biofilm growth program and architecture revealed by single-cell live imaging.

    PubMed

    Yan, Jing; Sharo, Andrew G; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

    2016-09-01

    Biofilms are surface-associated bacterial communities that are crucial in nature and during infection. Despite extensive work to identify biofilm components and to discover how they are regulated, little is known about biofilm structure at the level of individual cells. Here, we use state-of-the-art microscopy techniques to enable live single-cell resolution imaging of a Vibrio cholerae biofilm as it develops from one single founder cell to a mature biofilm of 10,000 cells, and to discover the forces underpinning the architectural evolution. Mutagenesis, matrix labeling, and simulations demonstrate that surface adhesion-mediated compression causes V. cholerae biofilms to transition from a 2D branched morphology to a dense, ordered 3D cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture in V. cholerae biofilms, and this growth pattern is controlled by a single gene, rbmA Competition analyses reveal that the dense growth mode has the advantage of providing the biofilm with superior mechanical properties. Our single-cell technology can broadly link genes to biofilm fine structure and provides a route to assessing cell-to-cell heterogeneity in response to external stimuli. PMID:27555592

  13. Vibrio cholerae biofilm growth program and architecture revealed by single-cell live imaging.

    PubMed

    Yan, Jing; Sharo, Andrew G; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

    2016-09-01

    Biofilms are surface-associated bacterial communities that are crucial in nature and during infection. Despite extensive work to identify biofilm components and to discover how they are regulated, little is known about biofilm structure at the level of individual cells. Here, we use state-of-the-art microscopy techniques to enable live single-cell resolution imaging of a Vibrio cholerae biofilm as it develops from one single founder cell to a mature biofilm of 10,000 cells, and to discover the forces underpinning the architectural evolution. Mutagenesis, matrix labeling, and simulations demonstrate that surface adhesion-mediated compression causes V. cholerae biofilms to transition from a 2D branched morphology to a dense, ordered 3D cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture in V. cholerae biofilms, and this growth pattern is controlled by a single gene, rbmA Competition analyses reveal that the dense growth mode has the advantage of providing the biofilm with superior mechanical properties. Our single-cell technology can broadly link genes to biofilm fine structure and provides a route to assessing cell-to-cell heterogeneity in response to external stimuli.

  14. Quantum Yield Heterogeneity among Single Nonblinking Quantum Dots Revealed by Atomic Structure-Quantum Optics Correlation.

    PubMed

    Orfield, Noah J; McBride, James R; Wang, Feng; Buck, Matthew R; Keene, Joseph D; Reid, Kemar R; Htoon, Han; Hollingsworth, Jennifer A; Rosenthal, Sandra J

    2016-02-23

    Physical variations in colloidal nanostructures give rise to heterogeneity in expressed optical behavior. This correlation between nanoscale structure and function demands interrogation of both atomic structure and photophysics at the level of single nanostructures to be fully understood. Herein, by conducting detailed analyses of fine atomic structure, chemical composition, and time-resolved single-photon photoluminescence data for the same individual nanocrystals, we reveal inhomogeneity in the quantum yields of single nonblinking "giant" CdSe/CdS core/shell quantum dots (g-QDs). We find that each g-QD possesses distinctive single exciton and biexciton quantum yields that result mainly from variations in the degree of charging, rather than from volume or structure inhomogeneity. We further establish that there is a very limited nonemissive "dark" fraction (<2%) among the studied g-QDs and present direct evidence that the g-QD core must lack inorganic passivation for the g-QD to be "dark". Therefore, in contrast to conventional QDs, ensemble photoluminescence quantum yield is principally defined by charging processes rather than the existence of dark g-QDs.

  15. Single Cell Wall Nonlinear Mechanics Revealed by a Multiscale Analysis of AFM Force-Indentation Curves.

    PubMed

    Digiuni, Simona; Berne-Dedieu, Annik; Martinez-Torres, Cristina; Szecsi, Judit; Bendahmane, Mohammed; Arneodo, Alain; Argoul, Françoise

    2015-05-01

    Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions. PMID:25954881

  16. Single Cell Wall Nonlinear Mechanics Revealed by a Multiscale Analysis of AFM Force-Indentation Curves

    PubMed Central

    Digiuni, Simona; Berne-Dedieu, Annik; Martinez-Torres, Cristina; Szecsi, Judit; Bendahmane, Mohammed; Arneodo, Alain; Argoul, Françoise

    2015-01-01

    Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions. PMID:25954881

  17. Strong coupling between adenine nucleobases in DNA single strands revealed by circular dichroism using synchrotron radiation

    NASA Astrophysics Data System (ADS)

    Kadhane, Umesh; Holm, Anne I. S.; Hoffmann, Søren Vrønning; Nielsen, Steen Brøndsted

    2008-02-01

    Circular dichroism (CD) experiments on DNA single strands (dAn) at the ASTRID synchrotron radiation facility reveal that eight adenine (A) bases electronically couple upon 190nm excitation. After n=8 , the CD signal increases linearly with n with a slope equal to the sum of the coupling terms. Nearest neighbor interactions account for only 24% of the CD signal whereas electronic communication is limited to nearest neighbors for two other exciton bands observed at 218 and 251nm (i.e., dimer excited states). Electronic coupling between bases in DNA is important for nonradiative deexcitation of electronically excited states since the hazardous energy is spread over a larger spatial region.

  18. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function

    PubMed Central

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  19. Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices

    PubMed Central

    Dupuis, Nicholas F.; Holmstrom, Erik D.; Nesbitt, David J.

    2013-01-01

    In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′on = 2.1(1) × 106 M−1 s−1) and 6 bp (k′on = 5.0(1) × 106 M−1 s−1) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s−1) to 6 bp (koff = 14 s−1) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, konincreases by >40-fold (kon = 0.10(1) s−1 to 4.0(4) s−1 between [NaCl] = 25 mM and 1 M), whereas in contrast, koffdecreases by fourfold (0.72(3) s−1 to 0.17(7) s−1) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation. PMID:23931323

  20. Revealing nonergodic dynamics in living cells from a single particle trajectory.

    PubMed

    Lanoiselée, Yann; Grebenkov, Denis S

    2016-05-01

    We propose the improved ergodicity and mixing estimators to identify nonergodic dynamics from a single particle trajectory. The estimators are based on the time-averaged characteristic function of the increments and can thus capture additional information on the process as compared to the conventional time-averaged mean-square displacement. The estimators are first investigated and validated for several models of anomalous diffusion, such as ergodic fractional Brownian motion and diffusion on percolating clusters, and nonergodic continuous-time random walks and scaled Brownian motion. The estimators are then applied to two sets of earlier published trajectories of mRNA molecules inside live Escherichia coli cells and of Kv2.1 potassium channels in the plasma membrane. These statistical tests did not reveal nonergodic features in the former set, while some trajectories of the latter set could be classified as nonergodic. Time averages along such trajectories are thus not representative and may be strongly misleading. Since the estimators do not rely on ensemble averages, the nonergodic features can be revealed separately for each trajectory, providing a more flexible and reliable analysis of single-particle tracking experiments in microbiology.

  1. Multi-omics maps of cotton fibre reveal epigenetic basis for staged single-cell differentiation

    PubMed Central

    Wang, Maojun; Wang, Pengcheng; Tu, Lili; Zhu, Sitao; Zhang, Lin; Li, Zhonghua; Zhang, Qinghua; Yuan, Daojun; Zhang, Xianlong

    2016-01-01

    Epigenetic modifications are highlighted for their great importance in regulating plant development, but their function associated with single-cell differentiation remains undetermined. Here, we used the cotton fibre, which is the epidermal hair on the cotton ovule, as a model to investigate the regulatory role of DNA methylation in cell differentiation. The level of CHH (H = A, T, or C) DNA methylation level was found to increase during fibre development, accompanied by a decrease in RNA-directed DNA methylation (RdDM). Examination of nucleosome positioning revealed a gradual transition from euchromatin to heterochromatin for chromatin dynamics in developing fibres, which could shape the DNA methylation landscape. The observed increase in DNA methylation in fibres, compared with other ovule tissue, was demonstrated to be mediated predominantly by an active H3K9me2-dependent pathway rather than the RdDM pathway, which was inactive. Furthermore, integrated multi-omics analyses revealed that dynamic DNA methylation played a role in the regulation of lipid biosynthesis and spatio-temporal modulation of reactive oxygen species during fibre differentiation. Our study illustrates two divergent pathways mediating a continuous increase of DNA methylation and also sheds further light on the epigenetic basis for single-cell differentiation in plants. These data and analyses are made available to the wider research community through a comprehensive web portal. PMID:27067544

  2. Revealing nonergodic dynamics in living cells from a single particle trajectory

    NASA Astrophysics Data System (ADS)

    Lanoiselée, Yann; Grebenkov, Denis S.

    2016-05-01

    We propose the improved ergodicity and mixing estimators to identify nonergodic dynamics from a single particle trajectory. The estimators are based on the time-averaged characteristic function of the increments and can thus capture additional information on the process as compared to the conventional time-averaged mean-square displacement. The estimators are first investigated and validated for several models of anomalous diffusion, such as ergodic fractional Brownian motion and diffusion on percolating clusters, and nonergodic continuous-time random walks and scaled Brownian motion. The estimators are then applied to two sets of earlier published trajectories of mRNA molecules inside live Escherichia coli cells and of Kv2.1 potassium channels in the plasma membrane. These statistical tests did not reveal nonergodic features in the former set, while some trajectories of the latter set could be classified as nonergodic. Time averages along such trajectories are thus not representative and may be strongly misleading. Since the estimators do not rely on ensemble averages, the nonergodic features can be revealed separately for each trajectory, providing a more flexible and reliable analysis of single-particle tracking experiments in microbiology.

  3. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin.

    PubMed

    Janssen, Aniek; Breuer, Gregory A; Brinkman, Eva K; van der Meulen, Annelot I; Borden, Sean V; van Steensel, Bas; Bindra, Ranjit S; LaRocque, Jeannine R; Karpen, Gary H

    2016-07-15

    Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context.Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains. PMID:27474442

  4. Multi-omics maps of cotton fibre reveal epigenetic basis for staged single-cell differentiation.

    PubMed

    Wang, Maojun; Wang, Pengcheng; Tu, Lili; Zhu, Sitao; Zhang, Lin; Li, Zhonghua; Zhang, Qinghua; Yuan, Daojun; Zhang, Xianlong

    2016-05-19

    Epigenetic modifications are highlighted for their great importance in regulating plant development, but their function associated with single-cell differentiation remains undetermined. Here, we used the cotton fibre, which is the epidermal hair on the cotton ovule, as a model to investigate the regulatory role of DNA methylation in cell differentiation. The level of CHH (H = A, T, or C) DNA methylation level was found to increase during fibre development, accompanied by a decrease in RNA-directed DNA methylation (RdDM). Examination of nucleosome positioning revealed a gradual transition from euchromatin to heterochromatin for chromatin dynamics in developing fibres, which could shape the DNA methylation landscape. The observed increase in DNA methylation in fibres, compared with other ovule tissue, was demonstrated to be mediated predominantly by an active H3K9me2-dependent pathway rather than the RdDM pathway, which was inactive. Furthermore, integrated multi-omics analyses revealed that dynamic DNA methylation played a role in the regulation of lipid biosynthesis and spatio-temporal modulation of reactive oxygen species during fibre differentiation. Our study illustrates two divergent pathways mediating a continuous increase of DNA methylation and also sheds further light on the epigenetic basis for single-cell differentiation in plants. These data and analyses are made available to the wider research community through a comprehensive web portal.

  5. Design principles of natural light-harvesting as revealed by single molecule spectroscopy

    NASA Astrophysics Data System (ADS)

    Krüger, T. P. J.; van Grondelle, R.

    2016-01-01

    Biology offers a boundless source of adaptation, innovation, and inspiration. A wide range of photosynthetic organisms exist that are capable of harvesting solar light in an exceptionally efficient way, using abundant and low-cost materials. These natural light-harvesting complexes consist of proteins that strongly bind a high density of chromophores to capture solar photons and rapidly transfer the excitation energy to the photochemical reaction centre. The amount of harvested light is also delicately tuned to the level of solar radiation to maintain a constant energy throughput at the reaction centre and avoid the accumulation of the products of charge separation. In this Review, recent developments in the understanding of light-harvesting by plants will be discussed, based on results obtained from single molecule spectroscopy studies. Three design principles of the main light-harvesting antenna of plants will be highlighted: (a) fine, photoactive control over the intrinsic protein disorder to efficiently use intrinsically available thermal energy dissipation mechanisms; (b) the design of the protein microenvironment of a low-energy chromophore dimer to control the amount of shade absorption; (c) the design of the exciton manifold to ensure efficient funneling of the harvested light to the terminal emitter cluster.

  6. Conformational selection in a protein-protein interaction revealed by dynamic pathway analysis

    SciTech Connect

    Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco; Otten, Renee; Higgins, Matthew K.; Schertler, Gebhard F. X.; Oprian, Daniel D.; Kern, Dorothee

    2015-12-24

    Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Lastly, protein dynamics in free recoverin limits the overall rate of binding.

  7. Conformational selection in a protein-protein interaction revealed by dynamic pathway analysis

    DOE PAGES

    Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco; Otten, Renee; Higgins, Matthew K.; Schertler, Gebhard F. X.; Oprian, Daniel D.; Kern, Dorothee

    2015-12-24

    Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsinmore » kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Lastly, protein dynamics in free recoverin limits the overall rate of binding.« less

  8. Conformational Selection in a Protein-Protein Interaction revealed by Dynamic Pathway Analysis

    PubMed Central

    Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco; Otten, Renee; Higgins, Matthew K.; Schertler, Gebhard F. X.; Oprian, Daniel D.; Kern, Dorothee

    2015-01-01

    SUMMARY Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding. PMID:26725117

  9. Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins.

    PubMed

    Narumi, Ryohei; Shimizu, Yoshihiro; Ukai-Tadenuma, Maki; Ode, Koji L; Kanda, Genki N; Shinohara, Yuta; Sato, Aya; Matsumoto, Katsuhiko; Ueda, Hiroki R

    2016-06-14

    Absolute values of protein expression levels in cells are crucial information for understanding cellular biological systems. Precise quantification of proteins can be achieved by liquid chromatography (LC)-mass spectrometry (MS) analysis of enzymatic digests of proteins in the presence of isotope-labeled internal standards. Thus, development of a simple and easy way for the preparation of internal standards is advantageous for the analyses of multiple target proteins, which will allow systems-level studies. Here we describe a method, termed MS-based Quantification By isotope-labeled Cell-free products (MS-QBiC), which provides the simple and high-throughput preparation of internal standards by using a reconstituted cell-free protein synthesis system, and thereby facilitates both multiplexed and sensitive quantification of absolute amounts of target proteins. This method was applied to a systems-level dynamic analysis of mammalian circadian clock proteins, which consist of transcription factors and protein kinases that govern central and peripheral circadian clocks in mammals. Sixteen proteins from 20 selected circadian clock proteins were successfully quantified from mouse liver over a 24-h time series, and 14 proteins had circadian variations. Quantified values were applied to detect internal body time using a previously developed molecular timetable method. The analyses showed that single time-point data from wild-type mice can predict the endogenous state of the circadian clock, whereas data from clock mutant mice are not applicable because of the disappearance of circadian variation. PMID:27247408

  10. The oral immunogenicity of BioProtein, a bacterial single-cell protein, is affected by its particulate nature.

    PubMed

    Christensen, Hanne R; Larsen, Linea C; Frøkiaer, Hanne

    2003-07-01

    The bacterial single-cell protein BioProtein (BP; Norferm Danmark, Odense, Denmark), produced by fermentation of natural gas with methanotrophic bacteria, is a potential protein source for man and animals. For human consumption, removal of the nucleic acid is necessary. Preliminary studies have shown that ingested BP induces a specific immune response. The objective of the present study was to characterize the type of response, its development over time and product-related causative factors. Mice were fed with diets containing 60 g nucleic acid-reduced BP/kg, 240 g nucleic acid-reduced BP/kg, 240 g untreated BP (basic BP)/kg or 240 g casein/kg (control). In another study, mice were fed 240 g basic BP/kg, whole cell-free BP-culture homogenate or control diet. The immune response was monitored using an ELISA for BP-specific immunoglobulin in blood and BP-specific immunoglobulin A in blood and saliva. Ingested BP induced a steady specific mucosal and systemic immune response, characterized by a dose-dependent production of immunoglobulin and immunoglobulin A in blood and immunoglobulin A in saliva. Basic BP and nucleic acid-reduced BP induced identical responses. However, feeding mice BP-culture homogenate induced immunoglobulin A in saliva but there was no systemic response. The antibodies from BP-fed mice cross-reacted with BP-culture homogenate revealing the presence of the same antigenic components in the two products despite the different oral immunogenicity. Thus, ingestion of BP induces a persistent mucosal and systemic immune response of which the systemic response can be avoided by ingesting a BP preparation free of whole cells. This indicates the importance of the non-particulate constitution of single-cell protein products intended for human or animal consumption.

  11. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    SciTech Connect

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  12. Single nucleotide polymorphism analysis reveals heterogeneity within a seedling tree population of a polyembryonic mango cultivar.

    PubMed

    Winterhagen, Patrick; Wünsche, Jens-Norbert

    2016-05-01

    Within a polyembryonic mango seedling tree population, the genetic background of individuals should be identical because vigorous plants for cultivation are expected to develop from nucellar embryos representing maternal clones. Due to the fact that the mango cultivar 'Hôi' is assigned to the polyembryonic ecotype, an intra-cultivar variability of ethylene receptor genes was unexpected. Ethylene receptors in plants are conserved, but the number of receptors or receptor isoforms is variable regarding different plant species. However, it is shown here that the ethylene receptor MiETR1 is present in various isoforms within the mango cultivar 'Hôi'. The investigation of single nucleotide polymorphisms revealed that different MiETR1 isoforms can not be discriminated simply by individual single nucleotide exchanges but by the specific arrangement of single nucleotide polymorphisms at certain positions in the exons of MiETR1. Furthermore, an MiETR1 isoform devoid of introns in the genomic sequence was identified. The investigation demonstrates some limitations of high resolution melting and ScreenClust analysis and points out the necessity of sequencing to identify individual isoforms and to determine the variability within the tree population. PMID:27093244

  13. Ecology of uncultured Prochlorococcus clades revealed through single-cell genomics and biogeographic analysis

    PubMed Central

    Malmstrom, Rex R; Rodrigue, Sébastien; Huang, Katherine H; Kelly, Libusha; Kern, Suzanne E; Thompson, Anne; Roggensack, Sara; Berube, Paul M; Henn, Matthew R; Chisholm, Sallie W

    2013-01-01

    Prochlorococcus is the numerically dominant photosynthetic organism throughout much of the world's oceans, yet little is known about the ecology and genetic diversity of populations inhabiting tropical waters. To help close this gap, we examined natural Prochlorococcus communities in the tropical Pacific Ocean using a single-cell whole-genome amplification and sequencing. Analysis of the gene content of just 10 single cells from these waters added 394 new genes to the Prochlorococcus pan-genome—that is, genes never before seen in a Prochlorococcus cell. Analysis of marker genes, including the ribosomal internal transcribed sequence, from dozens of individual cells revealed several representatives from two uncultivated clades of Prochlorococcus previously identified as HNLC1 and HNLC2. While the HNLC clades can dominate Prochlorococcus communities under certain conditions, their overall geographic distribution was highly restricted compared with other clades of Prochlorococcus. In the Atlantic and Pacific oceans, these clades were only found in warm waters with low Fe and high inorganic P levels. Genomic analysis suggests that at least one of these clades thrives in low Fe environments by scavenging organic-bound Fe, a process previously unknown in Prochlorococcus. Furthermore, the capacity to utilize organic-bound Fe appears to have been acquired horizontally and may be exchanged among other clades of Prochlorococcus. Finally, one of the single Prochlorococcus cells sequenced contained a partial genome of what appears to be a prophage integrated into the genome. PMID:22895163

  14. Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein

    SciTech Connect

    Arpino, James A. J.; Rizkallah, Pierre J.; Jones, D. Dafydd

    2014-08-01

    The beneficial engineered single-amino-acid deletion variants EGFP{sup D190Δ} and EGFP{sup A227Δ} have been studied. Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFP{sup D190Δ} containing a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFP{sup A227Δ} revealed that a ‘flipping’ mechanism was used to adjust for residue deletion at the end of a β-strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.

  15. Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids.

    PubMed Central

    Folmer, R H; Nilges, M; Konings, R N; Hilbers, C W

    1995-01-01

    The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins. Images PMID:7556054

  16. Rhabdovirus matrix protein structures reveal a novel mode of self-association.

    PubMed

    Graham, Stephen C; Assenberg, René; Delmas, Olivier; Verma, Anil; Gholami, Alireza; Talbi, Chiraz; Owens, Raymond J; Stuart, David I; Grimes, Jonathan M; Bourhy, Hervé

    2008-12-01

    The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.

  17. Sequential Layer Analysis of Protein Immunosensors based on Single Wall Carbon Nanotube Forests

    PubMed Central

    Malhotra, Ruchika; Papadimitrakopoulos, Fotios; Rusling, James F.

    2010-01-01

    Electrochemical immunosensors using vertically aligned single wall carbon nanotube (SWNT) forests can provide ultrasensitive, accurate cancer biomarker protein assays. Herein we report a systematic investigation of the structure, thickness and functionality of each layer of these immunosensors using atomic force microscopy (AFM), quartz crystal microbalance (QCM) and scanning white light interferometry (SWLI). This provides a detailed picture of the surface morphology of each layer along with surface concentration and thickness of each protein layer. Results reveal that the major reasons for sensitivity gain can be assigned to the dense packing of carboxylated SWNT forest tips, which translate to a large surface concentration of capture antibodies, together with the high quality of conductive SWNT forests. PMID:20731335

  18. Single-stranded DNA-binding proteins: multiple domains for multiple functions.

    PubMed

    Dickey, Thayne H; Altschuler, Sarah E; Wuttke, Deborah S

    2013-07-01

    The recognition of single-stranded DNA (ssDNA) is integral to myriad cellular functions. In eukaryotes, ssDNA is present stably at the ends of chromosomes and at some promoter elements. Furthermore, it is formed transiently by several cellular processes including telomere synthesis, transcription, and DNA replication, recombination, and repair. To coordinate these diverse activities, a variety of proteins have evolved to bind ssDNA in a manner specific to their function. Here, we review the recognition of ssDNA through the analysis of high-resolution structures of proteins in complex with ssDNA. This functionally diverse set of proteins arises from a limited set of structural motifs that can be modified and arranged to achieve distinct activities, including a range of ligand specificities. We also investigate the ways in which these domains interact in the context of large multidomain proteins/complexes. These comparisons reveal the structural features that define the range of functions exhibited by these proteins.

  19. Single-Stranded DNA-Binding Proteins: Multiple Domains for Multiple Functions

    PubMed Central

    Dickey, Thayne H.; Altschuler, Sarah E.; Wuttke, Deborah S.

    2013-01-01

    The recognition of single-stranded DNA (ssDNA) is integral to myriad cellular functions. In eukaryotes, ssDNA is present stably at the ends of chromosomes and at some promoter elements. Furthermore, it is formed transiently by several cellular processes including telomere synthesis, transcription, and DNA replication, recombination, and repair. To coordinate these diverse activities, a variety of proteins have evolved to bind ssDNA in a manner specific to their function. Here, we review the recognition of ssDNA through the analysis of high-resolution structures of proteins in complex with ssDNA. This functionally diverse set of proteins arises from a limited set of structural motifs that can be modified and arranged to achieve distinct activities, including a range of ligand specificities. We also investigate the ways in which these domains interact in the context of large multidomain proteins/complexes. These comparisons reveal the structural features that define the range of functions exhibited by these proteins. PMID:23823326

  20. Evidence for a single steroid-binding protein in the rabbit progesterone receptor.

    PubMed

    Lamb, D J; Kima, P E; Bullock, D W

    1986-10-01

    The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.

  1. Putative protein partners for the human CPI-17 protein revealed by bacterial two-hybrid screening.

    PubMed

    Kim, Kyung-mi; Adyshev, Djanybek M; Kása, Anita; Zemskov, Evgeny A; Kolosova, Irina A; Csortos, Csilla; Verin, Alexander D

    2013-07-01

    We have previously demonstrated that PKC-potentiated inhibitory protein of protein phosphatase-1 (CPI-17) is expressed in lung endothelium. CPI-17, a specific inhibitor of myosin light chain phosphatase (MLCP), is involved in the endothelial cytoskeletal and barrier regulation. In this paper, we report the identification of fourteen putative CPI-17 interacting proteins in the lung using BacterioMatch Two-Hybrid System. Five of them: plectin 1 isoform 1, alpha II spectrin, OK/SW-CL.16, gelsolin isoform a, and junction plakoglobin are involved in actin cytoskeleton organization and cell adhesion, suggesting possible significance of these binding partners in CPI-17-mediated cytoskeletal reorganization of endothelial cells. Furthermore, we confirmed the specific interaction between plakoglobin and CPI-17, which is affected by the phosphorylation status of CPI-17 in human lung microvascular endothelial cells. PMID:23583905

  2. Why do proteins aggregate? "Intrinsically insoluble proteins" and "dark mediators" revealed by studies on "insoluble proteins" solubilized in pure water.

    PubMed

    Song, Jianxing

    2013-01-01

    In 2008, I reviewed and proposed a model for our discovery in 2005 that unrefoldable and insoluble proteins could in fact be solubilized in unsalted water. Since then, this discovery has offered us and other groups a powerful tool to characterize insoluble proteins, and we have further addressed several fundamental and disease-relevant issues associated with this discovery. Here I review these results, which are conceptualized into several novel scenarios. 1) Unlike 'misfolded proteins', which still retain the capacity to fold into well-defined structures but are misled to 'off-pathway' aggregation, unrefoldable and insoluble proteins completely lack this ability and will unavoidably aggregate in vivo with ~150 mM ions, thus designated as 'intrinsically insoluble proteins (IIPs)' here. IIPs may largely account for the 'wastefully synthesized' DRiPs identified in human cells. 2) The fact that IIPs including membrane proteins are all soluble in unsalted water, but get aggregated upon being exposed to ions, logically suggests that ions existing in the background play a central role in mediating protein aggregation, thus acting as 'dark mediators'. Our study with 14 salts confirms that IIPs lack the capacity to fold into any well-defined structures. We uncover that salts modulate protein dynamics and anions bind proteins with high selectivity and affinity, which is surprisingly masked by pre-existing ions. Accordingly, I modified my previous model. 3) Insoluble proteins interact with lipids to different degrees. Remarkably, an ALS-causing P56S mutation transforms the β-sandwich MSP domain into a helical integral membrane protein. Consequently, the number of membrane-interacting proteins might be much larger than currently recognized. To attack biological membranes may represent a common mechanism by which aggregated proteins initiate human diseases. 4) Our discovery also implies a solution to the 'chicken-and-egg paradox' for the origin of primitive membranes embedded

  3. Proteomic Profiling of Cereal Aphid Saliva Reveals Both Ubiquitous and Adaptive Secreted Proteins

    PubMed Central

    Wilkinson, Tom L.

    2013-01-01

    The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113. PMID:23460852

  4. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn

    PubMed Central

    Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

    2015-01-01

    The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize. PMID:26587848

  5. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition.

    PubMed

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-28

    Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

  6. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery

    PubMed Central

    Liu, Jin

    2016-01-01

    Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier’s principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery. PMID:27115535

  7. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

    PubMed

    Dong, Yongbin; Wang, Qilei; Zhang, Long; Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

    2015-01-01

    The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize. PMID:26587848

  8. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition.

    PubMed

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-28

    Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context. PMID:25313039

  9. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

    PubMed

    Kalescky, Robert; Zhou, Hongyu; Liu, Jin; Tao, Peng

    2016-04-01

    Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery. PMID:27115535

  10. Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons.

    PubMed

    Hu, Ganlu; Huang, Kevin; Hu, Youjin; Du, Guizhen; Xue, Zhigang; Zhu, Xianmin; Fan, Guoping

    2016-01-01

    Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death, and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP), and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons. PMID:27558660

  11. Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons

    PubMed Central

    Hu, Ganlu; Huang, Kevin; Hu, Youjin; Du, Guizhen; Xue, Zhigang; Zhu, Xianmin; Fan, Guoping

    2016-01-01

    Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death, and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP), and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons. PMID:27558660

  12. Reducing multi-sensor data to a single time course that reveals experimental effects

    PubMed Central

    2013-01-01

    Background Multi-sensor technologies such as EEG, MEG, and ECoG result in high-dimensional data sets. Given the high temporal resolution of such techniques, scientific questions very often focus on the time-course of an experimental effect. In many studies, researchers focus on a single sensor or the average over a subset of sensors covering a “region of interest” (ROI). However, single-sensor or ROI analyses ignore the fact that the spatial focus of activity is constantly changing, and fail to make full use of the information distributed over the sensor array. Methods We describe a technique that exploits the optimality and simplicity of matched spatial filters in order to reduce experimental effects in multivariate time series data to a single time course. Each (multi-sensor) time sample of each trial is replaced with its projection onto a spatial filter that is matched to an observed experimental effect, estimated from the remaining trials (Effect-Matched Spatial filtering, or EMS filtering). The resulting set of time courses (one per trial) can be used to reveal the temporal evolution of an experimental effect, which distinguishes this approach from techniques that reveal the temporal evolution of an anatomical source or region of interest. Results We illustrate the technique with data from a dual-task experiment and use it to track the temporal evolution of brain activity during the psychological refractory period. We demonstrate its effectiveness in separating the means of two experimental conditions, and in significantly improving the signal-to-noise ratio at the single-trial level. It is fast to compute and results in readily-interpretable time courses and topographies. The technique can be applied to any data-analysis question that can be posed independently at each sensor, and we provide one example, using linear regression, that highlights the versatility of the technique. Conclusion The approach described here combines established techniques in a

  13. Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle

    PubMed Central

    Yang, Yalan; Tang, Zhonglin; Fan, Xinhao; Xu, Kui; Mu, Yulian; Zhou, Rong; Li, Kui

    2016-01-01

    Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals. PMID:27352850

  14. Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle.

    PubMed

    Yang, Yalan; Tang, Zhonglin; Fan, Xinhao; Xu, Kui; Mu, Yulian; Zhou, Rong; Li, Kui

    2016-01-01

    Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals. PMID:27352850

  15. Homologous Expression of the Caldicellulosiruptor bescii CelA Reveals that the Extracellular Protein Is Glycosylated

    PubMed Central

    Bomble, Yannick J.; Vander Wall, Todd A.; Groom, Joseph; Himmel, Michael E.; Westpheling, Janet

    2015-01-01

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure

  16. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated

    DOE PAGES

    Chung, Daehwan; Young, Jenna; Bomble, Yannick J.; Vander Wall, Todd A.; Groom, Joseph; Himmel, Michael E.; Westpheling, Janet

    2015-03-23

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm formore » cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. Finally, it will also allow the study of glycosylation of CelA itself and its role

  17. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    PubMed Central

    Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

    2015-01-01

    Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

  18. Identification of novel peptide substrates for protein farnesyltransferase reveals two substrate classes with distinct sequence selectivities

    PubMed Central

    Hougland, James L.; Hicks, Katherine A.; Hartman, Heather L.; Kelly, Rebekah A.; Watt, Terry J.; Fierke, Carol A.

    2010-01-01

    Prenylation is a post-translational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development and farnesyltransferase inhibitors (FTIs) are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for FTI efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover conditions. Based on these results, a second library was designed that yielded an additional 29 novel multiple-turnover FTase substrates and 45 single-turnover substrates. The two classes of substrates exhibit different specificity requirements. Efficient multiple-turnover reactivity correlates with the presence of a nonpolar amino acid at the a2 position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca1a2X sequence model. In contrast, the sequences of the single-turnover substrates vary significantly more at both the a2 and X residues and are not well-described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets

  19. Direct observation of TALE protein dynamics reveals a two-state search mechanism.

    PubMed

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M

    2015-06-01

    Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Here we use single-molecule techniques to directly observe TALE search dynamics along DNA templates. We find that TALE proteins are capable of rapid diffusion along DNA using a combination of sliding and hopping behaviour, which suggests that the TALE search process is governed in part by facilitated diffusion. We also observe that TALE proteins exhibit two distinct modes of action during the search process-a search state and a recognition state-facilitated by different subdomains in monomeric TALE proteins. Using TALE truncation mutants, we further demonstrate that the N-terminal region of TALEs is required for the initial non-specific binding and subsequent rapid search along DNA, whereas the central repeat domain is required for transitioning into the site-specific recognition state.

  20. Direct observation of TALE protein dynamics reveals a two-state search mechanism

    PubMed Central

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

    2015-01-01

    Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Here we use single-molecule techniques to directly observe TALE search dynamics along DNA templates. We find that TALE proteins are capable of rapid diffusion along DNA using a combination of sliding and hopping behaviour, which suggests that the TALE search process is governed in part by facilitated diffusion. We also observe that TALE proteins exhibit two distinct modes of action during the search process—a search state and a recognition state—facilitated by different subdomains in monomeric TALE proteins. Using TALE truncation mutants, we further demonstrate that the N-terminal region of TALEs is required for the initial non-specific binding and subsequent rapid search along DNA, whereas the central repeat domain is required for transitioning into the site-specific recognition state. PMID:26027871

  1. Direct observation of TALE protein dynamics reveals a two-state search mechanism

    NASA Astrophysics Data System (ADS)

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

    2015-06-01

    Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Here we use single-molecule techniques to directly observe TALE search dynamics along DNA templates. We find that TALE proteins are capable of rapid diffusion along DNA using a combination of sliding and hopping behaviour, which suggests that the TALE search process is governed in part by facilitated diffusion. We also observe that TALE proteins exhibit two distinct modes of action during the search process--a search state and a recognition state--facilitated by different subdomains in monomeric TALE proteins. Using TALE truncation mutants, we further demonstrate that the N-terminal region of TALEs is required for the initial non-specific binding and subsequent rapid search along DNA, whereas the central repeat domain is required for transitioning into the site-specific recognition state.

  2. Ideal probe single-molecule experiments reveal the intrinsic dynamic heterogeneity of a supercooled liquid

    PubMed Central

    Paeng, Keewook; Park, Heungman; Hoang, Dat Tien; Kaufman, Laura J.

    2015-01-01

    The concept of dynamic heterogeneity and the picture of the supercooled liquid as a mosaic of environments with distinct dynamics that interchange in time have been invoked to explain the nonexponential relaxations measured in these systems. The spatial extent and temporal persistence of these regions of distinct dynamics have remained challenging to identify. Here, single-molecule fluorescence measurements using a probe similar in size and mobility to the host o-terphenyl unambiguously reveal exponential relaxations distributed in time and space and directly demonstrate ergodicity of the system down to the glass transition temperature. In the temperature range probed, at least 200 times the structural relaxation time of the host is required to recover ensemble-averaged relaxation at every spatial region in the system. PMID:25825739

  3. Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment.

    PubMed

    van Wolfswinkel, Josien C; Wagner, Daniel E; Reddien, Peter W

    2014-09-01

    Planarians are flatworms capable of regenerating any missing body region. This capacity is mediated by neoblasts, a proliferative cell population that contains pluripotent stem cells. Although population-based studies have revealed many neoblast characteristics, whether functionally distinct classes exist within this population is unclear. Here, we used high-dimensional single-cell transcriptional profiling from over a thousand individual neoblasts to directly compare gene expression fingerprints during homeostasis and regeneration. We identified two prominent neoblast classes that we named ζ (zeta) and σ (sigma). Zeta-neoblasts encompass specified cells that give rise to an abundant postmitotic lineage, including epidermal cells, and are not required for regeneration. By contrast, sigma-neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta-neoblasts. These findings indicate that planarian neoblasts comprise two major and functionally distinct cellular compartments.

  4. Dichotomy of cellular inhibition by small-molecule inhibitors revealed by single-cell analysis

    PubMed Central

    Vogel, Robert M.; Erez, Amir; Altan-Bonnet, Grégoire

    2016-01-01

    Despite progress in drug development, a quantitative and physiological understanding of how small-molecule inhibitors act on cells is lacking. Here, we measure the signalling and proliferative response of individual primary T-lymphocytes to a combination of antigen, cytokine and drug. We uncover two distinct modes of signalling inhibition: digital inhibition (the activated fraction of cells diminishes upon drug treatment, but active cells appear unperturbed), versus analogue inhibition (the activated fraction is unperturbed whereas activation response is diminished). We introduce a computational model of the signalling cascade that accounts for such inhibition dichotomy, and test the model predictions for the phenotypic variability of cellular responses. Finally, we demonstrate that the digital/analogue dichotomy of cellular response as revealed on short (signal transduction) timescales, translates into similar dichotomy on longer (proliferation) timescales. Our single-cell analysis of drug action illustrates the strength of quantitative approaches to translate in vitro pharmacology into functionally relevant cellular settings. PMID:27687249

  5. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    PubMed Central

    Macaulay, Iain C.; Svensson, Valentine; Labalette, Charlotte; Ferreira, Lauren; Hamey, Fiona; Voet, Thierry; Teichmann, Sarah A.; Cvejic, Ana

    2016-01-01

    Summary The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment. PMID:26804912

  6. Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment

    PubMed Central

    van Wolfswinkel, Josien C.; Wagner, Daniel E.; Reddien, Peter W.

    2014-01-01

    Planarians are flatworms capable of regenerating any missing body region. This capacity is mediated by neoblasts, a proliferative cell population that contains pluripotent stem cells. Although population-based studies have revealed many neoblast characteristics, whether functionally distinct classes exist within this population is unclear. Here, we used high-dimensional single-cell transcriptional profiling from over a thousand individual neoblasts to directly compare gene expression fingerprints during homeostasis and regeneration. We identified two prominent neoblast classes that we named ζ (zeta) and σ (sigma). Zeta-neoblasts encompass specified cells that give rise to an abundant postmitotic lineage including epidermal cells, and are not required for regeneration. By contrast, sigma-neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta-neoblasts. These findings present a new view of planarian neoblasts, in which the population is comprised of two major and functionally distinct cellular compartments. PMID:25017721

  7. Imaging single endocytic events reveals diversity in clathrin, dynamin and vesicle dynamics.

    PubMed

    Mattheyses, Alexa L; Atkinson, Claire E; Simon, Sanford M

    2011-10-01

    The dynamics of clathrin-mediated endocytosis can be assayed using fluorescently tagged proteins and total internal reflection fluorescence microscopy. Many of these proteins, including clathrin and dynamin, are soluble and changes in fluorescence intensity can be attributed either to membrane/vesicle movement or to changes in the numbers of individual molecules. It is important for assays to discriminate between physical membrane events and the dynamics of molecules. Two physical events in endocytosis were investigated: vesicle scission from the plasma membrane and vesicle internalization. Single vesicle analysis allowed the characterization of dynamin and clathrin dynamics relative to scission and internalization. We show that vesicles remain proximal to the plasma membrane for variable amounts of time following scission, and that uncoating of clathrin can occur before or after vesicle internalization. The dynamics of dynamin also vary with respect to scission. Results from assays based on physical events suggest that disappearance of fluorescence from the evanescent field should be re-evaluated as an assay for endocytosis. These results illustrate the heterogeneity of behaviors of endocytic vesicles and the importance of establishing suitable evaluation criteria for biophysical processes.

  8. Raman spectroscopy of single extracellular vesicles reveals subpopulations with varying membrane content (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit S.; Saari, Heikki; Lazaro Ibañez, Elisa; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2016-03-01

    Exosomes are small (~100nm) membrane bound vesicles excreted by cells as part of their normal biological processes. These extracellular vesicles are currently an area of intense research, since they were recently found to carry functional mRNA that allows transfer of proteins and other cellular instructions between cells. Exosomes have been implicated in a wide range of diseases, including cancer. Cancer cells are known to have increased exosome production, and may use those exosomes to prepare remote environments for metastasis. Therefore, there is a strong need to develop characterization methods to help understand the structure and function of these vesicles. However, current techniques, such as proteomics and genomics technologies, rely on aggregating a large amount of exosome material and reporting on chemical content that is averaged over many millions of exosomes. Here we report on the use of laser-tweezers Raman spectroscopy (LTRS) to probe individual vesicles, discovering distinct heterogeneity among exosomes both within a cell line, as well as between different cell lines. Through principal components analysis followed by hierarchical clustering, we have identified four "subpopulations" of exosomes shared across seven cell lines. The key chemical differences between these subpopulations, as determined by spectral analysis of the principal component loadings, are primarily related to membrane composition. Specifically, the differences can be ascribed to cholesterol content, cholesterol to phospholipid ratio, and surface protein expression. Thus, we have shown LTRS to be a powerful method to probe the chemical content of single extracellular vesicles.

  9. Structure-function insights of membrane and soluble proteins revealed by electron crystallography.

    PubMed

    Dreaden, Tina M; Devarajan, Bharanidharan; Barry, Bridgette A; Schmidt-Krey, Ingeborg

    2013-01-01

    Electron crystallography is emerging as an important method in solving protein structures. While it has found extensive applications in the understanding of membrane protein structure and function at a wide range of resolutions, from revealing oligomeric arrangements to atomic models, electron crystallography has also provided invaluable information on the soluble α/β-tubulin which could not be obtained by any other method to date. Examples of critical insights from selected structures of membrane proteins as well as α/β-tubulin are described here, demonstrating the vast potential of electron crystallography that is first beginning to unfold.

  10. Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

    PubMed

    Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

    2005-07-01

    Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

  11. Genomewide Analysis Reveals Novel Pathways Affecting Endoplasmic Reticulum Homeostasis, Protein Modification and Quality Control

    PubMed Central

    Čopič, Alenka; Dorrington, Mariana; Pagant, Silvere; Barry, Justine; Lee, Marcus C. S.; Singh, Indira; Hartman, John L.; Miller, Elizabeth A.

    2009-01-01

    To gain new mechanistic insight into ER homeostasis and the biogenesis of secretory proteins, we screened a genomewide collection of yeast mutants for defective intracellular retention of the ER chaperone, Kar2p. We identified 87 Kar2p-secreting strains, including a number of known components in secretory protein modification and sorting. Further characterization of the 73 nonessential Kar2p retention mutants revealed roles for a number of novel gene products in protein glycosylation, GPI-anchor attachment, ER quality control, and retrieval of escaped ER residents. A subset of these mutants, required for ER retrieval, included the GET complex and two novel proteins that likely function similarly in membrane insertion of tail-anchored proteins. Finally, the variant histone, Htz1p, and its acetylation state seem to play an important role in maintaining ER retrieval pathways, suggesting a surprising link between chromatin remodeling and ER homeostasis. PMID:19433630

  12. Quasi-elastic neutron scattering reveals ligand-induced protein dynamics of a G-protein-coupled receptor

    DOE PAGES

    Shrestha, Utsab R.; Perera, Suchithranga M. D. C.; Bhowmik, Debsindhu; Chawla, Udeep; Mamontov, Eugene; Brown, Michael F.; Chu, Xiang -Qiang

    2016-09-15

    Light activation of the visual G-protein-coupled receptor (GPCR) rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane yielding the activation of cognate G-protein (transducin), which initiates biological signaling. Here, we report a quasi-elastic neutron scattering study of the activation of rhodopsin as a GPCR prototype. Our results reveal a broadly distributed relaxation of hydrogen atom dynamics of rhodopsin on a picosecond–nanosecond time scale, crucial for protein function, as only observed for globular proteins previously. Interestingly, the results suggest significant differences in the intrinsic protein dynamics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. These differencesmore » can be attributed to the influence of the covalently bound retinal ligand. Moreover, an idea of the generic free-energy landscape is used to explain the GPCR dynamics of ligand-binding and ligand-free protein conformations, which can be further applied to other GPCR systems.« less

  13. The Protein Architecture of Human Secretory Vesicles Reveals Differential Regulation of Signaling Molecule Secretion by Protein Kinases

    PubMed Central

    Taupenot, Laurent; Ziegler, Michael; O'Connor, Daniel T.; Ma, Qi; Smoot, Michael; Ideker, Trey; Hook, Vivian

    2012-01-01

    Secretory vesicles are required for release of chemical messengers to mediate intercellular signaling among human biological systems. It is necessary to define the organization of the protein architecture of the ‘human’ dense core secretory vesicles (DCSV) to understand mechanisms for secretion of signaling molecules essential for cellular regulatory processes. This study, therefore, conducted extensive quantitative proteomics and systems biology analyses of human DCSV purified from human pheochromocytoma. Over 600 human DCSV proteins were identified with quantitative evaluation of over 300 proteins, revealing that most proteins participate in producing peptide hormones and neurotransmitters, enzymes, and the secretory machinery. Systems biology analyses provided a model of interacting DCSV proteins, generating hypotheses for differential intracellular protein kinases A and C signaling pathways. Activation of cellular PKA and PKC pathways resulted in differential secretion of neuropeptides, catecholamines, and β-amyloid of Alzheimer's disease for mediating cell-cell communication. This is the first study to define a model of the protein architecture of human DCSV for human disease and health. PMID:22916103

  14. Structural basis for the modular recognition of single-stranded RNA by PPR proteins.

    PubMed

    Yin, Ping; Li, Quanxiu; Yan, Chuangye; Liu, Ying; Liu, Junjie; Yu, Feng; Wang, Zheng; Long, Jiafu; He, Jianhua; Wang, Hong-Wei; Wang, Jiawei; Zhu, Jian-Kang; Shi, Yigong; Yan, Nieng

    2013-12-01

    Pentatricopeptide repeat (PPR) proteins represent a large family of sequence-specific RNA-binding proteins that are involved in multiple aspects of RNA metabolism. PPR proteins, which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants, recognize single-stranded RNA (ssRNA) in a modular fashion. The maize chloroplast protein PPR10 binds to two similar RNA sequences from the ATPI-ATPH and PSAJ-RPL33 intergenic regions, referred to as ATPH and PSAJ, respectively. By protecting the target RNA elements from 5' or 3' exonucleases, PPR10 defines the corresponding 5' and 3' messenger RNA termini. Despite rigorous functional characterizations, the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated. Here we report the crystal structures of PPR10 in RNA-free and RNA-bound states at resolutions of 2.85 and 2.45 Å, respectively. In the absence of RNA binding, the nineteen repeats of PPR10 are assembled into a right-handed superhelical spiral. PPR10 forms an antiparallel, intertwined homodimer and exhibits considerable conformational changes upon binding to its target ssRNA, an 18-nucleotide PSAJ element. Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10 repeats following the predicted code. The molecular basis for the specific and modular recognition of RNA bases A, G and U is revealed. The structural elucidation of RNA recognition by PPR proteins provides an important framework for potential biotechnological applications of PPR proteins in RNA-related research areas.

  15. Structural basis for the modular recognition of single-stranded RNA by PPR proteins

    NASA Astrophysics Data System (ADS)

    Yin, Ping; Li, Quanxiu; Yan, Chuangye; Liu, Ying; Liu, Junjie; Yu, Feng; Wang, Zheng; Long, Jiafu; He, Jianhua; Wang, Hong-Wei; Wang, Jiawei; Zhu, Jian-Kang; Shi, Yigong; Yan, Nieng

    2013-12-01

    Pentatricopeptide repeat (PPR) proteins represent a large family of sequence-specific RNA-binding proteins that are involved in multiple aspects of RNA metabolism. PPR proteins, which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants, recognize single-stranded RNA (ssRNA) in a modular fashion. The maize chloroplast protein PPR10 binds to two similar RNA sequences from the ATPI-ATPH and PSAJ-RPL33 intergenic regions, referred to as ATPH and PSAJ, respectively. By protecting the target RNA elements from 5' or 3' exonucleases, PPR10 defines the corresponding 5' and 3' messenger RNA termini. Despite rigorous functional characterizations, the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated. Here we report the crystal structures of PPR10 in RNA-free and RNA-bound states at resolutions of 2.85 and 2.45Å, respectively. In the absence of RNA binding, the nineteen repeats of PPR10 are assembled into a right-handed superhelical spiral. PPR10 forms an antiparallel, intertwined homodimer and exhibits considerable conformational changes upon binding to its target ssRNA, an 18-nucleotide PSAJ element. Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10 repeats following the predicted code. The molecular basis for the specific and modular recognition of RNA bases A, G and U is revealed. The structural elucidation of RNA recognition by PPR proteins provides an important framework for potential biotechnological applications of PPR proteins in RNA-related research areas.

  16. Conserved evolutionary units in the heme-copper oxidase superfamily revealed by novel homologous protein families

    PubMed Central

    Pei, Jimin; Li, Wenlin; Kinch, Lisa N; Grishin, Nick V

    2014-01-01

    The heme-copper oxidase (HCO) superfamily includes HCOs in aerobic respiratory chains and nitric oxide reductases (NORs) in the denitrification pathway. The HCO/NOR catalytic subunit has a core structure consisting of 12 transmembrane helices (TMHs) arranged in three-fold rotational pseudosymmetry, with six conserved histidines for heme and metal binding. Using sensitive sequence similarity searches, we detected a number of novel HCO/NOR homologs and named them HCO Homology (HCOH) proteins. Several HCOH families possess only four TMHs that exhibit the most pronounced similarity to the last four TMHs (TMHs 9–12) of HCOs/NORs. Encoded by independent genes, four-TMH HCOH proteins represent a single evolutionary unit (EU) that relates to each of the three homologous EUs of HCOs/NORs comprising TMHs 1–4, TMHs 5–8, and TMHs 9–12. Single-EU HCOH proteins could form homotrimers or heterotrimers to maintain the general structure and ligand-binding sites defined by the HCO/NOR catalytic subunit fold. The remaining HCOH families, including NnrS, have 12-TMHs and three EUs. Most three-EU HCOH proteins possess two conserved histidines and could bind a single heme. Limited experimental studies and genomic context analysis suggest that many HCOH proteins could function in the denitrification pathway and in detoxification of reactive molecules such as nitric oxide. HCO/NOR catalytic subunits exhibit remarkable structural similarity to the homotrimers of MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) proteins. Gene duplication, fusion, and fission likely play important roles in the evolution of HCOs/NORs and HCOH proteins. PMID:24931479

  17. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

    PubMed Central

    Kohansal-Nodehi, Mahdokht; Chua, John JE; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. DOI: http://dx.doi.org/10.7554/eLife.14530.001 PMID:27115346

  18. Single particle tracking with sterol modulation reveals the cholesterol-mediated diffusion properties of integrin receptors

    NASA Astrophysics Data System (ADS)

    Arora, Neha; Syed, Aleem; Sander, Suzanne; Smith, Emily A.

    2014-12-01

    A combination of sterol modulation with cyclodextrins plus fluorescence microscopy revealed a biophysical mechanism behind cholesterol’s influence on the diffusion of a ubiquitous class of receptors called integrins. The heterogeneous diffusion of integrins bound to ligand-coated quantum dots was measured using single particle tracking (SPT), and the ensemble changes in integrin diffusion were measured by fluorescence recovery after photobleaching (FRAP). A 25 ± 1% reduction of membrane cholesterol resulted in three significant changes to the diffusion of ligand-bound αPS2CβPS integrins as measured by SPT. There was a 23% increase in ligand-bound mobile integrins; there was a statistically significant increase in the average diffusion coefficient inside zones of confined diffusion, and histograms of confined integrin trajectories showed an increased frequency in the range of 0.1-1 μm2 s-1 and a decreased frequency in the 0.001-0.1 μm2 s-1 range. No statistical change was measured in the duration of confinement nor the size of confined zones. Restoring the cholesterol-depleted cells with exogenous cholesterol or exogenous epicholesterol resulted in similar diffusion properties. Epicholesterol differs from cholesterol in the orientation of a single hydroxyl group. The ability of epicholesterol to substitute for cholesterol suggests a biophysical mechanism for cholesterol’s effect on integrin diffusion. Influences of bilayer thickness, viscosity and organization are discussed as possible explanations for the measured changes in integrin diffusion when the membrane cholesterol concentration is reduced.

  19. A single-nucleotide substitution mutator phenotype revealed by exome sequencing of human colon adenomas.

    PubMed

    Nikolaev, Sergey I; Sotiriou, Sotirios K; Pateras, Ioannis S; Santoni, Federico; Sougioultzis, Stavros; Edgren, Henrik; Almusa, Henrikki; Robyr, Daniel; Guipponi, Michel; Saarela, Janna; Gorgoulis, Vassilis G; Antonarakis, Stylianos E; Halazonetis, Thanos D

    2012-12-01

    Oncogene-induced DNA replication stress is thought to drive genomic instability in cancer. In particular, replication stress can explain the high prevalence of focal genomic deletions mapping within very large genes in human tumors. However, the origin of single-nucleotide substitutions (SNS) in nonfamilial cancers is strongly debated. Some argue that cancers have a mutator phenotype, whereas others argue that the normal DNA replication error rates are sufficient to explain the number of observed SNSs. Here, we sequenced the exomes of 24, mostly precancerous, colon polyps. Analysis of the sequences revealed mutations in the APC, CTNNB1, and BRAF genes as the presumptive cancer-initiating events and many passenger SNSs. We used the number of SNSs in the various lesions to calculate mutation rates for normal colon and adenomas and found that colon adenomas exhibit a mutator phenotype. Interestingly, the SNSs in the adenomas mapped more often than expected within very large genes, where focal deletions in response to DNA replication stress also map. We propose that single-stranded DNA generated in response to oncogene-induced replication stress compromises the repair of deaminated cytosines and other damaged bases, leading to the observed SNS mutator phenotype.

  20. Single-cell genomics reveal metabolic strategies for microbial growth and survival in an oligotrophic aquifer

    SciTech Connect

    Wilkins, Michael J.; Kennedy, David W.; Castelle, Cindy; Field, Erin; Stepanauskas, Ramunas; Fredrickson, Jim K.; Konopka, Allan

    2014-02-09

    Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site and have been shown to significantly change in abundance in response to the subsurface intrusion of Columbia River water. Here we employed single cell genomics techniques to shed light on the physiological niche of these microorganisms. Analysis of four Pedobacter single amplified genomes (SAGs) from Hanford Site sediments revealed a chemoheterotrophic lifestyle, with the potential to exist under both aerobic and microaerophilic conditions via expression of both aa3­-type and cbb3-type cytochrome c oxidases. These SAGs encoded a wide-range of both intra-and extra­-cellular carbohydrate-active enzymes, potentially enabling the degradation of recalcitrant substrates such as xylan and chitin, and the utilization of more labile sugars such as mannose and fucose. Coupled to these enzymes, a diversity of transporters and sugar-binding molecules were involved in the uptake of carbon from the extracellular local environment. The SAGs were enriched in TonB-dependent receptors (TBDRs), which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. CRISPR-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for persistence by heterotrophic microorganisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area subsurface and biogeochemical shifts associated with Columbia River water intrusion.

  1. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation

    NASA Astrophysics Data System (ADS)

    Shalek, Alex K.; Satija, Rahul; Shuga, Joe; Trombetta, John J.; Gennert, Dave; Lu, Diana; Chen, Peilin; Gertner, Rona S.; Gaublomme, Jellert T.; Yosef, Nir; Schwartz, Schraga; Fowler, Brian; Weaver, Suzanne; Wang, Jing; Wang, Xiaohui; Ding, Ruihua; Raychowdhury, Raktima; Friedman, Nir; Hacohen, Nir; Park, Hongkun; May, Andrew P.; Regev, Aviv

    2014-06-01

    High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a `core' module of antiviral genes is expressed very early by a few `precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced `peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.

  2. Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification

    PubMed Central

    Zdravkovic, Tamara; Nazor, Kristopher L.; Larocque, Nicholas; Gormley, Matthew; Donne, Matthew; Hunkapillar, Nathan; Giritharan, Gnanaratnam; Bernstein, Harold S.; Wei, Grace; Hebrok, Matthias; Zeng, Xianmin; Genbacev, Olga; Mattis, Aras; McMaster, Michael T.; Krtolica, Ana; Valbuena, Diana; Simón, Carlos; Laurent, Louise C.; Loring, Jeanne F.; Fisher, Susan J.

    2015-01-01

    Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active β-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines. PMID:26483210

  3. Global Phospholipidomics Analysis Reveals Selective Pulmonary Peroxidation Profiles Upon Inhalation of Single Walled Carbon Nanotubes

    PubMed Central

    Tyurina, Yulia Y.; Kisin, Elena R.; Murray, Ashley; Tyurin, Vladimir A.; Kapralova, Valentina I.; Sparvero, Louis J.; Amoscato, Andrew A.; Samhan-Arias, Alejandro K.; Swedin, Linda; Lahesmaa, Riitta; Fadeel, Bengt; Shvedova, Anna A.; Kagan, Valerian E.

    2011-01-01

    It is commonly believed that nanomaterials cause non-specific oxidative damage. Our mass spectrometry-based oxidative lipidomics analysis of all major phospholipid classes revealed highly selective patterns of pulmonary peroxidation after inhalation exposure of mice to single-walled carbon nanotubes. No oxidized molecular species were found in two most abundant phospholipid classes – phosphatidylcholine and phosphatidylethanolamine. Peroxidation products were identified in three relatively minor classes of anionic phospholipids, cardiolipin, phosphatidylserine and phosphatidylinositol whereby oxygenation of polyunsaturated fatty acid residues also showed unusual substrate specificity. This non-random peroxidation coincided with the accumulation of apoptotic cells in the lung. A similar selective phospholipid peroxidation profile was detected upon incubation of a mixture of total lung lipids with H2O2/cytochrome c known to catalyze cardiolipin and phosphatidylserine peroxidation in apoptotic cells. The characterized specific phospholipid peroxidation signaling pathways indicate new approaches to the development of mitochondria targeted regulators of cardiolipin peroxidation to protect against deleterious effects of pro-apoptotic effects of single-walled carbon nanotubes in the lung. PMID:21800898

  4. Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification.

    PubMed

    Zdravkovic, Tamara; Nazor, Kristopher L; Larocque, Nicholas; Gormley, Matthew; Donne, Matthew; Hunkapillar, Nathan; Giritharan, Gnanaratnam; Bernstein, Harold S; Wei, Grace; Hebrok, Matthias; Zeng, Xianmin; Genbacev, Olga; Mattis, Aras; McMaster, Michael T; Krtolica, Ana; Valbuena, Diana; Simón, Carlos; Laurent, Louise C; Loring, Jeanne F; Fisher, Susan J

    2015-12-01

    Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active β-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines.

  5. Function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations

    SciTech Connect

    Nutt, David; Smith, Jeremy C

    2008-10-01

    Atomistic molecular dynamics simulations are used to investigate the mechanism by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, binds to ice. Comparison of structural and dynamic properties of the water around the three faces of the triangular prism-shaped protein in aqueous solution reveals that at low temperature the water structure is ordered and the dynamics slowed down around the ice-binding face of the protein, with a disordering effect observed around the other two faces. These results suggest a dual role for the solvation water around the protein. The preconfigured solvation shell around the ice-binding face is involved in the initial recognition and binding of the antifreeze protein to ice by lowering the barrier for binding and consolidation of the protein:ice interaction surface. Thus, the antifreeze protein can bind to the molecularly rough ice surface by becoming actively involved in the formation of its own binding site. Also, the disruption of water structure around the rest of the protein helps prevent the adsorbed protein becoming covered by further ice growth.

  6. Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

    PubMed Central

    Tan, David W. M.; Jensen, Kim B.; Trotter, Matthew W. B.; Connelly, John T.; Broad, Simon; Watt, Fiona M.

    2013-01-01

    Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. PMID:23482486

  7. A Papillary Thyroid Microcarcinoma Revealed by a Single Bone Lesion with No Poor Prognostic Factors

    PubMed Central

    Godbert, Yann; Henriques-Figueiredo, Benedicte; Cazeau, Anne-Laure; Carrat, Xavier; Stegen, Marc; Soubeyran, Isabelle; Bonichon, Francoise

    2013-01-01

    Objectives. Thyroid carcinomas incidence, in particular papillary variants, is increasing. These cancers are generally considered to have excellent prognosis, and papillary microcarcinomas are usually noninvasive. Many prognostic histopathology factors have been described to guide therapeutic decisions. Most patients are treated with total thyroidectomy without radioiodine treatment or partial surgery. Case Summary. A 65-year-old man with no significant medical history presented with pain in the left chest wall that had been present for several months. A computed tomography (CT) found a large tissue mass of 4 cm responsible for lysis of the middle arch of the 4th rib on the left. It was a single lesion, highly hypermetabolic on the 18-FDG PET/CT. The histology analysis of the biopsy and surgical specimen favored an adenocarcinoma with immunostaining positive for TTF1 and thyroglobulin (Tg). The total thyroidectomy carried out subsequently revealed a 4 mm papillary microcarcinoma with vesicular architecture of the right lobe, well delimited and distant from the capsule without vascular embolisms. After two radioiodine treatments, the patient is in complete clinical, biological, and radiological remission. Conclusion. This extremely rare case of a singular bone metastasis revealing a papillary thyroid microcarcinoma illustrates the necessity of further research to better characterize the forms of papillary thyroid microcarcinomas with potentially poor prognosis. PMID:23509641

  8. Isolation, characterization, and bioinformatic analysis of calmodulin-binding protein cmbB reveals a novel tandem IP22 repeat common to many Dictyostelium and Mimivirus proteins.

    PubMed

    O'Day, Danton H; Suhre, Karsten; Myre, Michael A; Chatterjee-Chakraborty, Munmun; Chavez, Sara E

    2006-08-01

    A novel calmodulin-binding protein cmbB from Dictyostelium discoideum is encoded in a single gene. Northern analysis reveals two cmbB transcripts first detectable at 4 h during multicellular development. Western blotting detects an approximately 46.6 kDa protein. Sequence analysis and calmodulin-agarose binding studies identified a "classic" calcium-dependent calmodulin-binding domain (179IPKSLRSLFLGKGYNQPLEF198) but structural analyses suggest binding may not involve classic alpha-helical calmodulin-binding. The cmbB protein is comprised of tandem repeats of a newly identified IP22 motif ([I,L]Pxxhxxhxhxxxhxxxhxxxx; where h = any hydrophobic amino acid) that is highly conserved and a more precise representation of the FNIP repeat. At least eight Acanthamoeba polyphaga Mimivirus proteins and over 100 Dictyostelium proteins contain tandem arrays of the IP22 motif and its variants. cmbB also shares structural homology to YopM, from the plague bacterium Yersenia pestis. PMID:16777069

  9. Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling.

    PubMed Central

    Valentini, Sandro R; Casolari, Jason M; Oliveira, Carla C; Silver, Pamela A; McBride, Anne E

    2002-01-01

    The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIF5A domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIF5A may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes. PMID:11861547

  10. Differential Interaction Kinetics of a Bipolar Structure-Specific Endonuclease with DNA Flaps Revealed by Single-Molecule Imaging

    PubMed Central

    Rezgui, Rachid; Lestini, Roxane; Kühn, Joëlle; Fave, Xenia; McLeod, Lauren; Myllykallio, Hannu; Alexandrou, Antigoni; Bouzigues, Cedric

    2014-01-01

    As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5′ and 3′-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5′ or 3′ extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases. PMID:25412080

  11. Local Geometry and Evolutionary Conservation of Protein Surfaces Reveal the Multiple Recognition Patches in Protein-Protein Interactions.

    PubMed

    Laine, Elodie; Carbone, Alessandra

    2015-12-01

    Protein-protein interactions (PPIs) are essential to all biological processes and they represent increasingly important therapeutic targets. Here, we present a new method for accurately predicting protein-protein interfaces, understanding their properties, origins and binding to multiple partners. Contrary to machine learning approaches, our method combines in a rational and very straightforward way three sequence- and structure-based descriptors of protein residues: evolutionary conservation, physico-chemical properties and local geometry. The implemented strategy yields very precise predictions for a wide range of protein-protein interfaces and discriminates them from small-molecule binding sites. Beyond its predictive power, the approach permits to dissect interaction surfaces and unravel their complexity. We show how the analysis of the predicted patches can foster new strategies for PPIs modulation and interaction surface redesign. The approach is implemented in JET2, an automated tool based on the Joint Evolutionary Trees (JET) method for sequence-based protein interface prediction. JET2 is freely available at www.lcqb.upmc.fr/JET2.

  12. Local Geometry and Evolutionary Conservation of Protein Surfaces Reveal the Multiple Recognition Patches in Protein-Protein Interactions

    PubMed Central

    Laine, Elodie; Carbone, Alessandra

    2015-01-01

    Protein-protein interactions (PPIs) are essential to all biological processes and they represent increasingly important therapeutic targets. Here, we present a new method for accurately predicting protein-protein interfaces, understanding their properties, origins and binding to multiple partners. Contrary to machine learning approaches, our method combines in a rational and very straightforward way three sequence- and structure-based descriptors of protein residues: evolutionary conservation, physico-chemical properties and local geometry. The implemented strategy yields very precise predictions for a wide range of protein-protein interfaces and discriminates them from small-molecule binding sites. Beyond its predictive power, the approach permits to dissect interaction surfaces and unravel their complexity. We show how the analysis of the predicted patches can foster new strategies for PPIs modulation and interaction surface redesign. The approach is implemented in JET2, an automated tool based on the Joint Evolutionary Trees (JET) method for sequence-based protein interface prediction. JET2 is freely available at www.lcqb.upmc.fr/JET2. PMID:26690684

  13. Revealing the potential pathogenesis of glioma by utilizing a glioma associated protein-protein interaction network.

    PubMed

    Pan, Weiran; Li, Gang; Yang, Xiaoxiao; Miao, Jinming

    2015-04-01

    This study aims to explore the potential mechanism of glioma through bioinformatic approaches. The gene expression profile (GSE4290) of glioma tumor and non-tumor samples was downloaded from Gene Expression Omnibus database. A total of 180 samples were available, including 23 non-tumor and 157 tumor samples. Then the raw data were preprocessed using robust multiarray analysis, and 8,890 differentially expressed genes (DEGs) were identified by using t-test (false discovery rate < 0.0005). Furthermore, 16 known glioma related genes were abstracted from Genetic Association Database. After mapping 8,890 DEGs and 16 known glioma related genes to Human Protein Reference Database, a glioma associated protein-protein interaction network (GAPN) was constructed. In addition, 51 sub-networks in GAPN were screened out through Molecular Complex Detection (score ≥ 1), and sub-network 1 was found to have the closest interaction (score = 3). What' more, for the top 10 sub-networks, Gene Ontology (GO) enrichment analysis (p value < 0.05) was performed, and DEGs involved in sub-network 1 and 2, such as BRMS1L and CCNA1, were predicted to regulate cell growth, cell cycle, and DNA replication via interacting with known glioma related genes. Finally, the overlaps of DEGs and human essential, housekeeping, tissue-specific genes were calculated (p value = 1.0, 1.0, and 0.00014, respectively) and visualized by Venn Diagram package in R. About 61% of human tissue-specific genes were DEGs as well. This research shed new light on the pathogenesis of glioma based on DEGs and GAPN, and our findings might provide potential targets for clinical glioma treatment.

  14. Origin of worldwide cultivated barley revealed by NAM-1 gene and grain protein content.

    PubMed

    Wang, Yonggang; Ren, Xifeng; Sun, Dongfa; Sun, Genlou

    2015-01-01

    The origin, evolution, and distribution of cultivated barley provides powerful insights into the historic origin and early spread of agrarian culture. Here, population-based genetic diversity and phylogenetic analyses were performed to determine the evolution and origin of barley and how domestication and subsequent introgression have affected the genetic diversity and changes in cultivated barley on a worldwide scale. A set of worldwide cultivated and wild barleys from Asia and Tibet of China were analyzed using the sequences for NAM-1 gene and gene-associated traits-grain protein content (GPC). Our results showed Tibetan wild barley distinctly diverged from Near Eastern barley, and confirmed that Tibet is one of the origin and domestication centers for cultivated barley, and in turn supported a polyphyletic origin of domesticated barley. Comparison of haplotype composition among geographic regions revealed gene flow between Eastern and Western barley populations, suggesting that the Silk Road might have played a crucial role in the spread of genes. The GPC in the 118 cultivated and 93 wild barley accessions ranged from 6.73 to 12.35% with a mean of 9.43%. Overall, wild barley had higher averaged GPC (10.44%) than cultivated barley. Two unique haplotypes (Hap2 and Hap7) caused by a base mutations (at position 544) in the coding region of the NAM-1 gene might have a significant impact on the GPC. Single nucleotide polymorphisms and haplotypes of NAM-1 associated with GPC in barley could provide a useful method for screening GPC in barley germplasm. The Tibetan wild accessions with lower GPC could be useful for malt barley breeding. PMID:26483818

  15. Origin of worldwide cultivated barley revealed by NAM-1 gene and grain protein content

    PubMed Central

    Wang, Yonggang; Ren, Xifeng; Sun, Dongfa; Sun, Genlou

    2015-01-01

    The origin, evolution, and distribution of cultivated barley provides powerful insights into the historic origin and early spread of agrarian culture. Here, population-based genetic diversity and phylogenetic analyses were performed to determine the evolution and origin of barley and how domestication and subsequent introgression have affected the genetic diversity and changes in cultivated barley on a worldwide scale. A set of worldwide cultivated and wild barleys from Asia and Tibet of China were analyzed using the sequences for NAM-1 gene and gene-associated traits-grain protein content (GPC). Our results showed Tibetan wild barley distinctly diverged from Near Eastern barley, and confirmed that Tibet is one of the origin and domestication centers for cultivated barley, and in turn supported a polyphyletic origin of domesticated barley. Comparison of haplotype composition among geographic regions revealed gene flow between Eastern and Western barley populations, suggesting that the Silk Road might have played a crucial role in the spread of genes. The GPC in the 118 cultivated and 93 wild barley accessions ranged from 6.73 to 12.35% with a mean of 9.43%. Overall, wild barley had higher averaged GPC (10.44%) than cultivated barley. Two unique haplotypes (Hap2 and Hap7) caused by a base mutations (at position 544) in the coding region of the NAM-1 gene might have a significant impact on the GPC. Single nucleotide polymorphisms and haplotypes of NAM-1 associated with GPC in barley could provide a useful method for screening GPC in barley germplasm. The Tibetan wild accessions with lower GPC could be useful for malt barley breeding. PMID:26483818

  16. Origin of worldwide cultivated barley revealed by NAM-1 gene and grain protein content.

    PubMed

    Wang, Yonggang; Ren, Xifeng; Sun, Dongfa; Sun, Genlou

    2015-01-01

    The origin, evolution, and distribution of cultivated barley provides powerful insights into the historic origin and early spread of agrarian culture. Here, population-based genetic diversity and phylogenetic analyses were performed to determine the evolution and origin of barley and how domestication and subsequent introgression have affected the genetic diversity and changes in cultivated barley on a worldwide scale. A set of worldwide cultivated and wild barleys from Asia and Tibet of China were analyzed using the sequences for NAM-1 gene and gene-associated traits-grain protein content (GPC). Our results showed Tibetan wild barley distinctly diverged from Near Eastern barley, and confirmed that Tibet is one of the origin and domestication centers for cultivated barley, and in turn supported a polyphyletic origin of domesticated barley. Comparison of haplotype composition among geographic regions revealed gene flow between Eastern and Western barley populations, suggesting that the Silk Road might have played a crucial role in the spread of genes. The GPC in the 118 cultivated and 93 wild barley accessions ranged from 6.73 to 12.35% with a mean of 9.43%. Overall, wild barley had higher averaged GPC (10.44%) than cultivated barley. Two unique haplotypes (Hap2 and Hap7) caused by a base mutations (at position 544) in the coding region of the NAM-1 gene might have a significant impact on the GPC. Single nucleotide polymorphisms and haplotypes of NAM-1 associated with GPC in barley could provide a useful method for screening GPC in barley germplasm. The Tibetan wild accessions with lower GPC could be useful for malt barley breeding.

  17. Label-free, single protein detection on a near-infrared fluorescent single-walled carbon nanotube/protein microarray fabricated by cell-free synthesis.

    PubMed

    Ahn, Jin-Ho; Kim, Jong-Ho; Reuel, Nigel F; Barone, Paul W; Boghossian, Ardemis A; Zhang, Jingqing; Yoon, Hyeonseok; Chang, Alice C; Hilmer, Andrew J; Strano, Michael S

    2011-07-13

    Excessive sample volumes continue to be a major limitation in the analysis of protein-protein interactions, motivating the search for label-free detection methods of greater sensitivity. Herein, we report the first chemical approach for selective protein recognition using fluorescent single-walled carbon nanotubes (SWNTs) enabling label-free microarrays capable of single protein detection. Hexahistidine-tagged capture proteins directly expressed by cell-free synthesis on SWNT/chitosan microarray are bound to a Ni(2+) chelated by Nα,Nα-bis(carboxymethyl)-L-lysine grafted to chitosan surrounding the SWNT. The Ni(2+) acts as a proximity quencher with the Ni(2+)/SWNT distance altered upon docking of analyte proteins. This ability to discern single protein binding events decreases the apparent detection limit from 100 nM, for the ensemble average, to 10 pM for an observation time of 600 s. This first use of cell-free synthesis to functionalize a nanosensor extends this method to a virtually infinite number of capture proteins. To demonstrate this, the SWNT microarrays are used to analyze a network of 1156 protein-protein interactions in the staurosporine-induced apoptosis of SH-SY5Y cells, confirming literature predictions. PMID:21627102

  18. Single Molecule Force Measurement for Protein Synthesis on the Ribosome

    NASA Astrophysics Data System (ADS)

    Uemura, Sotaro

    2008-04-01

    The ribosome is a molecular machine that translates the genetic code described on the messenger RNA (mRNA) into an amino acid sequence through repetitive cycles of transfer RNA (tRNA) selection, peptide bond formation and translocation. Although the detailed interactions between the translation components have been revealed by extensive structural and biochemical studies, it is not known how the precise regulation of macromolecular movements required at each stage of translation is achieved. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine-Dalgarno (SD) sequence. The removal of the SD sequence significantly reduced the rupture force, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA in a pre-peptidyl transfer state. In contrast, the post-peptidyl transfer state weakened the rupture force as compared to the complex in a pre-peptidyl transfer state and it was the same for both the SD-containing and SD-deficient mRNAs. The results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step. In this article, we discuss about the above new results including the introduction of the ribosome translation mechanism and the optical tweezer method.

  19. Proteomic profiling of maize opaque endosperm mutants reveals selective accumulation of lysine-enriched proteins

    PubMed Central

    Morton, Kyla J.; Jia, Shangang; Zhang, Chi; Holding, David R.

    2016-01-01

    Reduced prolamin (zein) accumulation and defective endoplasmic reticulum (ER) body formation occurs in maize opaque endosperm mutants opaque2 (o2), floury2 (fl2), defective endosperm*B30 (DeB30), and Mucronate (Mc), whereas other opaque mutants such as opaque1 (o1) and floury1 (fl1) are normal in these regards. This suggests that other factors contribute to kernel texture. A liquid chromatography approach coupled with tandem mass spectrometry (LC-MS/MS) proteomics was used to compare non-zein proteins of nearly isogenic opaque endosperm mutants. In total, 2762 proteins were identified that were enriched for biological processes such as protein transport and folding, amino acid biosynthesis, and proteolysis. Principal component analysis and pathway enrichment suggested that the mutants partitioned into three groups: (i) Mc, DeB30, fl2 and o2; (ii) o1; and (iii) fl1. Indicator species analysis revealed mutant-specific proteins, and highlighted ER secretory pathway components that were enriched in selected groups of mutants. The most significantly changed proteins were related to stress or defense and zein partitioning into the soluble fraction for Mc, DeB30, o1, and fl1 specifically. In silico dissection of the most significantly changed proteins revealed novel qualitative changes in lysine abundance contributing to the overall lysine increase and the nutritional rebalancing of the o2 and fl2 endosperm. PMID:26712829

  20. Single-molecule measurements of proteins using carbon nanotube field-effect transistors

    NASA Astrophysics Data System (ADS)

    Sims, Patrick Craig

    Single-walled carbon nanotube (SWCNT) field-effect transistors (FETs) provide a promising platform for investigating proteins at the single-molecule level. Recently, we have demonstrated that SWCNT FETs have sufficient sensitivity and bandwidth to monitor the conformational motions and processivity of an individual T4 lysozyme molecule. This is accomplished by functionalizing a SWCNT FET device with a single protein and measuring the conductance versus time through the device as it is submerged in an electrolyte solution. To generalize this approach for the study of a wide variety of proteins at the single-molecule level, this dissertation investigates the conjugation process to determine and isolate the key parameters involved in functionalizing a SWCNT with a single protein, the physical basis for transducing conformational motion of a protein into an electrical signal, and finally, the general application of the technique to monitor the binary and ternary complex formation of cAMP-dependent protein kinase (PKA).

  1. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations.

    PubMed

    Hertig, Samuel; Latorraca, Naomi R; Dror, Ron O

    2016-06-01

    Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein's constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery-the fact that the two sites involved influence one another in a symmetrical manner-can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest. PMID:27285999

  2. Essential Strategies for Revealing Nanoscale Protein Dynamics by Neutron Spin Echo Spectroscopy.

    PubMed

    Callaway, David J E; Bu, Zimei

    2016-01-01

    Determining the internal motions of a protein on nanosecond-to-microsecond timescales and on nanometer length scales is challenging by experimental biophysical techniques. Neutron spin echo spectroscopy (NSE) offers a unique opportunity to determine such nanoscale protein domain motions. However, the major hurdle in applying NSE to determine nanoscale protein motion is that the time and length scales of internal protein motions tend to be comparable to that of the global motions of a protein. The signals detected by NSE tend to be dominated by rigid-body translational and rotational diffusion. Using theoretical analyses, our laboratory showed that selective deuteration of a protein domain or a subunit can enhance the capability of NSE to reveal the internal motions in a protein complex. Here, we discuss the essential theoretical analysis and experimental methodology in detail. Protein nanomachines are far more complex than any molecular motors that have been artificially constructed, and their skillful utilization likely represents the future of medicine. With selective deuteration, NSE will allow us to see these nanomachines in motion.

  3. Quantitative proteomics reveals the effect of protein glycosylation in soybean root under flooding stress

    PubMed Central

    Mustafa, Ghazala; Komatsu, Setsuko

    2014-01-01

    Flooding stress has a negative impact on soybean cultivation because it severely impairs growth and development. To understand the flooding responsive mechanism in early stage soybeans, a glycoproteomic technique was used. Two-day-old soybeans were treated with flooding for 2 days and roots were collected. Globally, the accumulation level of glycoproteins, as revealed by cross-reaction with concanavalin A decreased by 2 days of flooding stress. Glycoproteins were enriched from total protein extracts using concanavalin A lectin resin and analyzed using a gel-free proteomic technique. One-hundred eleven and 69 glycoproteins were identified without and with 2 days of flooding stress, respectively. Functional categorization of these identified glycoproteins indicated that the accumulation level of proteins related to protein degradation, cell wall, and glycolysis increased, while stress-related proteins decreased under flooding stress. Also the accumulation level of glycoproteins localized in the secretory pathway decreased under flooding stress. Out of 23 common glycoproteins between control and flooding conditions, peroxidases and glycosyl hydrolases were decreased by 2 days of flooding stress. mRNA expression levels of proteins in the endoplasmic reticulum and N-glycosylation related proteins were downregulated by flooding stress. These results suggest that flooding might negatively affect the process of N-glycosylation of proteins related to stress and protein degradation; however glycoproteins involved in glycolysis are activated. PMID:25477889

  4. Revealing Linear Aggregates of Light Harvesting Antenna Proteins in Photosynthetic Membranes

    PubMed Central

    He, Yufan; Zeng, Xiaohua; Mukherjee, Saptarshi; Rajapaksha, Suneth; Kaplan, Samuel; Lu, H. Peter

    2010-01-01

    How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under ambient conditions by using atomic force microscopy (AFM) imaging and spectroscopic analysis. Our results suggest that the light harvesting protein, LH2, can exist as linear aggregates of 4±2 proteins in the photosynthetic membranes and that the protein distributions are highly heterogeneous. In the photosynthetic membranes examined in our measurements, the ratio of the aggregated to the non-aggregated LH2 proteins is about 3:1 to 5:1 depending on the intensity of the illumination used during sample incubation and on the bacterial species. AFM images further identify that the LH2 proteins in the linear aggregates are monotonically tilted at an angle 4°±2° from the plane of the photosynthetic membranes. The aggregates result in red-shifted absorption and emission spectra that are measured using various mutant membranes, including an LH2 knock-out, LH1 knock-out, and LH2 at different population densities. Measuring the fluorescence lifetimes of purified LH2 and LH2 in membranes, we have observed that the LH2 proteins in membranes exhibit biexponential lifetime decays whereas the purified LH2 proteins gave single exponential lifetime decays. We attribute that the two lifetime components originate from the existence of both aggregated and non-aggregated LH2 proteins in the photosynthetic membranes. PMID:19572507

  5. Single-molecule pull-down for investigating protein-nucleic acid interactions.

    PubMed

    Fareh, Mohamed; Loeff, Luuk; Szczepaniak, Malwina; Haagsma, Anna C; Yeom, Kyu-Hyeon; Joo, Chirlmin

    2016-08-01

    The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes. PMID:27017911

  6. A six-plex proteome quantification strategy reveals the dynamics of protein turnover.

    PubMed

    Wang, Fangjun; Cheng, Kai; Wei, Xiaoluan; Qin, Hongqiang; Chen, Rui; Liu, Jing; Zou, Hanfa

    2013-01-01

    MS1 full scan based quantification is one of the most popular approaches for large-scale proteome quantification. Typically only three different samples can be differentially labeled and quantified in a single experiment. Here we present a two stages stable isotope labeling strategy which allows six different protein samples (six-plex) to be reliably labeled and simultaneously quantified at MS1 level. Briefly in the first stage, isotope lysine-d0 (K0) and lysine-d4 (K4) are in vivo incorporated into different protein samples during cell culture. Then in the second stage, three of K0 and K4 labeled protein samples are digested by lysine C and in vitro labeled with light (2CH3), medium (2CD2H), and heavy (2(13)CD3) dimethyl groups, respectively. We demonstrated that this six-plex isotope labeling strategy could successfully investigate the dynamics of protein turnover in a high throughput manner. PMID:23661174

  7. Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism

    PubMed Central

    Özdemir, Emre; McKinney, John D.

    2015-01-01

    ABSTRACT ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening. PMID:25691591

  8. Single-Molecule FISH Reveals Non-selective Packaging of Rift Valley Fever Virus Genome Segments.

    PubMed

    Wichgers Schreur, Paul J; Kortekaas, Jeroen

    2016-08-01

    The bunyavirus genome comprises a small (S), medium (M), and large (L) RNA segment of negative polarity. Although genome segmentation confers evolutionary advantages by enabling genome reassortment events with related viruses, genome segmentation also complicates genome replication and packaging. Accumulating evidence suggests that genomes of viruses with eight or more genome segments are incorporated into virions by highly selective processes. Remarkably, little is known about the genome packaging process of the tri-segmented bunyaviruses. Here, we evaluated, by single-molecule RNA fluorescence in situ hybridization (FISH), the intracellular spatio-temporal distribution and replication kinetics of the Rift Valley fever virus (RVFV) genome and determined the segment composition of mature virions. The results reveal that the RVFV genome segments start to replicate near the site of infection before spreading and replicating throughout the cytoplasm followed by translocation to the virion assembly site at the Golgi network. Despite the average intracellular S, M and L genome segments approached a 1:1:1 ratio, major differences in genome segment ratios were observed among cells. We also observed a significant amount of cells lacking evidence of M-segment replication. Analysis of two-segmented replicons and four-segmented viruses subsequently confirmed the previous notion that Golgi recruitment is mediated by the Gn glycoprotein. The absence of colocalization of the different segments in the cytoplasm and the successful rescue of a tri-segmented variant with a codon shuffled M-segment suggested that inter-segment interactions are unlikely to drive the copackaging of the different segments into a single virion. The latter was confirmed by direct visualization of RNPs inside mature virions which showed that the majority of virions lack one or more genome segments. Altogether, this study suggests that RVFV genome packaging is a non-selective process. PMID:27548280

  9. Single-Molecule FISH Reveals Non-selective Packaging of Rift Valley Fever Virus Genome Segments

    PubMed Central

    Wichgers Schreur, Paul J.; Kortekaas, Jeroen

    2016-01-01

    The bunyavirus genome comprises a small (S), medium (M), and large (L) RNA segment of negative polarity. Although genome segmentation confers evolutionary advantages by enabling genome reassortment events with related viruses, genome segmentation also complicates genome replication and packaging. Accumulating evidence suggests that genomes of viruses with eight or more genome segments are incorporated into virions by highly selective processes. Remarkably, little is known about the genome packaging process of the tri-segmented bunyaviruses. Here, we evaluated, by single-molecule RNA fluorescence in situ hybridization (FISH), the intracellular spatio-temporal distribution and replication kinetics of the Rift Valley fever virus (RVFV) genome and determined the segment composition of mature virions. The results reveal that the RVFV genome segments start to replicate near the site of infection before spreading and replicating throughout the cytoplasm followed by translocation to the virion assembly site at the Golgi network. Despite the average intracellular S, M and L genome segments approached a 1:1:1 ratio, major differences in genome segment ratios were observed among cells. We also observed a significant amount of cells lacking evidence of M-segment replication. Analysis of two-segmented replicons and four-segmented viruses subsequently confirmed the previous notion that Golgi recruitment is mediated by the Gn glycoprotein. The absence of colocalization of the different segments in the cytoplasm and the successful rescue of a tri-segmented variant with a codon shuffled M-segment suggested that inter-segment interactions are unlikely to drive the copackaging of the different segments into a single virion. The latter was confirmed by direct visualization of RNPs inside mature virions which showed that the majority of virions lack one or more genome segments. Altogether, this study suggests that RVFV genome packaging is a non-selective process. PMID:27548280

  10. Experimental single-strain mobilomics reveals events that shape pathogen emergence

    DOE PAGES

    Schoeniger, Joseph S.; Hudson, Corey M.; Bent, Zachary W.; Sinha, Anupama; Williams, Kelly P.

    2016-07-04

    Virulence and resistance genes carried on mobile DNAs such as genomic islands (GIs) and plasmids promote bacterial pathogen emergence. An early step in the mobilization of GIs is their excision, which produces both a circular form of the GI and a deletion site in the chromosome; circular forms have also been described for some bacterial insertion sequences (ISs). We demonstrate that the recombinant sequence produced at the junction of such circles, and their corresponding deletion sites, can be detected sensitively in high throughput sequencing data, using new computational methods that enable empirical discovery of new mobile DNAs. Applied to themore » rich mobilome of a single strain (Kpn2146) of the emerging multidrug-resistant pathogen Klebsiella pneumoniae, our approach detected circular junctions for six GIs and seven IS types (several of the latter not previously known to circularize). Our methods further revealed differential biology of multiple mobile DNAs, imprecision of integrases and transposases, and differential activity among identical IS copies for IS26, ISKpn18 and ISKpn21. Exonuclease was used to enrich for circular dsDNA molecules, and internal calibration with the native Kpn2146 plasmids showed that not all molecules bearing GI and IS circular junctions were circular dsDNAs. Transposition events were also detected, revealing replicon preference (ISKpn18 preferring a conjugative IncA/C2 plasmid), local action (IS26), regional preferences, selection (against capsule synthesis), and left-right IS end swapping. Efficient discovery and global characterization of numerous mobile elements per experiment will allow detailed accounting of bacterial evolution, explaining the new gene combinations that arise in emerging pathogens.« less

  11. STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA (presentation video)

    NASA Astrophysics Data System (ADS)

    Heller, Iddo; Sitters, Gerrit; Broekmans, Onno D.; Farge, Géraldine; Menges, Carolin; Wende, Wolfgang; Hell, Stefan W.; Peterman, Erwin J.; Wuite, Gijs J.

    2014-09-01

    Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication and repair. We present a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in the presence of high protein concentrations. Our approach combines optical tweezers with multicolor confocal and stimulated emission depletion (STED) fluorescence microscopy. Proteins on DNA are visualized at a resolution of 50 nm, a sixfold resolution improvement over that of confocal microscopy. High temporal resolution (<50 ms) is ensured by fast one-dimensional beam scanning. Individual trajectories of proteins translocating on DNA can thus be distinguished and tracked with high precision. We demonstrate our multimodal approach by visualizing the assembly of dense nucleoprotein filaments with unprecedented spatial resolution in real time. Experimental access to the force-dependent kinetics and motility of DNA-associating proteins at biologically relevant protein densities is essential for linking idealized in vitro experiments with the in vivo situation.

  12. Single-Molecule Study of Proteins by Biological Nanopore Sensors

    PubMed Central

    Wu, Dongmei; Bi, Sheng; Zhang, Liyu; Yang, Jun

    2014-01-01

    Nanopore technology has been developed for detecting properties of proteins through monitoring of ionic current modulations as protein passes via a nanosize pore. As a real-time, sensitive, selective and stable technology, biological nanopores are of widespread concern. Here, we introduce the background of nanopore researches in the area of α-hemolysin (α-HL) nanopores in protein conformation detections and protein–ligand interactions. Moreover, several original biological nanopores are also introduced with various features and functions. PMID:25268917

  13. Single-molecule RNA observation in vivo reveals dynamics of co-transcriptional splicing

    NASA Astrophysics Data System (ADS)

    Ferguson, M. L.; Coulon, A.; de Turris, V.; Palangat, M.; Chow, C. C.; Singer, R. H.; Larson, D. R.

    2013-03-01

    The synthesis of pre-mRNA and the splicing of that pre-mRNA to form completed transcripts requires coordination between two large multi-subunit complexes (the transcription elongation complex and the spliceosome). How this coordination occurs in vivo is unknown. Here we report the first experimental observation of transcription and splicing occurring at the same gene in living cells. By utilizing the PP7/MS2 fluorescent RNA reporter system, we can directly observe two distinct regions of the nascent RNA, allowing us to measure the rise and fall time of the intron and exon of a reporter gene stably integrated into a human cell line. The reporter gene consists of a beta globin gene where we have inserted a 24 RNA hairpin cassette into the intron/exon. Upon synthesis, the RNA hairpins are tightly bound by fluorescently-labeled PP7/MS2 bacteriophage coat proteins. After gene induction, a single locus of active transcription in the nucleus shows fluorescence intensity changes characteristic of the synthesis and excision of the intron/exon. Using fluctuation analysis, we determine the elongation rate to be 1.5 kb/min. From the temporal cross correlation function, we determine that splicing of this gene must be co-transcriptional with a splicing time of ~100 seconds before termination and a ~200 second pause at termination. We propose that dual-color RNA imaging may be extended to investigate other mechanisms of transcription, gene regulation, and RNA processing.

  14. Single-Molecule Tracking of Inositol Trisphosphate Receptors Reveals Different Motilities and Distributions

    PubMed Central

    Smith, Ian F.; Swaminathan, Divya; Dickinson, George D.; Parker, Ian

    2014-01-01

    Puffs are local Ca2+ signals that arise by Ca2+ liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP3Rs). The locations of puff sites observed by Ca2+ imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP3Rs have shown that the majority of IP3Rs are freely motile. To address this discrepancy, we applied single-molecule imaging to locate and track type 1 IP3Rs tagged with a photoswitchable fluorescent protein and expressed in COS-7 cells. We found that ∼70% of the IP3R1 molecules were freely motile, undergoing random walk motility with an apparent diffusion coefficient of ∼0.095 μm s−1, whereas the remaining molecules were essentially immotile. A fraction of the immotile IP3Rs were organized in clusters, with dimensions (a few hundred nanometers across) comparable to those previously estimated for the IP3R clusters underlying functional puff sites. No short-term (seconds) changes in overall motility or in clustering of immotile IP3Rs were apparent following activation of IP3/Ca2+ signaling. We conclude that stable clusters of small numbers of immotile IP3Rs may underlie local Ca2+ release sites, whereas the more numerous motile IP3Rs appear to be functionally silent. PMID:25140418

  15. Solution single-vesicle assay reveals PIP2-mediated sequential actions of synaptotagmin-1 on SNAREs

    PubMed Central

    Kim, Jae-Yeol; Choi, Bong-Kyu; Choi, Mal-Gi; Kim, Sun-Ae; Lai, Ying; Shin, Yeon-Kyun; Lee, Nam Ki

    2012-01-01

    Synaptotagmin-1 (Syt1) is a major Ca2+ sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t-) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single-molecule technique, alternating-laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca2+, Syt1 mediates vesicle docking by directly binding to t-SNARE/phosphatidylinositol 4,5-biphosphate (PIP2) complex and increases the docking rate by 103 times. Second, synaptobrevin-2 binding to t-SNARE displaces Syt1 from SNAREpin. Third, with Ca2+, Syt1 rebinds to SNAREpin, which again requires PIP2. Thus without Ca2+, Syt1 may bring vesicles to the plasma membrane in proximity via binding to t-SNARE/PIP2 to help SNAREpin formation and then, upon Ca2+ influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway. PMID:22407297

  16. Stabilization of protein crystals by electrostatic interactions as revealed by a numerical approach.

    PubMed

    Takahashi, T; Endo, S; Nagayama, K

    1993-11-20

    We developed a novel algorithm to solve numerically the Poisson-Boltzmann equations under a periodic boundary condition. By employing this algorithm to calculate the electrostatic potentials in two different types of protein crystals, a bovine pancreatic trypsin inhibitor (BPTI) orthorhombic crystal and a pig-insulin cubic crystal, the energy contributions of the electrostatic interactions to the crystals' stability were evaluated. At a high ionic strength, the condensed state of proteins in the crystal was stabilized electrostatically compared with that isolated in dilute solution because the attractive electrostatic interactions between neighboring protein molecules overcame the repulsive forces that originated from the same net charges of the equivalent protein molecules. On the other hand, at a low ionic strength the electrostatic interactions destabilized the crystalline state of both proteins, although a different dependence on the ionic strength was found between them. Here, the insulin crystal was more stable than the BPTI one because of the higher charge density in the BPTI crystal. In all of the solvent ionic strengths investigated, the attractive electrostatic interactions between charge pairs separated by less than 5 A on the respective protein molecules prominently stabilize the protein crystals. Therefore, two protein molecules in the crystals are oriented to compensate each other for their opposite charges on the surfaces. We also found a specific role for bound phosphate ions in the stabilization of the BPTI crystal, based on comparison of the electrostatic energies of the two crystals with and without the ions. By determining the contribution of each atomic charge in the crystals to the electrostatic energy, it was revealed that several electrostatic pairs specifically contributed to the crystal's stability. On the basis of our numerical calculation results, we propose a new method to design protein molecules that adopt stable crystals by replacing

  17. Observation of HIV-1 Nucleocapsid Protein induced TAR DNA melting at the single molecule level

    NASA Astrophysics Data System (ADS)

    Cosa, Gonzalo; Harbron, Elizabeth; O'Connor, Donald; Musier-Forsyth, Karin; Barbara, Paul

    2003-03-01

    Reverse transcription of the HIV-1 RNA genome involves several nucleic acid rearrangement steps, and the HIV-1 nucleocapsid protein (NC) plays a key role in this process. NC is a nucleic acid chaperone protein, which facilitates the formation of the most stable nucleic acid structures. Single molecule fluorescence resonance energy transfer (SM-FRET) measurements enable us to observe the NC-induced conformational fluctuations of a transactivation response region (TAR) DNA hairpin, which is part of the initial product of reverse transcription known as minus-strand strong-stop DNA. SM-FRET studies show that the majority of conformational fluctuations of the fluorescently-labeled TAR DNA hairpin in the presence of NC occur in <100 ms. A single molecule explores a wide range of confomations unpon NC binding, with fluctuations encompassing as many as 40 bases in both arms of the hairpin. No conformational fluctuations are observed with the dye-labeled TAR DNA hairpin in the absence of NC or when a labeled TAR DNA hairpin variant lacking bulges and internal loops is analyzed in the presence of NC. This study represents the first real-time observation of NC-mediated nucleic acid conformational fluctuations, revealing new insights into NC's nucleic acid chaperone activity.

  18. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations

    PubMed Central

    Hertig, Samuel

    2016-01-01

    Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein’s constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery—the fact that the two sites involved influence one another in a symmetrical manner—can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest. PMID:27285999

  19. Oligosaccharide Binding Proteins from Bifidobacterium longum subsp. infantis Reveal a Preference for Host Glycans

    PubMed Central

    Garrido, Daniel; Kim, Jae Han; German, J. Bruce; Raybould, Helen E.; Mills, David A.

    2011-01-01

    Bifidobacterium longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota, and it has been characterized by its foraging capacity for human milk oligosaccharides (HMO). Its genome sequence revealed an overabundance of the Family 1 of solute binding proteins (F1SBPs), part of ABC transporters and associated with the import of oligosaccharides. In this study we have used the Mammalian Glycan Array to determine the specific affinities of these proteins. This was correlated with binding protein expression induced by different prebiotics including HMO. Half of the F1SBPs in B. infantis were determined to bind mammalian oligosaccharides. Their affinities included different blood group structures and mucin oligosaccharides. Related to HMO, other proteins were specific for oligomers of lacto-N-biose (LNB) and polylactosamines with different degrees of fucosylation. Growth on HMO induced the expression of specific binding proteins that import HMO isomers, but also bind blood group and mucin oligosaccharides, suggesting coregulated transport mechanisms. The prebiotic inulin induced other family 1 binding proteins with affinity for intestinal glycans. Most of the host glycan F1SBPs in B. infantis do not have homologs in other bifidobacteria. Finally, some of these proteins were found to be adherent to intestinal epithelial cells in vitro. In conclusion, this study represents further evidence for the particular adaptations of B. infantis to the infant gut environment, and helps to understand the molecular mechanisms involved in this process. PMID:21423604

  20. Proteomic analysis of a segregant population reveals candidate proteins linked to mealiness in peach.

    PubMed

    Almeida, Andréa Miyasaka; Urra, Claudio; Moraga, Carol; Jego, Marcela; Flores, Alejandra; Meisel, Lee; González, Mauricio; Infante, Rodrigo; Defilippi, Bruno G; Campos-Vargas, Reinaldo; Orellana, Ariel

    2016-01-10

    Peaches are stored at low temperatures to delay ripening and increase postharvest life. However some varieties are susceptible to chilling injury,which leads to fruit mealiness, browning and flesh bleeding. In order to identify potentialmarkers associated with chilling injury,we performed proteomic analyses on a segregating population with contrasting susceptibility to chilling-induced mealiness. Chilling-induced mealiness was assessed by measuring juiciness in fruits that have been stored in cold and then allowed to ripen. Fruitmesocarp and leaf proteome from contrasting segregants were analyzed using 2-DE gels. Comparison of protein abundance between segregants revealed 133 spots from fruit mesocarp and 36 from leaf. Thirty four fruit mesocarp proteins were identified from these spots. Most of these proteins were related to ethylene synthesis, ABA response and stress response. Leaf protein analyses identified 22 proteins, most of which related to energy metabolism. Some of the genes that code for these proteins have been previously correlated with chilling injury through transcript analyses and co-segregation with mealiness QTLs. The results from this study, further deciphers the molecular mechanisms associated with chilling response in peach fruit, and identifies candidate proteins linked to mealiness in peach which may be used as putative markers for this trait. PMID:26459401

  1. Structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein Rec1-resilin

    NASA Astrophysics Data System (ADS)

    Balu, Rajkamal; Knott, Robert; Cowieson, Nathan P.; Elvin, Christopher M.; Hill, Anita J.; Choudhury, Namita R.; Dutta, Naba K.

    2015-06-01

    Rec1-resilin is the first recombinant resilin-mimetic protein polymer, synthesized from exon-1 of the Drosophila melanogaster gene CG15920 that has demonstrated unusual multi-stimuli responsiveness in aqueous solution. Crosslinked hydrogels of Rec1-resilin have also displayed remarkable mechanical properties including near-perfect rubber-like elasticity. The structural basis of these extraordinary properties is not clearly understood. Here we combine a computational and experimental investigation to examine structural ensembles of Rec1-resilin in aqueous solution. The structure of Rec1-resilin in aqueous solutions is investigated experimentally using circular dichroism (CD) spectroscopy and small angle X-ray scattering (SAXS). Both bench-top and synchrotron SAXS are employed to extract structural data sets of Rec1-resilin and to confirm their validity. Computational approaches have been applied to these experimental data sets in order to extract quantitative information about structural ensembles including radius of gyration, pair-distance distribution function, and the fractal dimension. The present work confirms that Rec1-resilin is an intrinsically disordered protein (IDP) that displays equilibrium structural qualities between those of a structured globular protein and a denatured protein. The ensemble optimization method (EOM) analysis reveals a single conformational population with partial compactness. This work provides new insight into the structural ensembles of Rec1-resilin in solution.

  2. Structure of a Nudix protein from Pyrobaculum aerophilum reveals a dimer with two intersubunit beta-sheets.

    PubMed

    Wang, Shuishu; Mura, Cameron; Sawaya, Michael R; Cascio, Duilio; Eisenberg, David

    2002-04-01

    Nudix proteins, formerly called MutT homolog proteins, are a large family of proteins that play an important role in reducing the accumulation of potentially toxic compounds inside the cell. They hydrolyze a wide variety of substrates that are mainly composed of a nucleoside diphosphate linked to some other moiety X and thus are called Nudix hydrolases. Here, the crystal structure of a Nudix hydrolase from the hyperthermophilic archaeon Pyrobaculum aerophilum is reported. The structure was determined by the single-wavelength anomalous scattering method with data collected at the peak anomalous wavelength of an iridium-derivatized crystal. It reveals an extensive dimer interface, with each subunit contributing two strands to the beta-sheet of the other subunit. Individual subunits consist of a mixed highly twisted and curved beta-sheet of 11 beta-strands and two alpha-helices, forming an alpha-beta-alpha sandwich. The conserved Nudix box signature motif, which contains the essential catalytic residues, is located at the first alpha-helix and the beta-strand and loop preceding it. The unusually short connections between secondary-structural elements, together with the dimer form of the structure, are likely to contribute to the thermostability of the P. aerophilum Nudix protein.

  3. Structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein Rec1-resilin

    PubMed Central

    Balu, Rajkamal; Knott, Robert; Cowieson, Nathan P.; Elvin, Christopher M.; Hill, Anita J.; Choudhury, Namita R.; Dutta, Naba K.

    2015-01-01

    Rec1-resilin is the first recombinant resilin-mimetic protein polymer, synthesized from exon-1 of the Drosophila melanogaster gene CG15920 that has demonstrated unusual multi-stimuli responsiveness in aqueous solution. Crosslinked hydrogels of Rec1-resilin have also displayed remarkable mechanical properties including near-perfect rubber-like elasticity. The structural basis of these extraordinary properties is not clearly understood. Here we combine a computational and experimental investigation to examine structural ensembles of Rec1-resilin in aqueous solution. The structure of Rec1-resilin in aqueous solutions is investigated experimentally using circular dichroism (CD) spectroscopy and small angle X-ray scattering (SAXS). Both bench-top and synchrotron SAXS are employed to extract structural data sets of Rec1-resilin and to confirm their validity. Computational approaches have been applied to these experimental data sets in order to extract quantitative information about structural ensembles including radius of gyration, pair-distance distribution function, and the fractal dimension. The present work confirms that Rec1-resilin is an intrinsically disordered protein (IDP) that displays equilibrium structural qualities between those of a structured globular protein and a denatured protein. The ensemble optimization method (EOM) analysis reveals a single conformational population with partial compactness. This work provides new insight into the structural ensembles of Rec1-resilin in solution. PMID:26042819

  4. Systematic protein interactome analysis of glycosaminoglycans revealed YcbS as a novel bacterial virulence factor.

    PubMed

    Hsiao, Felix Shih-Hsiang; Sutandy, Fx Reymond; Syu, Guan-Da; Chen, Yi-Wen; Lin, Jun-Mu; Chen, Chien-Sheng

    2016-01-01

    Microbial pathogens have evolved several strategies for interacting with host cell components, such as glycosaminoglycans (GAGs). Some microbial proteins involved in host-GAG binding have been described; however, a systematic study on microbial proteome-mammalian GAG interactions has not been conducted. Here, we used Escherichia coli proteome chips to probe four typical mammalian GAGs, heparin, heparan sulphate (HS), chondroitin sulphate B (CSB), and chondroitin sulphate C (CSC), and identified 185 heparin-, 62 HS-, 98 CSB-, and 101 CSC-interacting proteins. Bioinformatics analyses revealed the unique functions of heparin- and HS-specific interacting proteins in glycine, serine, and threonine metabolism. Among all the GAG-interacting proteins, three were outer membrane proteins (MbhA, YcbS, and YmgH). Invasion assays confirmed that mutant E. coli lacking ycbS could not invade the epithelial cells. Introducing plasmid carrying ycbS complemented the invading defects at ycbS lacking E. coli mutant, that can be further improved by overexpressing ycbS. Preblocking epithelial cells with YcbS reduced the percentage of E. coli invasions. Moreover, we observed that whole components of the ycb operon were crucial for invasion. The displacement assay revealed that YcbS binds to the laminin-binding site of heparin and might affect the host extracellular matrix structure by displacing heparin from laminin. PMID:27323865

  5. Systematic protein interactome analysis of glycosaminoglycans revealed YcbS as a novel bacterial virulence factor

    PubMed Central

    Hsiao, Felix Shih-Hsiang; Sutandy, FX Reymond; Syu, Guan-Da; Chen, Yi-Wen; Lin, Jun-Mu; Chen, Chien-Sheng

    2016-01-01

    Microbial pathogens have evolved several strategies for interacting with host cell components, such as glycosaminoglycans (GAGs). Some microbial proteins involved in host–GAG binding have been described; however, a systematic study on microbial proteome–mammalian GAG interactions has not been conducted. Here, we used Escherichia coli proteome chips to probe four typical mammalian GAGs, heparin, heparan sulphate (HS), chondroitin sulphate B (CSB), and chondroitin sulphate C (CSC), and identified 185 heparin-, 62 HS-, 98 CSB-, and 101 CSC-interacting proteins. Bioinformatics analyses revealed the unique functions of heparin- and HS-specific interacting proteins in glycine, serine, and threonine metabolism. Among all the GAG-interacting proteins, three were outer membrane proteins (MbhA, YcbS, and YmgH). Invasion assays confirmed that mutant E. coli lacking ycbS could not invade the epithelial cells. Introducing plasmid carrying ycbS complemented the invading defects at ycbS lacking E. coli mutant, that can be further improved by overexpressing ycbS. Preblocking epithelial cells with YcbS reduced the percentage of E. coli invasions. Moreover, we observed that whole components of the ycb operon were crucial for invasion. The displacement assay revealed that YcbS binds to the laminin-binding site of heparin and might affect the host extracellular matrix structure by displacing heparin from laminin. PMID:27323865

  6. Protein turnover methods in single-celled organisms: dynamic SILAC.

    PubMed

    Claydon, Amy J; Beynon, Robert J

    2011-01-01

    Early achievements in proteomics were qualitative, typified by the identification of very small quantities of proteins. However, as the subject has developed, there has been a pressure to develop approaches to define the amounts of each protein--whether in a relative or an absolute sense. A further dimension to quantitative proteomics embeds the behavior of each protein in terms of its turnover. Virtually every protein in the cell is in a dynamic state, subject to continuous synthesis and degradation, the relative rates of which control the expansion or the contraction of the protein pool, and the absolute values of which dictate the temporal responsiveness of the protein pool. Strategies must therefore be developed to assess the turnover of individual proteins in the proteome. Because a protein can be turning over rapidly even when the protein pool is in steady state, the only acceptable approach to measure turnover is to use metabolic labels that are incorporated or lost from the protein pool as it is replaced. Using metabolic labeling on a proteome-wide scale in turn requires metabolic labels that contain stable isotopes, the incorporation or loss of which can be assessed by mass spectrometry. A typical turnover experiment is complex. The choice of metabolic label is dictated by several factors, including abundance in the proteome, metabolic redistribution of the label in the precursor pool, and the downstream mass spectrometric analytical protocols. Key issues include the need to control and understand the relative isotope abundance of the precursor, the optimization of label flux into and out of the protein pool, and a sampling strategy that ensures the coverage of the greatest range of turnover rates. Finally, the informatics approaches to data analysis will not be as straightforward as in other areas of proteomics. In this chapter, we will discuss the principles and practice of workflow development for turnover analysis, exemplified by the development of

  7. Phylogenomics Controlling for Base Compositional Bias Reveals a Single Origin of Eusociality in Corbiculate Bees.

    PubMed

    Romiguier, Jonathan; Cameron, Sydney A; Woodard, S Hollis; Fischman, Brielle J; Keller, Laurent; Praz, Christophe J

    2016-03-01

    As increasingly large molecular data sets are collected for phylogenomics, the conflicting phylogenetic signal among gene trees poses challenges to resolve some difficult nodes of the Tree of Life. Among these nodes, the phylogenetic position of the honey bees (Apini) within the corbiculate bee group remains controversial, despite its considerable importance for understanding the emergence and maintenance of eusociality. Here, we show that this controversy stems in part from pervasive phylogenetic conflicts among GC-rich gene trees. GC-rich genes typically have a high nucleotidic heterogeneity among species, which can induce topological conflicts among gene trees. When retaining only the most GC-homogeneous genes or using a nonhomogeneous model of sequence evolution, our analyses reveal a monophyletic group of the three lineages with a eusocial lifestyle (honey bees, bumble bees, and stingless bees). These phylogenetic relationships strongly suggest a single origin of eusociality in the corbiculate bees, with no reversal to solitary living in this group. To accurately reconstruct other important evolutionary steps across the Tree of Life, we suggest removing GC-rich and GC-heterogeneous genes from large phylogenomic data sets. Interpreted as a consequence of genome-wide variations in recombination rates, this GC effect can affect all taxa featuring GC-biased gene conversion, which is common in eukaryotes. PMID:26576851

  8. Mitochondrial specialization revealed by single muscle fiber proteomics: focus on the Krebs cycle.

    PubMed

    Schiaffino, S; Reggiani, C; Kostrominova, T Y; Mann, M; Murgia, M

    2015-12-01

    We have developed a highly sensitive mass spectrometry-based proteomic workflow to examine the proteome of single muscle fibers. This study revealed significant differences in the mitochondrial proteome of the four major fiber types present in mouse skeletal muscle. Here, we focus on Krebs cycle enzymes and in particular on the differential distribution of the two mitochondrial isocitrate dehydrogenases, IDH2 and IDH3. Type 1/slow fibers contain high levels of IDH2 and relatively low levels of IDH3, whereas fast 2X and 2B fibers show an opposite expression pattern. The findings suggest that in skeletal muscle, IDH2 functions in the forward direction of the Krebs cycle and that substrate flux along the cycle occurs predominantly via IDH2 in type 1 fibers and via IDH3 in 2X and 2B fibers. IDH2-mediated conversion of isocitrate to α-ketoglutarate leads to the generation of NADPH, which is critical to buffering the H2O2 produced by the respiratory chain. Nicotinamide nucleotide transhydrogenase (NNT), the other major mitochondrial enzyme involved in NADPH generation, is also more abundant in type 1 fibers. We suggest that the continuously active type 1 fibers are endowed with a more efficient H2O2 scavenging capacity to cope with the higher levels of reactive oxygen species production.

  9. Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy.

    PubMed

    Pozzi, Nicola; Bystranowska, Dominika; Zuo, Xiaobing; Di Cera, Enrico

    2016-08-26

    The coagulation factor prothrombin has a complex spatial organization of its modular assembly that comprises the N-terminal Gla domain, kringle-1, kringle-2, and the C-terminal protease domain connected by three intervening linkers. Here we use single molecule Förster resonance energy transfer to access the conformational landscape of prothrombin in solution and uncover structural features of functional significance that extend recent x-ray crystallographic analysis. Prothrombin exists in equilibrium between two alternative conformations, open and closed. The closed conformation predominates (70%) and features an unanticipated intramolecular collapse of Tyr(93) in kringle-1 onto Trp(547) in the protease domain that obliterates access to the active site and protects the zymogen from autoproteolytic conversion to thrombin. The open conformation (30%) is more susceptible to chymotrypsin digestion and autoactivation, and features a shape consistent with recent x-ray crystal structures. Small angle x-ray scattering measurements of prothrombin wild type stabilized 70% in the closed conformation and of the mutant Y93A stabilized 80% in the open conformation directly document two envelopes that differ 50 Å in length. These findings reveal important new details on the conformational plasticity of prothrombin in solution and the drastic structural difference between its alternative conformations. Prothrombin uses the intramolecular collapse of kringle-1 onto the active site in the closed form to prevent autoactivation. The open-closed equilibrium also defines a new structural framework for the mechanism of activation of prothrombin by prothrombinase. PMID:27435675

  10. Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity

    PubMed Central

    Chiu, Isaac M; Barrett, Lee B; Williams, Erika K; Strochlic, David E; Lee, Seungkyu; Weyer, Andy D; Lou, Shan; Bryman, Gregory S; Roberson, David P; Ghasemlou, Nader; Piccoli, Cara; Ahat, Ezgi; Wang, Victor; Cobos, Enrique J; Stucky, Cheryl L; Ma, Qiufu; Liberles, Stephen D; Woolf, Clifford J

    2014-01-01

    The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4+SNS-Cre/TdTomato+, 2) IB4−SNS-Cre/TdTomato+, and 3) Parv-Cre/TdTomato+ cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation. DOI: http://dx.doi.org/10.7554/eLife.04660.001 PMID:25525749

  11. Coarse grained simulation reveals antifreeze properties of hyperactive antifreeze protein from Antarctic bacterium Colwellia sp.

    NASA Astrophysics Data System (ADS)

    Nguyen, Hung; Van, Thanh Dac; Le, Ly

    2015-10-01

    The novel hyperactive antifreeze protein (AFP) of Antarctic sea ice bacterium Colwellia sp. provides a target for studying the protection of psychrophilic microgoranisms against freezing environment. Interestingly, the Colwellia sp. hyperactive antifreeze protein (ColAFP) was crystallized without the structural dynamic characteristics. Here, the result indicated, through coarse grained simulation of ColAFP under various subfreezing temperature, that ColAFP remains active at temperature of equal and greater than 275 K (∼2 °C). Extensive simulation analyses also revealed the adaptive mechanism of ColAFP in subfreezing environment. Our result provides a structural dynamic understanding of the ColAFP.

  12. Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry.

    PubMed

    Asara, John M; Schweitzer, Mary H; Freimark, Lisa M; Phillips, Matthew; Cantley, Lewis C

    2007-04-13

    Fossilized bones from extinct taxa harbor the potential for obtaining protein or DNA sequences that could reveal evolutionary links to extant species. We used mass spectrometry to obtain protein sequences from bones of a 160,000- to 600,000-year-old extinct mastodon (Mammut americanum) and a 68-million-year-old dinosaur (Tyrannosaurus rex). The presence of T. rex sequences indicates that their peptide bonds were remarkably stable. Mass spectrometry can thus be used to determine unique sequences from ancient organisms from peptide fragmentation patterns, a valuable tool to study the evolution and adaptation of ancient taxa from which genomic sequences are unlikely to be obtained.

  13. Massively parallel sampling of lattice proteins reveals foundations of thermal adaptation

    NASA Astrophysics Data System (ADS)

    Venev, Sergey V.; Zeldovich, Konstantin B.

    2015-08-01

    Evolution of proteins in bacteria and archaea living in different conditions leads to significant correlations between amino acid usage and environmental temperature. The origins of these correlations are poorly understood, and an important question of protein theory, physics-based prediction of types of amino acids overrepresented in highly thermostable proteins, remains largely unsolved. Here, we extend the random energy model of protein folding by weighting the interaction energies of amino acids by their frequencies in protein sequences and predict the energy gap of proteins designed to fold well at elevated temperatures. To test the model, we present a novel scalable algorithm for simultaneous energy calculation for many sequences in many structures, targeting massively parallel computing architectures such as graphics processing unit. The energy calculation is performed by multiplying two matrices, one representing the complete set of sequences, and the other describing the contact maps of all structural templates. An implementation of the algorithm for the CUDA platform is available at http://www.github.com/kzeldovich/galeprot and calculates protein folding energies over 250 times faster than a single central processing unit. Analysis of amino acid usage in 64-mer cubic lattice proteins designed to fold well at different temperatures demonstrates an excellent agreement between theoretical and simulated values of energy gap. The theoretical predictions of temperature trends of amino acid frequencies are significantly correlated with bioinformatics data on 191 bacteria and archaea, and highlight protein folding constraints as a fundamental selection pressure during thermal adaptation in biological evolution.

  14. Single-cell genome and metatranscriptome sequencing reveal metabolic interactions of an alkane-degrading methanogenic community

    PubMed Central

    Embree, Mallory; Nagarajan, Harish; Movahedi, Narjes; Chitsaz, Hamidreza; Zengler, Karsten

    2014-01-01

    Microbial interactions have a key role in global geochemical cycles. Although we possess significant knowledge about the general biochemical processes occurring in microbial communities, we are often unable to decipher key functions of individual microorganisms within the environment in part owing to the inability to cultivate or study them in isolation. Here, we circumvent this shortcoming through the use of single-cell genome sequencing and a novel low-input metatranscriptomics protocol to reveal the intricate metabolic capabilities and microbial interactions of an alkane-degrading methanogenic community. This methanogenic consortium oxidizes saturated hydrocarbons under anoxic conditions through a thus-far-uncharacterized biochemical process. The genome sequence of a dominant bacterial member of this community, belonging to the genus Smithella, was sequenced and served as the basis for subsequent analysis through metabolic reconstruction. Metatranscriptomic data generated from less than 500 pg of mRNA highlighted metabolically active genes during anaerobic alkane oxidation in comparison with growth on fatty acids. These data sets suggest that Smithella is not activating hexadecane by fumarate addition. Differential expression assisted in the identification of hypothetical proteins with no known homology that may be involved in hexadecane activation. Additionally, the combination of 16S rDNA sequence and metatranscriptomic data enabled the study of other prevalent organisms within the consortium and their interactions with Smithella, thus yielding a comprehensive characterization of individual constituents at the genome scale during methanogenic alkane oxidation. PMID:24152715

  15. Single-molecule studies of the lysine riboswitch reveal effector-dependent conformational dynamics of the aptamer domain.

    PubMed

    Fiegland, Larry R; Garst, Andrew D; Batey, Robert T; Nesbitt, David J

    2012-11-13

    The lysine riboswitch is a cis-acting RNA genetic regulatory element found in the leader sequence of bacterial mRNAs coding for proteins related to biosynthesis or transport of lysine. Structural analysis of the lysine-binding aptamer domain of this RNA has revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing upon interaction with lysine. In this work, single-molecule fluorescence resonance energy transfer (FRET) methods are used to monitor these ligand-induced structural transitions that are central to lysine riboswitch function. Specifically, a model FRET system has been developed for characterizing the lysine dissociation constant as well as the opening/closing rate constants for the Bacillus subtilis lysC aptamer domain. These techniques permit measurement of the dissociation constant (K(D)) for lysine binding of 1.7(5) mM and opening/closing rate constants of 1.4(3) s(-1) and 0.203(7) s(-1), respectively. These rates predict an apparent dissociation constant for lysine binding (K(D,apparent)) of 0.25(9) mM at near physiological ionic strength, which differs markedly from previous reports. PMID:23067368

  16. Single-Molecule Studies of the Lysine Riboswitch Reveal Effector Dependent Conformational Dynamics of the Aptamer Domain

    PubMed Central

    Fiegland, Larry R.; Garst, Andrew D.; Batey, Robert T.; Nesbitt, David J.

    2013-01-01

    The lysine riboswitch is a cis-acting RNA genetic regulatory element found in the leader sequence of bacterial mRNAs coding for proteins related to biosynthesis or transport of lysine. Structural analysis of the lysine-binding aptamer domain of this RNA has revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing upon interaction with lysine. In this work, single-molecule fluorescence resonance energy transfer (FRET) methods are used to monitor these ligand-induced structural transitions that are central to lysine riboswitch function. Specifically, a model FRET system has been developed for characterizing the lysine dissociation constant, as well as the opening/closing rate constants for the Bacillus subtilis lysC aptamer domain. These techniques permit measurement of the dissociation constant (KD) for lysine binding of 1.7(5) mM, and opening/closing rate constants of 1.4(3) s−1 and 0.203(7) s−1, respectively. These rates predict an apparent dissociation constant for lysine binding (KD, apparent) of 0.25(9) mM at near physiological ionic strength, which differs markedly from previous reports. PMID:23067368

  17. Hindered submicron mobility and long-term storage of presynaptic dense-core granules revealed by single-particle tracking

    PubMed Central

    Scalettar, B. A.; Jacobs, C.; Fulwiler, A.; Prahl, L.; Simon, A.; Hilken, L.; Lochner, J. E.

    2012-01-01

    Dense-core granules (DCGs) are organelles found in neuroendocrine cells and neurons that house, transport, and release a number of important peptides and proteins. In neurons, DCG cargo can include the secreted neuromodulatory proteins tissue plasminogen activator (tPA) and/or brain-derived neurotrophic factor (BDNF), which play a key role in modulating synaptic efficacy in the hippocampus. This function has spurred interest in DCGs that localize to synaptic contacts between hippocampal neurons, and several studies recently have established that DCGs localize to, and undergo regulated exocytosis from, postsynaptic sites. To complement this work, we have studied presynaptically-localized DCGs in hippocampal neurons, which are much more poorly understood than their postsynaptic analogs. Moreover, to enhance relevance, we visualized DCGs via fluorescence labeling of exogenous and endogenous tPA and BDNF. Using single-particle tracking, we determined trajectories of more than 150 presynaptically-localized DCGs. These trajectories reveal that mobility of DCGs in presynaptic boutons is highly hindered and that storage is long-lived. We also computed mean-squared displacement curves, which can be used to elucidate mechanisms of transport. Over shorter time windows, most curves are linear, demonstrating that DCG transport in boutons is driven predominantly by diffusion. The remaining curves plateau with time, consistent with motion constrained by a submicron-sized corral. These results have relevance to recent models of presynaptic organization and to recent hypotheses about DCG cargo function. The results also provide estimates for transit times to the presynaptic plasma membrane that are consistent with measured times for onset of neurotrophin release from synaptically-localized DCGs. PMID:21976424

  18. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    DOE PAGES

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-13

    Re-engineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test thismore » strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. The context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.« less

  19. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    SciTech Connect

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-13

    Re-engineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. The context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

  20. Crystal structures of flax rust avirulence proteins AvrL567-A and -D reveal details of the structural basis for flax disease resistance specificity.

    PubMed

    Wang, Ching-I A; Guncar, Gregor; Forwood, Jade K; Teh, Trazel; Catanzariti, Ann-Maree; Lawrence, Gregory J; Loughlin, Fionna E; Mackay, Joel P; Schirra, Horst Joachim; Anderson, Peter A; Ellis, Jeffrey G; Dodds, Peter N; Kobe, Bostjan

    2007-09-01

    The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-A resolution, respectively. The structures of both proteins are very similar and reveal a beta-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

  1. Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization

    PubMed Central

    Ariza, Antonio; Tanner, Sian J.; Walter, Cheryl T.; Dent, Kyle C.; Shepherd, Dale A.; Wu, Weining; Matthews, Susan V.; Hiscox, Julian A.; Green, Todd J.; Luo, Ming; Elliott, Richard M.; Fooks, Anthony R.; Ashcroft, Alison E.; Stonehouse, Nicola J.; Ranson, Neil A.; Barr, John N.; Edwards, Thomas A.

    2013-01-01

    All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N–RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses. PMID:23595147

  2. Revealing the amino acid composition of proteins within an expanded genetic code

    PubMed Central

    Aerni, Hans R.; Shifman, Mark A.; Rogulina, Svetlana; O'Donoghue, Patrick; Rinehart, Jesse

    2015-01-01

    The genetic code can be manipulated to reassign codons for the incorporation of non-standard amino acids (NSAA). Deletion of release factor 1 in Escherichia coli enhances translation of UAG (Stop) codons, yet may also extended protein synthesis at natural UAG terminated messenger RNAs. The fidelity of protein synthesis at reassigned UAG codons and the purity of the NSAA containing proteins produced require careful examination. Proteomics would be an ideal tool for these tasks, but conventional proteomic analyses cannot readily identify the extended proteins and accurately discover multiple amino acid (AA) insertions at a single UAG. To address these challenges, we created a new proteomic workflow that enabled the detection of UAG readthrough in native proteins in E. coli strains in which UAG was reassigned to encode phosphoserine. The method also enabled quantitation of NSAA and natural AA incorporation at UAG in a recombinant reporter protein. As a proof-of-principle, we measured the fidelity and purity of the phosphoserine orthogonal translation system (OTS) and used this information to improve its performance. Our results show a surprising diversity of natural AAs at reassigned stop codons. Our method can be used to improve OTSs and to quantify amino acid purity at reassigned codons in organisms with expanded genetic codes. PMID:25378305

  3. Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging.

    PubMed

    Khan, Shahid; Conte, Ianina; Carter, Tom; Bayer, K Ulrich; Molloy, Justin E

    2016-07-26

    Localization of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to dendritic spine synapses is determined in part by the actin cytoskeleton. We determined binding of GFP-tagged CaMKII to tag-RFP-labeled actin cytoskeleton within live cells using total internal reflection fluorescence microscopy and single-molecule tracking. Stepwise photobleaching showed that CaMKII formed oligomeric complexes. Photoactivation experiments demonstrated that diffusion out of the evanescent field determined the track lifetimes. Latrunculin treatment triggered a coupled loss of actin stress fibers and the colocalized, long-lived CaMKII tracks. The CaMKIIα (α) isoform, which was previously thought to lack F-actin interactions, also showed binding, but this was threefold weaker than that observed for CaMKIIβ (β). The βE' splice variant bound more weakly than α, showing that binding by β depends critically on the interdomain linker. The mutations βT287D and αT286D, which mimic autophosphorylation states, also abolished F-actin binding. Autophosphorylation triggers autonomous CaMKII activity, but does not impair GluN2B binding, another important synaptic protein interaction of CaMKII. The CaMKII inhibitor tatCN21 or CaMKII mutations that inhibit GluN2B association by blocking binding of ATP (βK43R and αK42M) or Ca(2+)/calmodulin (βA303R) had no effect on the interaction with F-actin. These results provide the first rationale for the reduced synaptic spine localization of the αT286D mutant, indicating that transient F-actin binding contributes to the synaptic localization of the CaMKIIα isoform. The track lifetime distributions had a stretched exponential form consistent with a heterogeneously diffusing population. This heterogeneity suggests that CaMKII adopts different F-actin binding modes, which is most easily rationalized by multiple subunit contacts between the CaMKII dodecamer and the F-actin cytoskeleton that stabilize the initial weak (micromolar

  4. Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces

    PubMed Central

    Engin, H. Billur; Kreisberg, Jason F.; Carter, Hannah

    2016-01-01

    Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10−4) and oncogenes (Odds Ratio 1.17, P-value < 10−3). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10−8). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer

  5. Quantitative proteomics reveals novel insights into isoniazid susceptibility in mycobacteria mediated by a universal stress protein.

    PubMed

    Hu, Xinling; Li, Xiaojing; Huang, Lige; Chan, John; Chen, Yuling; Deng, Haiteng; Mi, Kaixia

    2015-03-01

    Tuberculosis (TB) is caused by the ancient pathogen, Mycobacterium tuberculosis, and is one of the most serious infectious diseases in the world. Isoniazid (INH) is an important first-line drug for the treatment of active and latent TB. INH resistance is an increasing problem in the treatment of TB. Phenotypic resistance to INH, however, is poorly understood. In this study, we constructed a strain of Mycobacterium bovis BCG that overexpresses the latency-related universal stress protein (USP), BCG_2013, and designated this strain BCG-2013. BCG_2013 overexpression increased susceptibility to INH compared with that of the wild-type strain, BCG-pMV261. Quantitative proteomic analysis revealed that BCG_2013 overexpression resulted in the upregulation of 50 proteins and the downregulation of 26 proteins among the 1500 proteins identified. Upregulation of catalase-peroxidase KatG expression in BCG-2013 was observed and confirmed by qPCR, whereas expression of other INH resistance-related proteins did not change. In addition, differential expression of the mycobacterial persistence regulator MprA and its regulatory proteins was observed. BCG_2013 and katG mRNA levels increased in a Wayne dormancy model, whereas MprA mRNA levels decreased. Taken together, our results suggest that the increase in KatG levels induced by increased BCG_2013 levels underlies the phenotypic susceptibility of mycobacteria to INH.

  6. Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

    PubMed

    Hamsten, C; Häggmark, A; Grundström, J; Mikus, M; Lindskog, C; Konradsen, J R; Eklund, A; Pershagen, G; Wickman, M; Grunewald, J; Melén, E; Hedlin, G; Nilsson, P; van Hage, M

    2016-09-01

    Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma. PMID:27145233

  7. A simple atomic-level hydrophobicity scale reveals protein interfacial structure.

    PubMed

    Kapcha, Lauren H; Rossky, Peter J

    2014-01-23

    Many amino acid residue hydrophobicity scales have been created in an effort to better understand and rapidly characterize water-protein interactions based only on protein structure and sequence. There is surprisingly low consistency in the ranking of residue hydrophobicity between scales, and their ability to provide insightful characterization varies substantially across subject proteins. All current scales characterize hydrophobicity based on entire amino acid residue units. We introduce a simple binary but atomic-level hydrophobicity scale that allows for the classification of polar and non-polar moieties within single residues, including backbone atoms. This simple scale is first shown to capture the anticipated hydrophobic character for those whole residues that align in classification among most scales. Examination of a set of protein binding interfaces establishes good agreement between residue-based and atomic-level descriptions of hydrophobicity for five residues, while the remaining residues produce discrepancies. We then show that the atomistic scale properly classifies the hydrophobicity of functionally important regions where residue-based scales fail. To illustrate the utility of the new approach, we show that the atomic-level scale rationalizes the hydration of two hydrophobic pockets and the presence of a void in a third pocket within a single protein and that it appropriately classifies all of the functionally important hydrophilic sites within two otherwise hydrophobic pores. We suggest that an atomic level of detail is, in general, necessary for the reliable depiction of hydrophobicity for all protein surfaces. The present formulation can be implemented simply in a manner no more complex than current residue-based approaches.

  8. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

    PubMed

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D

    2016-03-22

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  9. Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic.

    PubMed

    Zhang, Hai-Nan; Yang, Lina; Ling, Jian-Ya; Czajkowsky, Daniel M; Wang, Jing-Fang; Zhang, Xiao-Wei; Zhou, Yi-Ming; Ge, Feng; Yang, Ming-Kun; Xiong, Qian; Guo, Shu-Juan; Le, Huang-Ying; Wu, Song-Fang; Yan, Wei; Liu, Bingya; Zhu, Heng; Chen, Zhu; Tao, Sheng-Ce

    2015-12-01

    Arsenic is highly effective for treating acute promyelocytic leukemia (APL) and has shown significant promise against many other tumors. However, although its mechanistic effects in APL are established, its broader anticancer mode of action is not understood. In this study, using a human proteome microarray, we identified 360 proteins that specifically bind arsenic. Among the most highly enriched proteins in this set are those in the glycolysis pathway, including the rate-limiting enzyme in glycolysis, hexokinase-1. Detailed biochemical and metabolomics analyses of the highly homologous hexokinase-2 (HK2), which is overexpressed in many cancers, revealed significant inhibition by arsenic. Furthermore, overexpression of HK2 rescued cells from arsenic-induced apoptosis. Our results thus strongly implicate glycolysis, and HK2 in particular, as a key target of arsenic. Moreover, the arsenic-binding proteins identified in this work are expected to serve as a valuable resource for the development of synergistic antitumor therapeutic strategies.

  10. Mass Spectrometry and Next-Generation Sequencing Reveal an Abundant and Rapidly Evolving Abalone Sperm Protein

    PubMed Central

    Palmer, Melody R.; McDowall, Margo H.; Stewart, Lia; Ouaddi, Aleena; MacCoss, Michael J.; Swanson, Willie J.

    2014-01-01

    SUMMARY Abalone, a broadcast spawning marine mollusk, is an important model for molecular interactions and positive selection in fertilization, but the focus has previously been on only two sperm proteins, lysin and sp18.We used genomic and proteomic techniques to bring new insights to this model by characterizing the testis transcriptome and sperm proteome of the Red abalone Haliotis rufescens. One pair of homologous, testis-specific proteins contains a secretion signal and is small, abundant, and associated with the acrosome. Comparative analysis revealed that homologs are extremely divergent between species, and show strong evidence for positive selection. The acrosomal localization and rapid evolution of these proteins indicates that they play an important role in fertilization, and could be involved in the species-specificity of sperm-egg interactions in abalone. Our genomic and proteomic characterization of abalone fertilization resulted in the identification of interesting, novel peptides that have eluded detection in this important model system for 20 years. PMID:23585193

  11. Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic

    PubMed Central

    Zhang, Hai-nan; Yang, Lina; Ling, Jian-ya; Czajkowsky, Daniel M.; Wang, Jing-Fang; Zhang, Xiao-Wei; Zhou, Yi-Ming; Ge, Feng; Yang, Ming-kun; Xiong, Qian; Guo, Shu-Juan; Le, Huang-Ying; Wu, Song-Fang; Yan, Wei; Liu, Bingya; Zhu, Heng; Chen, Zhu; Tao, Sheng-ce

    2015-01-01

    Arsenic is highly effective for treating acute promyelocytic leukemia (APL) and has shown significant promise against many other tumors. However, although its mechanistic effects in APL are established, its broader anticancer mode of action is not understood. In this study, using a human proteome microarray, we identified 360 proteins that specifically bind arsenic. Among the most highly enriched proteins in this set are those in the glycolysis pathway, including the rate-limiting enzyme in glycolysis, hexokinase-1. Detailed biochemical and metabolomics analyses of the highly homologous hexokinase-2 (HK2), which is overexpressed in many cancers, revealed significant inhibition by arsenic. Furthermore, overexpression of HK2 rescued cells from arsenic-induced apoptosis. Our results thus strongly implicate glycolysis, and HK2 in particular, as a key target of arsenic. Moreover, the arsenic-binding proteins identified in this work are expected to serve as a valuable resource for the development of synergistic antitumor therapeutic strategies. PMID:26598702

  12. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    PubMed

    Huesgen, Pitter F; Alami, Meriem; Lange, Philipp F; Foster, Leonard J; Schröder, Wolfgang P; Overall, Christopher M; Green, Beverley R

    2013-01-01

    In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  13. Fluorescence Anisotropy Reveals Order and Disorder of Protein Domains in the Nuclear Pore Complex

    PubMed Central

    Mattheyses, Alexa L.; Kampmann, Martin; Atkinson, Claire E.; Simon, Sanford M.

    2010-01-01

    We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport. PMID:20858414

  14. Structures of replication initiation proteins from staphylococcal antibiotic resistance plasmids reveal protein asymmetry and flexibility are necessary for replication

    PubMed Central

    Carr, Stephen B.; Phillips, Simon E.V.; Thomas, Christopher D.

    2016-01-01

    Antibiotic resistance in pathogenic bacteria is a continual threat to human health, often residing in extrachromosomal plasmid DNA. Plasmids of the pT181 family are widespread and confer various antibiotic resistances to Staphylococcus aureus. They replicate via a rolling circle mechanism that requires a multi-functional, plasmid-encoded replication protein to initiate replication, recruit a helicase to the site of initiation and terminate replication after DNA synthesis is complete. We present the first atomic resolution structures of three such replication proteins that reveal distinct, functionally relevant conformations. The proteins possess a unique active site and have been shown to contain a catalytically essential metal ion that is bound in a manner distinct from that of any other rolling circle replication proteins. These structures are the first examples of the Rep_trans Pfam family providing insights into the replication of numerous antibiotic resistance plasmids from Gram-positive bacteria, Gram-negative phage and the mobilisation of DNA by conjugative transposons. PMID:26792891

  15. Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

    PubMed

    De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

    2016-02-01

    Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. PMID:26031839

  16. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions.

    PubMed

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N; Flood, Beverly E; Bailey, Jake V; Mußmann, Marc

    2016-01-01

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na(+)-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of "Ca. T. nelsonii Thio36" provides additional insight into the ecology of giant sulfur

  17. Single cell analysis reveals gametic and tissue-specific instability of the SCA1 CAG repeat

    SciTech Connect

    Chong, S.S.; McCall, A.E.; Cota, J.

    1994-09-01

    Spinocerebellar ataxia type 1 is an autosomal dominant neurodegenerative disease caused by expansion of a CAG trinucleotide repeat within the SCA1 gene on chromosome 6p22-23. We performed a comparative analysis of the SCA1 CAG repeat from blood and sperm of an affected male. Genomic amplification revealed a broader smear of the SCA1 allele product from sperm compared to that from peripheral blood leukocytes (PBL). To resolve this observed difference, we analyzed single sperm directly and demonstrate that the SCA1 allele in PBL is also heterogeneous, although the range of variability in allele sizes is much less than that observed in sperm. Limited genome analysis was also performed on PBL DNA from an unaffected individual with an upper normal allele of 36 repeats in parallel with an affected individual with an expanded allele of 40 repeats. The 36 repeat normal allele, which contains a CAT interruption, was completely stable compared to the uninterrupted repeat of the SCA1 allele, demonstrating a direct correlation between absence of a CAT interruption and somatic instability of the repeat. We also analyzed the size of the CAG repeat in tissues derived from various brain regions from a patient with juvenile-onset disease to determine if the size of the expansion correlated with the site of neuropathology. The results clearly show tissue-specific differences in mosaicism of repeat length. More importantly, the pattern of tissue-specific differences in repeat-length mosaicism in SCA1 within the brain parallels those seen in Huntington disease. In both disorders the expanded alleles are smaller in cerebellar tissue. These results suggest that the observed tissue-specific differences in instability of the SCA1 CAG repeat, either within the brain or between blood and sperm, are a function of the intracellular milieu or the intrinsic replicative potential of the various celltypes.

  18. Genome-wide single nucleotide polymorphisms reveal population history and adaptive divergence in wild guppies.

    PubMed

    Willing, Eva-Maria; Bentzen, Paul; van Oosterhout, Cock; Hoffmann, Margarete; Cable, Joanne; Breden, Felix; Weigel, Detlef; Dreyer, Christine

    2010-03-01

    Adaptation of guppies (Poecilia reticulata) to contrasting upland and lowland habitats has been extensively studied with respect to behaviour, morphology and life history traits. Yet population history has not been studied at the whole-genome level. Although single nucleotide polymorphisms (SNPs) are the most abundant form of variation in many genomes and consequently very informative for a genome-wide picture of standing natural variation in populations, genome-wide SNP data are rarely available for wild vertebrates. Here we use genetically mapped SNP markers to comprehensively survey genetic variation within and among naturally occurring guppy populations from a wide geographic range in Trinidad and Venezuela. Results from three different clustering methods, Neighbor-net, principal component analysis (PCA) and Bayesian analysis show that the population substructure agrees with geographic separation and largely with previously hypothesized patterns of historical colonization. Within major drainages (Caroni, Oropouche and Northern), populations are genetically similar, but those in different geographic regions are highly divergent from one another, with some indications of ancient shared polymorphisms. Clear genomic signatures of a previous introduction experiment were seen, and we detected additional potential admixture events. Headwater populations were significantly less heterozygous than downstream populations. Pairwise F(ST) values revealed marked differences in allele frequencies among populations from different regions, and also among populations within the same region. F(ST) outlier methods indicated some regions of the genome as being under directional selection. Overall, this study demonstrates the power of a genome-wide SNP data set to inform for studies on natural variation, adaptation and evolution of wild populations.

  19. Compensatory evolution reveals functional interactions between ribosomal proteins S12, L14 and L19.

    PubMed

    Maisnier-Patin, Sophie; Paulander, Wilhelm; Pennhag, Alexandra; Andersson, Dan I

    2007-02-01

    Certain mutations in S12, a ribosomal protein involved in translation elongation rate and translation accuracy, confer resistance to the aminoglycoside streptomycin. Previously we showed in Salmonella typhimurium that the fitness cost, i.e. reduced growth rate, due to the amino acid substitution K42N in S12 could be compensated by at least 35 different mutations located in the ribosomal proteins S4, S5 and L19. Here, we have characterized in vivo the fitness, translation speed and translation accuracy of four different L19 mutants. When separated from the resistance mutation located in S12, the three different compensatory amino acid substitutions in L19 at position 40 (Q40H, Q40L and Q40R) caused a decrease in fitness while the G104A change had no effect on bacterial growth. The rate of protein synthesis was unaffected or increased by the mutations at position 40 and the level of read-through of a UGA nonsense codon was increased in vivo, indicating a loss of translational accuracy. The mutations in L19 increased sensitivity to aminoglycosides active at the A-site, further indicating a perturbation of the decoding step. These phenotypes are similar to those of the classical S4 and S5 ram (ribosomal ambiguity) mutants. By evolving low-fitness L19 mutants by serial passage, we showed that the fitness cost conferred by the L19 mutations could be compensated by additional mutations in the ribosomal protein L19 itself, in S12 and in L14, a protein located close to L19. Our results reveal a novel functional role for the 50 S ribosomal protein L19 during protein synthesis, supporting published structural data suggesting that the interaction of L14 and L19 with 16 S rRNA could influence function of the 30 S subunit. Moreover, our study demonstrates how compensatory fitness-evolution can be used to discover new molecular functions of ribosomal proteins.

  20. Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins.

    PubMed

    Markov, Alexander G; Falchuk, Evgeny L; Kruglova, Natalia M; Rybalchenko, Oksana V; Fromm, Michael; Amasheh, Salah

    2014-11-01

    Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (ISC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of ISC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of ISC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.

  1. Entrapment of DNA in an intersubunit tunnel system of a single-stranded DNA-binding protein

    PubMed Central

    Ghalei, Homa; von Moeller, Holger; Eppers, Detlef; Sohmen, Daniel; Wilson, Daniel N.; Loll, Bernhard; Wahl, Markus C.

    2014-01-01

    Instead of a classical single-stranded deoxyribonuleic acid (DNA)-binding protein (SSB), some hyperthermophilic crenarchaea harbor a non-canonical SSB termed ThermoDBP. Two related but poorly characterized groups of proteins, which share the ThermoDBP N-terminal DNA-binding domain, have a broader phylogenetic distribution and co-exist with ThermoDBPs and/or other SSBs. We have investigated the nucleic acid binding properties and crystal structures of representatives of these groups of ThermoDBP-related proteins (ThermoDBP-RPs) 1 and 2. ThermoDBP-RP 1 and 2 oligomerize by different mechanisms and only ThermoDBP-RP2 exhibits strong single-stranded DNA affinity in vitro. A crystal structure of ThermoDBP-RP2 in complex with DNA reveals how the NTD common to ThermoDBPs and ThermoDBP-RPs can contact the nucleic acid in a manner that allows a symmetric homotetrameric protein complex to bind single-stranded DNA molecules asymmetrically. While single-stranded DNA wraps around the surface or binds along channels of previously investigated SSBs, it traverses an internal, intersubunit tunnel system of a ThermoDBP-RP2 tetramer. Our results indicate that some archaea have acquired special SSBs for genome maintenance in particularly challenging environments. PMID:24744237

  2. Single-Cell Co-expression Analysis Reveals Distinct Functional Modules, Co-regulation Mechanisms and Clinical Outcomes

    PubMed Central

    Wang, Jie; Xia, Shuli; Arand, Brian; Zhu, Heng; Machiraju, Raghu; Huang, Kun; Ji, Hongkai; Qian, Jiang

    2016-01-01

    Co-expression analysis has been employed to predict gene function, identify functional modules, and determine tumor subtypes. Previous co-expression analysis was mainly conducted at bulk tissue level. It is unclear whether co-expression analysis at the single-cell level will provide novel insights into transcriptional regulation. Here we developed a computational approach to compare glioblastoma expression profiles at the single-cell level with those obtained from bulk tumors. We found that the co-expressed genes observed in single cells and bulk tumors have little overlap and show distinct characteristics. The co-expressed genes identified in bulk tumors tend to have similar biological functions, and are enriched for intrachromosomal interactions with synchronized promoter activity. In contrast, single-cell co-expressed genes are enriched for known protein-protein interactions, and are regulated through interchromosomal interactions. Moreover, gene members of some protein complexes are co-expressed only at the bulk level, while those of other complexes are co-expressed at both single-cell and bulk levels. Finally, we identified a set of co-expressed genes that can predict the survival of glioblastoma patients. Our study highlights that comparative analyses of single-cell and bulk gene expression profiles enable us to identify functional modules that are regulated at different levels and hold great translational potential. PMID:27100869

  3. Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins.

    PubMed

    Cappellini, Enrico; Jensen, Lars J; Szklarczyk, Damian; Ginolhac, Aurélien; da Fonseca, Rute A R; Stafford, Thomas W; Holen, Steven R; Collins, Matthew J; Orlando, Ludovic; Willerslev, Eske; Gilbert, M Thomas P; Olsen, Jesper V

    2012-02-01

    We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth ( Mammuthus primigenius ) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance extracellular matrix and plasma proteins, were confidently identified by solid molecular evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African ( Loxodonta africana ) and Indian ( Elephas maximus ) elephants. Strong evidence was observed of amino acid modifications due to post-mortem hydrolytic and oxidative damage. A consistent subset of this permafrost bone proteome was also identified in more recent Columbian mammoth ( Mammuthus columbi ) samples from temperate latitudes, extending the potential of the approach described beyond subpolar environments. Mass spectrometry-based ancient protein sequencing offers new perspectives for future molecular phylogenetic inference and physiological studies on samples not amenable to ancient DNA investigation. This approach therefore represents a further step into the ongoing integration of different high-throughput technologies for identification of ancient biomolecules, unleashing the field of paleoproteomics.

  4. Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins.

    PubMed

    Cappellini, Enrico; Jensen, Lars J; Szklarczyk, Damian; Ginolhac, Aurélien; da Fonseca, Rute A R; Stafford, Thomas W; Holen, Steven R; Collins, Matthew J; Orlando, Ludovic; Willerslev, Eske; Gilbert, M Thomas P; Olsen, Jesper V

    2012-02-01

    We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth ( Mammuthus primigenius ) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance extracellular matrix and plasma proteins, were confidently identified by solid molecular evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African ( Loxodonta africana ) and Indian ( Elephas maximus ) elephants. Strong evidence was observed of amino acid modifications due to post-mortem hydrolytic and oxidative damage. A consistent subset of this permafrost bone proteome was also identified in more recent Columbian mammoth ( Mammuthus columbi ) samples from temperate latitudes, extending the potential of the approach described beyond subpolar environments. Mass spectrometry-based ancient protein sequencing offers new perspectives for future molecular phylogenetic inference and physiological studies on samples not amenable to ancient DNA investigation. This approach therefore represents a further step into the ongoing integration of different high-throughput technologies for identification of ancient biomolecules, unleashing the field of paleoproteomics. PMID:22103443

  5. Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality.

    PubMed

    Wu, Nicholas C; Olson, C Anders; Du, Yushen; Le, Shuai; Tran, Kevin; Remenyi, Roland; Gong, Danyang; Al-Mawsawi, Laith Q; Qi, Hangfei; Wu, Ting-Ting; Sun, Ren

    2015-07-01

    Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available.

  6. Purification and characterization of a mitochondrial, single-stranded-DNA-binding protein from Paracentrotus lividus eggs.

    PubMed

    Roberti, M; Musicco, C; Loguercio Polosa, P; Gadaleta, M N; Quagliariello, E; Cantatore, P

    1997-07-01

    A binding protein for single-stranded DNA was purified from Paracentrotus lividus egg mitochondria to near homogeneity by chromatography on DEAE-Sephacel and single-stranded-DNA-cellulose. The protein consists of a single polypeptide of about 15 kDa. Glycerol gradient sedimentation analysis suggested that P. lividus mitochondrial single-stranded-DNA-binding protein exists as a homo-oligomer, possibly a tetramer, in solution. The protein shows a stronger preference for poly(dT) with respect to single-stranded M13, poly(dI) and poly(dC). Binding to poly(dA) takes place with much lower affinity. The binding-site size, determined by gel mobility-shift experiments with oligonucleotides of different length, is approximately 45 nucleotides. The binding to single-stranded DNA occurs with low or no cooperativity and is not influenced by ionic strength. The protein has a very high affinity for the DNA: its apparent macroscopic association constant is 2x10(9) M(-1), a value which is the highest among the mitochondrial single-stranded-DNA-binding proteins characterized to date. The lack of cooperativity and the high association constant represent distinctive features of this protein and might be related to the peculiar mechanism of sea urchin mitochondrial DNA replication.

  7. Comparative analysis reveals conserved protein phosphorylation networks implicated in multiple diseases.

    PubMed

    Tan, Chris Soon Heng; Bodenmiller, Bernd; Pasculescu, Adrian; Jovanovic, Marko; Hengartner, Michael O; Jørgensen, Claus; Bader, Gary D; Aebersold, Ruedi; Pawson, Tony; Linding, Rune

    2009-01-01

    Protein kinases enable cellular information processing. Although numerous human phosphorylation sites and their dynamics have been characterized, the evolutionary history and physiological importance of many signaling events remain unknown. Using target phosphoproteomes determined with a similar experimental and computational pipeline, we investigated the conservation of human phosphorylation events in distantly related model organisms (fly, worm, and yeast). With a sequence-alignment approach, we identified 479 phosphorylation events in 344 human proteins that appear to be positionally conserved over approximately 600 million years of evolution and hence are likely to be involved in fundamental cellular processes. This sequence-alignment analysis suggested that many phosphorylation sites evolve rapidly and therefore do not display strong evolutionary conservation in terms of sequence position in distantly related organisms. Thus, we devised a network-alignment approach to reconstruct conserved kinase-substrate networks, which identified 778 phosphorylation events in 698 human proteins. Both methods identified proteins tightly regulated by phosphorylation as well as signal integration hubs, and both types of phosphoproteins were enriched in proteins encoded by disease-associated genes. We analyzed the cellular functions and structural relationships for these conserved signaling events, noting the incomplete nature of current phosphoproteomes. Assessing phosphorylation conservation at both site and network levels proved useful for exploring both fast-evolving and ancient signaling events. We reveal that multiple complex diseases seem to converge within the conserved networks, suggesting that disease development might rely on common molecular networks.

  8. Tissue-Based Proteogenomics Reveals that Human Testis Endows Plentiful Missing Proteins.

    PubMed

    Zhang, Yao; Li, Qidan; Wu, Feilin; Zhou, Ruo; Qi, Yingzi; Su, Na; Chen, Lingsheng; Xu, Shaohang; Jiang, Tao; Zhang, Chengpu; Cheng, Gang; Chen, Xinguo; Kong, Degang; Wang, Yujia; Zhang, Tao; Zi, Jin; Wei, Wei; Gao, Yuan; Zhen, Bei; Xiong, Zhi; Wu, Songfeng; Yang, Pengyuan; Wang, Quanhui; Wen, Bo; He, Fuchu; Xu, Ping; Liu, Siqi

    2015-09-01

    Investigations of missing proteins (MPs) are being endorsed by many bioanalytical strategies. We proposed that proteogenomics of testis tissue was a feasible approach to identify more MPs because testis tissues have higher gene expression levels. Here we combined proteomics and transcriptomics to survey gene expression in human testis tissues from three post-mortem individuals. Proteins were extracted and separated with glycine- and tricine-SDS-PAGE. A total of 9597 protein groups were identified; of these, 166 protein groups were listed as MPs, including 138 groups (83.1%) with transcriptional evidence. A total of 2948 proteins are designated as MPs, and 5.6% of these were identified in this study. The high incidence of MPs in testis tissue indicates that this is a rich resource for MPs. Functional category analysis revealed that the biological processes that testis MPs are mainly involved in are sexual reproduction and spermatogenesis. Some of the MPs are potentially involved in tumorgenesis in other tissues. Therefore, this proteogenomics analysis of individual testis tissues provides convincing evidence of the discovery of MPs. All mass spectrometry data from this study have been deposited in the ProteomeXchange (data set identifier PXD002179).

  9. Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction

    PubMed Central

    Teramoto, Takamasa; Fujikawa, Yukari; Kawaguchi, Yoshirou; Kurogi, Katsuhisa; Soejima, Masayuki; Adachi, Rumi; Nakanishi, Yuichi; Mishiro-Sato, Emi; Liu, Ming-Cheh; Sakakibara, Yoichi; Suiko, Masahito; Kimura, Makoto; Kakuta, Yoshimitsu

    2013-01-01

    Post-translational protein modification by tyrosine-sulfation plays an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalyzed by the Golgi-enzyme called the tyrosylprotein sulfotransferase (TPST). To date, no crystal structure is available for TPST. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human TPST isoform 2 (TPST2) complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3’-phosphoadenosine-5’-phosphate (PAP) at 1.9Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. TPST2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel β-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the TPST2 structure resembles that observed for the receptor type tyrosine kinases. PMID:23481380

  10. Principal Component Analysis reveals correlation of cavities evolution and functional motions in proteins.

    PubMed

    Desdouits, Nathan; Nilges, Michael; Blondel, Arnaud

    2015-02-01

    Protein conformation has been recognized as the key feature determining biological function, as it determines the position of the essential groups specifically interacting with substrates. Hence, the shape of the cavities or grooves at the protein surface appears to drive those functions. However, only a few studies describe the geometrical evolution of protein cavities during molecular dynamics simulations (MD), usually with a crude representation. To unveil the dynamics of cavity geometry evolution, we developed an approach combining cavity detection and Principal Component Analysis (PCA). This approach was applied to four systems subjected to MD (lysozyme, sperm whale myoglobin, Dengue envelope protein and EF-CaM complex). PCA on cavities allows us to perform efficient analysis and classification of the geometry diversity explored by a cavity. Additionally, it reveals correlations between the evolutions of the cavities and structures, and can even suggest how to modify the protein conformation to induce a given cavity geometry. It also helps to perform fast and consensual clustering of conformations according to cavity geometry. Finally, using this approach, we show that both carbon monoxide (CO) location and transfer among the different xenon sites of myoglobin are correlated with few cavity evolution modes of high amplitude. This correlation illustrates the link between ligand diffusion and the dynamic network of internal cavities.

  11. Principal Component Analysis reveals correlation of cavities evolution and functional motions in proteins.

    PubMed

    Desdouits, Nathan; Nilges, Michael; Blondel, Arnaud

    2015-02-01

    Protein conformation has been recognized as the key feature determining biological function, as it determines the position of the essential groups specifically interacting with substrates. Hence, the shape of the cavities or grooves at the protein surface appears to drive those functions. However, only a few studies describe the geometrical evolution of protein cavities during molecular dynamics simulations (MD), usually with a crude representation. To unveil the dynamics of cavity geometry evolution, we developed an approach combining cavity detection and Principal Component Analysis (PCA). This approach was applied to four systems subjected to MD (lysozyme, sperm whale myoglobin, Dengue envelope protein and EF-CaM complex). PCA on cavities allows us to perform efficient analysis and classification of the geometry diversity explored by a cavity. Additionally, it reveals correlations between the evolutions of the cavities and structures, and can even suggest how to modify the protein conformation to induce a given cavity geometry. It also helps to perform fast and consensual clustering of conformations according to cavity geometry. Finally, using this approach, we show that both carbon monoxide (CO) location and transfer among the different xenon sites of myoglobin are correlated with few cavity evolution modes of high amplitude. This correlation illustrates the link between ligand diffusion and the dynamic network of internal cavities. PMID:25424655

  12. Dynamic Network-Based Relevance Score Reveals Essential Proteins and Functional Modules in Directed Differentiation

    PubMed Central

    Wu, Chia-Chou; Lin, Che

    2015-01-01

    The induction of stem cells toward a desired differentiation direction is required for the advancement of stem cell-based therapies. Despite successful demonstrations of the control of differentiation direction, the effective use of stem cell-based therapies suffers from a lack of systematic knowledge regarding the mechanisms underlying directed differentiation. Using dynamic modeling and the temporal microarray data of three differentiation stages, three dynamic protein-protein interaction networks were constructed. The interaction difference networks derived from the constructed networks systematically delineated the evolution of interaction variations and the underlying mechanisms. A proposed relevance score identified the essential components in the directed differentiation. Inspection of well-known proteins and functional modules in the directed differentiation showed the plausibility of the proposed relevance score, with the higher scores of several proteins and function modules indicating their essential roles in the directed differentiation. During the differentiation process, the proteins and functional modules with higher relevance scores also became more specific to the neuronal identity. Ultimately, the essential components revealed by the relevance scores may play a role in controlling the direction of differentiation. In addition, these components may serve as a starting point for understanding the systematic mechanisms of directed differentiation and for increasing the efficiency of stem cell-based therapies. PMID:25977693

  13. Effects of pressure on the dynamics of a hyperthermophilic protein revealed by quasielastic neutron scattering

    NASA Astrophysics Data System (ADS)

    Shrestha, U. R.; Bhowmik, D.; Copley, J. R. D.; Tyagi, M.; Leao, J. B.; Chu, X.-Q.

    Inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens is a large oligomeric protein derived from hyperthermophilic microorganism that is found near hydrothermal vents deep under the sea, where the pressure is nearly 100 MPa. Here we study the effects of pressure on the conformational flexibility and relaxation dynamics of IPPase over a wide temperature range using quasielastic neutron scattering (QENS) technique. Two spectrometers were used to investigate the β-relaxation dynamics of proteins in time ranges from 2 to 25 ps, and from 100 ps to 2 ns. Our results reveal that, under the pressure of 100 MPa, IPPase displays much faster relaxation dynamics than a mesophilic model protein, hen egg white lysozyme (HEWL), opposite to what we observed previously under the ambient pressure. These contradictory observations imply that high pressure affects the dynamical properties of proteins by distorting their energy landscapes. Accordingly, we derived a general schematic denaturation phase diagram that can be used as a general picture to understand the effects of pressure on protein dynamics and activities Wayne State Univ Startup Fund.

  14. Novel proteomic tools reveal essential roles of SRP and importance of proper membrane protein biogenesis.

    PubMed

    Zhang, Dawei; Sweredoski, Michael J; Graham, Robert L J; Hess, Sonja; Shan, Shu-ou

    2012-02-01

    The signal recognition particle (SRP), which mediates cotranslational protein targeting to cellular membranes, is universally conserved and essential for bacterial and mammalian cells. However, the current understanding of the role of SRP in cell physiology and pathology is still poor, and the reasons behind its essential role in cell survival remain unclear. Here, we systematically analyzed the consequences of SRP loss in E. coli using time-resolved quantitative proteomic analyses. A series of snapshots of the steady-state and newly synthesized proteome unveiled three stages of cellular responses to SRP depletion, and demonstrated essential roles of SRP in metabolism, membrane potential, and protein and energy homeostasis in both the membrane and cytoplasm. We also identified a group of periplasmic proteins, including key molecular chaperones, whose localization was impaired by the loss of SRP; this and additional results showed that SRP is crucial for protein homeostasis in the bacterial envelope. These results reveal the extensive roles that SRP plays in bacterial physiology, emphasize the importance of proper membrane protein biogenesis, and demonstrate the ability of time-resolved quantitative proteomic analysis to provide new biological insights.

  15. Tryptophan fluorescence reveals induced folding of Vibrio harveyi acyl carrier protein upon interaction with partner enzymes.

    PubMed

    Gong, Huansheng; Murphy, Peter W; Langille, Gavin M; Minielly, Sarah J; Murphy, Anne; McMaster, Christopher R; Byers, David M

    2008-11-01

    We have introduced tryptophan as a local fluorescent probe to monitor the conformation of Vibrio harveyi acyl carrier protein (ACP), a small flexible protein that is unfolded at neutral pH but must undergo reversible conformational change during the synthesis and delivery of bacterial fatty acids. Consistent with known 3D structures of ACP, steady-state fluorescence and quenching experiments indicated that Trp at positions 46, 50, and 72 are buried in the hydrophobic core upon Mg(2+)-induced ACP folding, whereas residues 25 and 45 remain in a hydrophilic environment on the protein surface. Attachment of fatty acids to the phosphopantetheine prosthetic group progressively stabilized the folded conformation of all Trp-substituted ACPs, but longer chains (14:0) were less effective than medium chains (8:0) in shielding Trp from acrylamide quenching in the L46W protein. Interaction with ACP-dependent enzymes LpxA and holo-ACP synthase also caused folding of L46W; fluorescence quenching indicated proximity of Trp-45 in helix II of ACP in LpxA binding. Our results suggest that divalent cations and fatty acylation produce differing environments in the ACP core and also reveal enzyme partner-induced folding of ACP, a key feature of "natively unfolded" proteins.

  16. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

    PubMed

    Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

    2016-06-01

    Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria. PMID:27183143

  17. Optical recording of signal-mediated protein transport through single nuclear pore complexes.

    PubMed

    Keminer, O; Siebrasse, J P; Zerf, K; Peters, R

    1999-10-12

    Optical single-transporter recording, a recently established fluorescence microscopic method, was used to study the selective transport of proteins through single nuclear pore complexes (NPCs) of Xenopus oocytes. Recombinant proteins containing either a nuclear localization signal (import protein) or a nuclear export signal (export protein) were generated as transport substrates. To approximate in vivo conditions as closely as possible, a Xenopus egg extract was applied to the cytosolic side and a Xenopus oocyte nuclear extract to the nuclear side of the NPCs. It was found that protein transport through functionally isolated, "patched" NPCs depended on signal sequences, extracts, and metabolic energy, as in vivo. All NPCs were competent for both import and export. The transport direction was strictly determined by the transport signal, and at none of the conditions explored was the import protein exported or the export protein imported, even when the application sides of the extracts were reversed. The mean transport rates of the single NPC were approximately 2 dimers/s for the import protein and approximately 4 dimers/s for the export protein ( approximately 15 microM substrate concentration, 22-24 degrees C), in good agreement with in vivo rates estimated for mammalian cells by microinjection experiments. The study shows that optical single-transporter recording permits the analysis of membrane transport processes not previously accessible to single-transporter recording and thus provides additional possibilities for the elucidation of nucleocytoplasmic transport mechanisms. PMID:10518538

  18. Optical recording of signal-mediated protein transport through single nuclear pore complexes

    PubMed Central

    Keminer, Oliver; Siebrasse, Jan-Peter; Zerf, Katja; Peters, Reiner

    1999-01-01

    Optical single-transporter recording, a recently established fluorescence microscopic method, was used to study the selective transport of proteins through single nuclear pore complexes (NPCs) of Xenopus oocytes. Recombinant proteins containing either a nuclear localization signal (import protein) or a nuclear export signal (export protein) were generated as transport substrates. To approximate in vivo conditions as closely as possible, a Xenopus egg extract was applied to the cytosolic side and a Xenopus oocyte nuclear extract to the nuclear side of the NPCs. It was found that protein transport through functionally isolated, “patched” NPCs depended on signal sequences, extracts, and metabolic energy, as in vivo. All NPCs were competent for both import and export. The transport direction was strictly determined by the transport signal, and at none of the conditions explored was the import protein exported or the export protein imported, even when the application sides of the extracts were reversed. The mean transport rates of the single NPC were ≈2 dimers/s for the import protein and ≈4 dimers/s for the export protein (≈15 μM substrate concentration, 22–24°C), in good agreement with in vivo rates estimated for mammalian cells by microinjection experiments. The study shows that optical single-transporter recording permits the analysis of membrane transport processes not previously accessible to single-transporter recording and thus provides additional possibilities for the elucidation of nucleocytoplasmic transport mechanisms. PMID:10518538

  19. Photoconversion of organic materials into single-cell protein

    DOEpatents

    Weaver, Paul F.

    2001-01-01

    A process is described for converting organic materials (such as biomass wastes) into sterile, high-grade bacterial protein suitable for use an animal feed or human food supplements. In a preferred embodiment the process involves thermally gasifying the organic material into primarily carbon monoxide, hydrogen and nitrogen products, followed by photosynthetic bacterial assimilation of the gases into cell material, which can be as high as 65% protein. The process is ideally suited for waste recycling and for food production under zero-gravity or extra-terrestrial conditions.

  20. Photoconversion of organic materials into single-cell protein

    SciTech Connect

    Weaver, P.F.

    1991-12-31

    A process is described for converting organic materials (such as biomass wastes) into sterile, high-grade bacterial protein suitable for use an animal feed or human food supplements. In a preferred embodiment the process involves thermally gasifying the organic material into primarily carbon monoxide, hydrogen and nitrogen products, followed by photosynthetic bacterial assimilation of the gases into cell material, which can be high as 65% protein. The process is ideally suited for waste recycling and for food production under zero-gravity or extra-terrestrial conditions.

  1. Single metallic nanoparticle imaging for protein detection in cells.

    PubMed

    Cognet, L; Tardin, C; Boyer, D; Choquet, D; Tamarat, P; Lounis, B

    2003-09-30

    We performed a visualization of membrane proteins labeled with 10-nm gold nanoparticles in cells, using an all-optical method based on photothermal interference contrast. The high sensitivity of the method and the stability of the signals allows 3D imaging of individual nanoparticles without the drawbacks of photobleaching and blinking inherent to fluorescent markers. A simple analytical model is derived to account for the measurements of the signal amplitude and the spatial resolution. The photothermal interference contrast method provides an efficient, reproducible, and promising way to visualize low amounts of proteins in cells by optical means. PMID:13679586

  2. Modulating non-native aggregation and electrostatic protein-protein interactions with computationally designed single-point mutations.

    PubMed

    O'Brien, C J; Blanco, M A; Costanzo, J A; Enterline, M; Fernandez, E J; Robinson, A S; Roberts, C J

    2016-06-01

    Non-native protein aggregation is a ubiquitous challenge in the production, storage and administration of protein-based biotherapeutics. This study focuses on altering electrostatic protein-protein interactions as a strategy to modulate aggregation propensity in terms of temperature-dependent aggregation rates, using single-charge variants of human γ-D crystallin. Molecular models were combined to predict amino acid substitutions that would modulate protein-protein interactions with minimal effects on conformational stability. Experimental protein-protein interactions were quantified by the Kirkwood-Buff integrals (G22) from laser scattering, and G22 showed semi-quantitative agreement with model predictions. Experimental initial-rates for aggregation showed that increased (decreased) repulsive interactions led to significantly increased (decreased) aggregation resistance, even based solely on single-point mutations. However, in the case of a particular amino acid (E17), the aggregation mechanism was altered by substitution with R or K, and this greatly mitigated improvements in aggregation resistance. The results illustrate that predictions based on native protein-protein interactions can provide a useful design target for engineering aggregation resistance; however, this approach needs to be balanced with consideration of how mutations can impact aggregation mechanisms. PMID:27160179

  3. Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus.

    PubMed

    Huang, Mingwei; Hebert, Alexander S; Coon, Joshua J; Hull, Christina M

    2015-08-01

    Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.

  4. Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus

    PubMed Central

    Huang, Mingwei; Hebert, Alexander S.; Coon, Joshua J.; Hull, Christina M.

    2015-01-01

    Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote. PMID:26313153

  5. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    PubMed

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  6. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters

    PubMed Central

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  7. New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

    PubMed

    Moroz, Yurii S; Dunston, Tiffany T; Makhlynets, Olga V; Moroz, Olesia V; Wu, Yibing; Yoon, Jennifer H; Olsen, Alissa B; McLaughlin, Jaclyn M; Mack, Korrie L; Gosavi, Pallavi M; van Nuland, Nico A J; Korendovych, Ivan V

    2015-12-01

    Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

  8. New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

    PubMed

    Moroz, Yurii S; Dunston, Tiffany T; Makhlynets, Olga V; Moroz, Olesia V; Wu, Yibing; Yoon, Jennifer H; Olsen, Alissa B; McLaughlin, Jaclyn M; Mack, Korrie L; Gosavi, Pallavi M; van Nuland, Nico A J; Korendovych, Ivan V

    2015-12-01

    Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution. PMID:26555770

  9. Single neuron transcriptome analysis can reveal more than cell type classification

    PubMed Central

    Harbom, Lise J.; Chronister, William D.

    2016-01-01

    A recent single cell mRNA sequencing study by Dueck et al. compares neuronal transcriptomes to the transcriptomes of adipocytes and cardiomyocytes. Single cell ‘omic approaches such as those used by the authors are at the leading edge of molecular and biophysical measurement. Many groups are currently employing single cell sequencing approaches to understand cellular heterogeneity in cancer and during normal development. These single cell approaches also are beginning to address long‐standing questions regarding nervous system diversity. Beyond an innate interest in cataloging cell type diversity in the brain, single cell neuronal diversity has important implications for neurotypic neural circuit function and for neurological disease. Herein, we review the authors’ methods and findings, which most notably include evidence of unique expression profiles in some single neurons. PMID:26749010

  10. Revealing the binding mode between respiratory syncytial virus fusion protein and benzimidazole-based inhibitors.

    PubMed

    Ji, Dingjue; Ye, Wei; Chen, HaiFeng

    2015-07-01

    Human respiratory syncytial virus (HRSV) is a major respiratory pathogen in newborn infants and young children and can also be a threat to some elderly and high-risk adults with chronic pulmonary disease and the severely immunocompromised. The RSV fusion (RSVF) protein has been an attractive target for vaccine and drug development. Experimental results indicate a series of benzimidazole-based inhibitors which target RSVF protein to inhibit the viral entry of RSV. To reveal the binding mode between these inhibitors and RSVF protein, molecular docking and molecular dynamics simulations were used to investigate the interactions between the inhibitors and the core domain of RSVF protein. MD results suggest that the active molecules have stronger π-π stacking, cation-π, and other interactions than less active inhibitors. The binding free energy between the active inhibitor and RSVF protein is also found to be significantly lower than that of the less active one using MM/GBSA. Then, Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) methods were used to construct three dimensional quantitative structure-activity (3D-QSAR) models. The cross-validated q(2) values are found to be 0.821 and 0.795 for CoMFA and CoMSIA, respectively. And the non-cross-validated r(2) values are 0.973 and 0.961. Ninety-two test set compounds validated these models. The results suggest that these models are robust with good prediction abilities. Furthermore, these models reveal possible methods to improve the bioactivity of inhibitors. PMID:25872614

  11. Human replication protein A binds single-stranded DNA in two distinct complexes.

    PubMed Central

    Blackwell, L J; Borowiec, J A

    1994-01-01

    Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes. Images PMID:8196638

  12. A single mutation at residue 25 populates the folding intermediate of E. coli RNase H and reveals a highly dynamic partially-folded ensemble

    PubMed Central

    Connell, Katelyn B.; Horner, Geoffrey A.; Marqusee, Susan

    2010-01-01

    Understanding the nature of partially folded intermediates transiently populated during protein folding is important for understanding both protein folding and misfolding. These ephemeral species, however, often elude direct experimental characterization. The well-characterized protein ribonuclease H from E. coli populates an on-pathway intermediate identified in both bulk studies and single molecule mechanical unfolding experiments. Here, we set out to trap the transient intermediate of RNase H at equilibrium by selectively destabilizing the region of the protein known to be unfolded in this species. Surprisingly, a single change at Ile 25 (I25A) resulted in the equilibrium population of the intermediate under near-native conditions. The intermediate was undetectable in a series of HSQC's, revealing the dynamic nature of this partially unfolded form on the timescale of NMR detection. This result is in contrast to studies in which the structures of trapped intermediates are solved by NMR, indicating that the they are well-packed and native-like. The dynamic nature of the RNase H intermediate may be important for its role as an on-pathway, productive species that promotes efficient folding. PMID:19505477

  13. The Volumetric Diversity of Misfolded Prion Protein Oligomers Revealed by Pressure Dissociation*

    PubMed Central

    Torrent, Joan; Lange, Reinhard; Rezaei, Human

    2015-01-01

    Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct β-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation of one specific PrP oligomer, we reveal a large negative activation volume and an increase in both apparent activation enthalpy and entropy. This suggests a transition state ensemble that is less structured and significantly more hydrated than the oligomeric state. Finally, we found that site-specific fluorescent labeling allows monitoring of the transient population of a kinetic intermediate in the dissociation reaction. Our results indicate that defects in atomic packing may deserve consideration as a new factor that influences differences between PrP assemblies and that could be relevant also for explaining the origin of prion strains. PMID:26126829

  14. Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane

    PubMed Central

    Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

    2016-01-01

    Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers. DOI: http://dx.doi.org/10.7554/eLife.12125.001 PMID:26824389

  15. Single-Cell (Meta-)Genomics of a Dimorphic Candidatus Thiomargarita nelsonii Reveals Genomic Plasticity

    PubMed Central

    Flood, Beverly E.; Fliss, Palmer; Jones, Daniel S.; Dick, Gregory J.; Jain, Sunit; Kaster, Anne-Kristin; Winkel, Matthias; Mußmann, Marc; Bailey, Jake

    2016-01-01

    The genus Thiomargarita includes the world's largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group

  16. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions.

    PubMed

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N; Flood, Beverly E; Bailey, Jake V; Mußmann, Marc

    2016-01-01

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na(+)-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of "Ca. T. nelsonii Thio36" provides additional insight into the ecology of giant sulfur

  17. Single-Cell (Meta-)Genomics of a Dimorphic Candidatus Thiomargarita nelsonii Reveals Genomic Plasticity.

    PubMed

    Flood, Beverly E; Fliss, Palmer; Jones, Daniel S; Dick, Gregory J; Jain, Sunit; Kaster, Anne-Kristin; Winkel, Matthias; Mußmann, Marc; Bailey, Jake

    2016-01-01

    The genus Thiomargarita includes the world's largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group

  18. Single-Cell (Meta-)Genomics of a Dimorphic Candidatus Thiomargarita nelsonii Reveals Genomic Plasticity.

    PubMed

    Flood, Beverly E; Fliss, Palmer; Jones, Daniel S; Dick, Gregory J; Jain, Sunit; Kaster, Anne-Kristin; Winkel, Matthias; Mußmann, Marc; Bailey, Jake

    2016-01-01

    The genus Thiomargarita includes the world's largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group

  19. Single-cell sequencing of Thiomargarita reveals genomic flexibility for adaptation to dynamic redox conditions

    DOE PAGES

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N.; Flood, Beverly E.; Bailey, Jake V.; Mußmann, Marc

    2016-06-21

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiornargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of amore » chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na+-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In conclusion, the genome of "Ca. T. nelsonii Thio36" provides additional insight into the ecology of giant sulfur

  20. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions

    PubMed Central

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N.; Flood, Beverly E.; Bailey, Jake V.; Mußmann, Marc

    2016-01-01

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming “Candidatus Thiomargarita nelsonii Thio36”, and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. “Ca. T. nelsonii Thio36” is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that “Ca. T. nelsonii Thio36” can function as a chemolithoautotroph. Carbon can be fixed via the Calvin–Benson–Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, “Ca. T. nelsonii Thio36” also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na+-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae “Ca. T. nelsonii Thio36” encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of “Ca. T. nelsonii Thio36” provides additional insight into the ecology of

  1. The structure of the BfrB-Bfd complex reveals protein-protein interactions enabling iron release from bacterioferritin

    PubMed Central

    Yao, Huili; Wang, Yan; Lovell, Scott; Kumar, Ritesh; Ruvinsky, Anatoly M.; Battaile, Kevin P.; Vakser, Ilya A.; Rivera, Mario

    2012-01-01

    Ferritin-like molecules are unique to cellular iron homeostasis because they can store iron at concentrations much higher than those dictated by the solubility of Fe3+. Very little is known about the protein interactions that deliver iron for storage, or promote the mobilization of stored iron from ferritin-like molecules. Here, we report the X-ray crystal structure of Pseudomonas aeruginosa bacterioferritin (Pa-BfrB) in complex with bacterioferritin-associated ferredoxin (Pa-Bfd) at 2.0 Å resolution. As the first example of a ferritin-like molecule in complex with a cognate partner, the structure provides unprecedented insight into the complementary interface that enables the [2Fe-2S] cluster of Pa-Bfd to promote heme-mediated electron transfer through the BfrB protein dielectric (~18 Å), a process that is necessary to reduce the core ferric mineral and facilitate mobilization of Fe2+. The Pa-BfrB-Bfd complex also revealed the first structure of a Bfd, thus providing a first view to what appears to be a versatile metal binding domain ubiquitous to the large Fer2_BFD family of proteins and enzymes with diverse functions. Residues at the Pa-BfrB-Bfd interface are highly conserved in Bfr and Bfd sequences from a number of pathogenic bacteria, suggesting that the specific recognition between Pa-BfrB and Pa-Bfd is of widespread significance to the understanding of bacterial iron homeostasis. PMID:22812654

  2. The Structure of the BfrB-Bfd Complex Reveals Protein-Protein Interactions Enabling Iron Release from Bacterioferritin

    SciTech Connect

    Yao, Huili; Wang, Yan; Lovell, Scott; Kumar, Ritesh; Ruvinsky, Anatoly M; Battaile, Kevin P; Vakser, Ilya A; Rivera, Mario

    2012-09-11

    Ferritin-like molecules are unique to cellular iron homeostasis because they can store iron at concentrations much higher than those dictated by the solubility of Fe3+. Very little is known about the protein interactions that deliver iron for storage or promote the mobilization of stored iron from ferritin-like molecules. Here, we report the X-ray crystal structure of Pseudomonas aeruginosa bacterioferritin (Pa-BfrB) in complex with bacterioferritin-associated ferredoxin (Pa-Bfd) at 2.0 Å resolution. As the first example of a ferritin-like molecule in complex with a cognate partner, the structure provides unprecedented insight into the complementary interface that enables the [2Fe-2S] cluster of Pa-Bfd to promote heme-mediated electron transfer through the BfrB protein dielectric (~18 Å), a process that is necessary to reduce the core ferric mineral and facilitate mobilization of Fe2+. The Pa-BfrB-Bfd complex also revealed the first structure of a Bfd, thus providing a first view to what appears to be a versatile metal binding domain ubiquitous to the large Fer2_BFD family of proteins and enzymes with diverse functions. Residues at the Pa-BfrB-Bfd interface are highly conserved in Bfr and Bfd sequences from a number of pathogenic bacteria, suggesting that the specific recognition between Pa-BfrB and Pa-Bfd is of widespread significance to the understanding of bacterial iron homeostasis.

  3. The RecOR proteins modulate RecA protein function at 5' ends of single-stranded DNA.

    PubMed

    Bork, J M; Cox, M M; Inman, R B

    2001-12-17

    The Escherichia coli RecF, RecO and RecR pro teins have previously been implicated in bacterial recombinational DNA repair at DNA gaps. The RecOR-facilitated binding of RecA protein to single-stranded DNA (ssDNA) that is bound by single-stranded DNA-binding protein (SSB) is much faster if the ssDNA is linear, suggesting that a DNA end (rather than a gap) facilitates binding. In addition, the RecOR complex facilitates RecA protein-mediated D-loop formation at the 5' ends of linear ssDNAs. RecR protein remains associated with the RecA filament and its continued presence is required to prevent filament disassembly. RecF protein competes with RecO protein for RecR protein association and its addition destabilizes RecAOR filaments. An enhanced function of the RecO and RecR proteins can thus be seen in vitro at the 5' ends of linear ssDNA that is not as evident in DNA gaps. This function is countered by the RecF/RecO competition for association with the RecR protein. PMID:11743007

  4. Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.

    PubMed

    Schlyer, B D; Schauerte, J A; Steel, D G; Gafni, A

    1994-09-01

    The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that

  5. Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.

    PubMed Central

    Schlyer, B D; Schauerte, J A; Steel, D G; Gafni, A

    1994-01-01

    The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that

  6. The Lipid-Droplet Proteome Reveals that Droplets Are a Protein-Storage Depot

    SciTech Connect

    Cermelli, Silvia; Guo, Yi; Gross, Steven P.; Welte, Michael

    2006-09-19

    Lipid droplets are ubiquitous organelles that are among the basic building blocks of eukaryotic cells. Despite central roles for cholesterol homeostasis and lipid metabolism, their function and protein composition are poorly understood. Results: We purified lipid droplets from Drosophila embryos and analyzed the associated proteins by capillary LC-MS-MS. Important functional groups include enzymes involved in lipid metabolism, signaling molecules, and proteins related to membrane trafficking. Unexpectedly, histones H2A, H2Av, and H2B were present. Using biochemistry, genetics, real-time imaging, and cell biology, we confirm that roughly 50% of certain embryonic histones are physically attached to lipid droplets, a localization conserved in other fly species. Histone association with droplets starts during oogenesis and is prominent in early embryos, but it is undetectable in later stages or in cultured cells. Histones on droplets are not irreversibly trapped; quantitation of droplet histone levels and transplantation experiments suggest that histones are transferred from droplets to nuclei as development proceeds. When this maternal store of histones is unavailable because lipid droplets are mislocalized, zygotic histone production starts prematurely. Conclusions: Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.

  7. Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

    PubMed

    Borges, Chad R; Rehder, Douglas S

    2016-09-15

    Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo.

  8. Evolutionarily Conserved Pattern of Interactions in a Protein Revealed by Local Thermal Expansion Properties.

    PubMed

    Dellarole, Mariano; Caro, Jose A; Roche, Julien; Fossat, Martin; Barthe, Philippe; García-Moreno E, Bertrand; Royer, Catherine A; Roumestand, Christian

    2015-07-29

    The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function.

  9. Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

    PubMed

    Borges, Chad R; Rehder, Douglas S

    2016-09-15

    Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. PMID:27503803

  10. Nullspace Sampling with Holonomic Constraints Reveals Molecular Mechanisms of Protein Gαs

    PubMed Central

    Pachov, Dimitar V.; van den Bedem, Henry

    2015-01-01

    Proteins perform their function or interact with partners by exchanging between conformational substates on a wide range of spatiotemporal scales. Structurally characterizing these exchanges is challenging, both experimentally and computationally. Large, diffusional motions are often on timescales that are difficult to access with molecular dynamics simulations, especially for large proteins and their complexes. The low frequency modes of normal mode analysis (NMA) report on molecular fluctuations associated with biological activity. However, NMA is limited to a second order expansion about a minimum of the potential energy function, which limits opportunities to observe diffusional motions. By contrast, kino-geometric conformational sampling (KGS) permits large perturbations while maintaining the exact geometry of explicit conformational constraints, such as hydrogen bonds. Here, we extend KGS and show that a conformational ensemble of the α subunit Gαs of heterotrimeric stimulatory protein Gs exhibits structural features implicated in its activation pathway. Activation of protein Gs by G protein-coupled receptors (GPCRs) is associated with GDP release and large conformational changes of its α-helical domain. Our method reveals a coupled α-helical domain opening motion while, simultaneously, Gαs helix α5 samples an activated conformation. These motions are moderated in the activated state. The motion centers on a dynamic hub near the nucleotide-binding site of Gαs, and radiates to helix α4. We find that comparative NMA-based ensembles underestimate the amplitudes of the motion. Additionally, the ensembles fall short in predicting the accepted direction of the full activation pathway. Taken together, our findings suggest that nullspace sampling with explicit, holonomic constraints yields ensembles that illuminate molecular mechanisms involved in GDP release and protein Gs activation, and further establish conformational coupling between key structural

  11. Logic implementations using a single nanoparticle-protein hybrid

    NASA Astrophysics Data System (ADS)

    Medalsy, Izhar; Klein, Michael; Heyman, Arnon; Shoseyov, Oded; Remacle, F.; Levine, R. D.; Porath, Danny

    2010-06-01

    A Set-Reset machine is the simplest logic circuit with a built-in memory. Its output is a (nonlinear) function of the input and of the state stored in the machine's memory. Here, we report a nanoscale Set-Reset machine operating at room temperature that is based on a 5-nm silicon nanoparticle attached to the inner pore of a stable circular protein. The nanoparticle-protein hybrid can also function as a balanced ternary multiplier. Conductive atomic force microscopy is used to implement the logic input and output operations, and the processing of the logic Set and Reset operations relies on the finite capacitance of the nanoparticle provided by the good electrical isolation given by the protein, thus enabling stability of the logic device states. We show that the machine can be cycled, such that in every successive cycle, the previous state in the memory is retained as the present state. The energy cost of one cycle of computation is minimized to the cost of charging this state.

  12. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

    PubMed Central

    Colson, Brett A.; Thompson, Andrew R.; Espinoza-Fonseca, L. Michel; Thomas, David D.

    2016-01-01

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron–electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0–C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein’s flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein–protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  13. Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse

    PubMed Central

    Zampieri, Alexandre; Champagne, Julien; Auzemery, Baptiste; Fuentes, Ivanna; Maurel, Benjamin; Bienvenu, Frédéric

    2015-01-01

    We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed “Tandem-HTRF”, can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material. PMID:26503526

  14. The crystal structure of choline kinase reveals a eukaryotic protein kinase fold

    SciTech Connect

    Peisach, D.; Gee, P.; Kent, K.; Xu, Z.

    2010-03-08

    Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 {angstrom} crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.

  15. Structure of the Yeast DEAD Box Protein Mss116p Reveals Two Wedges that Crimp RNA

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2010-01-12

    The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF{sub 3}, or ADP-AlF{sub 4}{sup -}. The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge {alpha} helix bending RNA 3' of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5' of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which a protein uses two wedges to act as a molecular crimper.

  16. Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications.

    PubMed

    Halim, Adnan; Carlsson, Michael C; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L; Wandall, Hans H

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.

  17. Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications*

    PubMed Central

    Halim, Adnan; Carlsson, Michael C.; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y.; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L.; Wandall, Hans H.

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM1 characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines. PMID:25389185

  18. Proteomic Investigation of Aphid Honeydew Reveals an Unexpected Diversity of Proteins

    PubMed Central

    Haubruge, Eric; Hance, Thierry; Thonart, Philippe; De Pauw, Edwin; Francis, Frédéric

    2013-01-01

    Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige) approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora). Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu), and flagellin that might act as mediators in the plant-aphid interaction. In addition to providing the first aphid honeydew proteome analysis, we propose to reconsider the importance of this substance, mainly acknowledged to be a waste product, from the aphid ecology perspective. PMID:24086359

  19. The evolutionary analysis reveals domain fusion of proteins with Frizzled-like CRD domain.

    PubMed

    Yan, Jun; Jia, Haibo; Ma, Zhaowu; Ye, Huashan; Zhou, Mi; Su, Li; Liu, Jianfeng; Guo, An-Yuan

    2014-01-01

    Frizzleds (FZDs) are transmembrane receptors in the Wnt signaling pathway and they play pivotal roles in developments. The Frizzled-like extracellular Cysteine-rich domain (Fz-CRD) has been identified in FZDs and other proteins. The origin and evolution of these proteins with Fz-CRD is the main interest of this study. We found that the Fz-CRD exists in FZD, SFRP, RTK, MFRP, CPZ, CORIN, COL18A1 and other proteins. Our systematic analysis revealed that the Fz-CRD domain might have originated in protists and then fused with the Frizzled-like seven-transmembrane domain (7TM) to form the FZD receptors, which duplicated and diversified into about 11 members in Vertebrates. The SFRPs and RTKs with the Fz-CRD were found in sponge and expanded in Vertebrates. Other proteins with Fz-CRD may have emerged during Vertebrate evolution through domain fusion. Moreover, we found a glycosylation site and several conserved motifs in FZDs, which may be related to Wnt interaction. Based on these results, we proposed a model showing that the domain fusion and expansion of Fz-CRD genes occurred in Metazoa and Vertebrates. Our study may help to pave the way for further research on the conservation and diversification of Wnt signaling functions during evolution.

  20. Proteomic analysis of mice fed methionine and choline deficient diet reveals marker proteins associated with steatohepatitis.

    PubMed

    Lee, Su Jin; Kang, Jeong Han; Iqbal, Waqas; Kwon, Oh-Shin

    2015-01-01

    The mechanisms underlying the progression of simple steatosis to steatohepatitis are yet to be elucidated. To identify the proteins involved in the development of liver tissue inflammation, we performed comparative proteomic analysis of non-alcoholic steatohepatitis (NASH). Mice fed a methionine and choline deficient diet (MCD) developed hepatic steatosis characterized by increased free fatty acid (FFA) and triglyceride levels as well as alpha-SMA. Two-dimensional proteomic analysis revealed that the change from the normal diet to the MCD diet affected the expressions of 50 proteins. The most-pronounced changes were observed in the expression of proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in NASH model animals. Peroxiredoxin (Prx) is the most interesting among the modulated proteins identified in this study. In particular, cross-regulated Prx1 and Prx6 are likely to participate in cellular defense against the development of hepatitis. Thus, these Prx isoforms may be a useful new marker for early stage steatohepatitis. Moreover, curcumin treatment results in alleviation of the severity of hepatic inflammation in steatohepatitis. Notably, curcumin administration in MCD-fed mice dramatically reduced CYP2E1 as well as Prx1 expression, while upregulating Prx6 expression. These findings suggest that curcumin may have a protective role against MCD fed-induced oxidative stress.

  1. Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

    PubMed

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

    2014-11-27

    In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

  2. In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes[OPEN

    PubMed Central

    Husbands, Aman Y.; Aggarwal, Vasudha; Ha, Taekjip; Timmermans, Marja C.P.

    2016-01-01

    Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies. PMID:27385814

  3. In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes.

    PubMed

    Husbands, Aman Y; Aggarwal, Vasudha; Ha, Taekjip; Timmermans, Marja C P

    2016-08-01

    Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies.

  4. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.

    PubMed

    Gao, Xing-Huang; Krokowski, Dawid; Guan, Bo-Jhih; Bederman, Ilya; Majumder, Mithu; Parisien, Marc; Diatchenko, Luda; Kabil, Omer; Willard, Belinda; Banerjee, Ruma; Wang, Benlian; Bebek, Gurkan; Evans, Charles R; Fox, Paul L; Gerson, Stanton L; Hoppel, Charles L; Liu, Ming; Arvan, Peter; Hatzoglou, Maria

    2015-01-01

    The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. PMID:26595448

  5. Ultrasensitive detection of proteins and sugars at single-cell level

    PubMed Central

    Watabe, Satoshi; Morikawa, Mika; Kaneda, Mugiho; Nakaishi, Kazunari; Nakatsuma, Akira; Ninomiya, Masaki; Yoshimura, Teruki; Miura, Toshiaki; Ito, Etsuro

    2016-01-01

    ABSTRACT Each cell produces its own responses even if it appears identical to other cells. To analyze these individual cell characteristics, we need to measure trace amounts of molecules in a single cell. Nucleic acids in a single cell can be easily amplified by polymerase chain reaction, but single-cell measurement of proteins and sugars will require de novo techniques. In the present study, we outline the techniques we have developed toward this end. For proteins, our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide cycling can detect proteins at subattomoles per assay. For sugars, fluorescence correlation spectroscopy coupled with glucose oxidase-catalyzed reaction allows us to measure glucose at tens of nM. Our methods thus offer versatile techniques for single-cell-level analyses, and they are hoped to strongly promote single-cell biology as well as to develop noninvasive tests in clinical medicine. PMID:27064305

  6. The Structure of DdrB from Deinococcus: a New Fold for Single-stranded DNA Binding Proteins

    SciTech Connect

    Sugiman-Marangos, S.; Junop, M

    2010-01-01

    Deinococcus spp. are renowned for their amazing ability to recover rapidly from severe genomic fragmentation as a result of exposure to extreme levels of ionizing radiation or desiccation. Despite having been originally characterized over 50 years ago, the mechanism underlying this remarkable repair process is still poorly understood. Here, we report the 2.8 {angstrom} structure of DdrB, a single-stranded DNA (ssDNA) binding protein unique to Deinococcus spp. that is crucial for recovery following DNA damage. DdrB forms a pentameric ring capable of binding single-stranded but not double-stranded DNA. Unexpectedly, the crystal structure reveals that DdrB comprises a novel fold that is structurally and topologically distinct from all other single-stranded binding (SSB) proteins characterized to date. The need for a unique ssDNA binding function in response to severe damage, suggests a distinct role for DdrB which may encompass not only standard SSB protein function in protection of ssDNA, but also more specialized roles in protein recruitment or DNA architecture maintenance. Possible mechanisms of DdrB action in damage recovery are discussed.

  7. Systematic Analysis of Compositional Order of Proteins Reveals New Characteristics of Biological Functions and a Universal Correlate of Macroevolution

    PubMed Central

    Persi, Erez; Horn, David

    2013-01-01

    We present a novel analysis of compositional order (CO) based on the occurrence of Frequent amino-acid Triplets (FTs) that appear much more than random in protein sequences. The method captures all types of proteomic compositional order including single amino-acid runs, tandem repeats, periodic structure of motifs and otherwise low complexity amino-acid regions. We introduce new order measures, distinguishing between ‘regularity’, ‘periodicity’ and ‘vocabulary’, to quantify these phenomena and to facilitate the identification of evolutionary effects. Detailed analysis of representative species across the tree-of-life demonstrates that CO proteins exhibit numerous functional enrichments, including a wide repertoire of particular patterns of dependencies on regularity and periodicity. Comparison between human and mouse proteomes further reveals the interplay of CO with evolutionary trends, such as faster substitution rate in mouse leading to decrease of periodicity, while innovation along the human lineage leads to larger regularity. Large-scale analysis of 94 proteomes leads to systematic ordering of all major taxonomic groups according to FT-vocabulary size. This is measured by the count of Different Frequent Triplets (DFT) in proteomes. The latter provides a clear hierarchical delineation of vertebrates, invertebrates, plants, fungi and prokaryotes, with thermophiles showing the lowest level of FT-vocabulary. Among eukaryotes, this ordering correlates with phylogenetic proximity. Interestingly, in all kingdoms CO accumulation in the proteome has universal characteristics. We suggest that CO is a genomic-information correlate of both macroevolution and various protein functions. The results indicate a mechanism of genomic ‘innovation’ at the peptide level, involved in protein elongation, shaped in a universal manner by mutational and selective forces. PMID:24278003

  8. Single cell genomics reveals activation signatures of endogenous SCAR's networks in aneuploid human embryos and clinically intractable malignant tumors.

    PubMed

    Glinsky, Gennadi V

    2016-10-10

    Somatic mutations and chromosome instability are hallmarks of genomic aberrations in cancer cells. Aneuploidies represent common manifestations of chromosome instability, which is frequently observed in human embryos and malignant solid tumors. Activation of human endogenous retroviruses (HERV)-derived loci is documented in preimplantation human embryos, hESC, and multiple types of human malignancies. It remains unknown whether the HERV activation may highlight a common molecular pathway contributing to the frequent occurrence of chromosome instability in the early stages of human embryonic development and the emergence of genomic aberrations in cancer. Single cell RNA sequencing analysis of human preimplantation embryos reveals activation of specific LTR7/HERVH loci during the transition from the oocytes to zygotes and identifies HERVH network signatures associated with the aneuploidy in human embryos. The correlation patterns' analysis links transcriptome signatures of the HERVH network activation of the in vivo matured human oocytes with gene expression profiles of clinical samples of prostate tumors supporting the existence of a cancer progression pathway from putative precursor lesions (prostatic intraepithelial neoplasia) to localized and metastatic prostate cancers. Tracking signatures of HERVH networks' activation in tumor samples from cancer patients with known long-term therapy outcomes enabled patients' stratification into sub-groups with markedly distinct likelihoods of therapy failure and death from cancer. Genome-wide analyses of human-specific genetic elements of stem cell-associated retroviruses (SCARs)-regulated networks in 12,093 clinical tumor samples across 29 cancer types revealed pan-cancer genomic signatures of clinically-lethal therapy resistant disease defined by the presence of somatic non-silent mutations (SNMs), gene-level copy number changes, and transcripts and proteins' expression of SCARs-regulated host genes. More than 73% of all

  9. Direct Probe of Spectral Inhomogeneity Reveals Synthetic Tunability of Single-Nanocrystal Spectral Linewidths

    PubMed Central

    Cui, Jian; Beyler, Andrew P.; Marshall, Lisa F.; Chen, Ou; Harris, Daniel K.; Wanger, Darcy D.; Brokmann, Xavier; Bawendi, Moungi G.

    2013-01-01

    The spectral linewidth of an ensemble of fluorescent emitters is dictated by a combination of the single emitter linewidths and sample inhomogeneities. For semiconductor nanocrystals, efforts to tune ensemble linewidths for optical applications have focused primarily on eliminating sample inhomogeneities because conventional single-molecule methods cannot reliably build accurate ensemble-level statistics for single-particle linewidths. Photon-correlation Fourier spectroscopy in solution (S-PCFS) offers a unique approach to investigating single-nanocrystal spectra with large sample statistics, without user selection bias, with high signal-to-noise ratios, and at fast timescales. With S-PCFS, we directly and quantitatively deconstruct the ensemble linewidth into contributions from the average single-particle linewidth and from sample inhomogeneity. We demonstrate that single-particle linewidths vary significantly from batch to batch and can be synthetically controlled. These findings crystallize our understanding of the synthetic challenges facing underdeveloped nanomaterials such as InP and InAs core/shell particles and introduce new avenues for the synthetic optimization of fluorescent nanoparticles. PMID:23787751

  10. Yersinia pestis Ail: multiple roles of a single protein

    PubMed Central

    Kolodziejek, Anna M.; Hovde, Carolyn J.; Minnich, Scott A.

    2012-01-01

    Yersinia pestis is one of the most virulent bacteria identified. It is the causative agent of plague—a systemic disease that has claimed millions of human lives throughout history. Y. pestis survival in insect and mammalian host species requires fine-tuning to sense and respond to varying environmental cues. Multiple Y. pestis attributes participate in this process and contribute to its pathogenicity and highly efficient transmission between hosts. These include factors inherited from its enteric predecessors; Y. enterocolitica and Y. pseudotuberculosis, as well as phenotypes acquired or lost during Y. pestis speciation. Representatives of a large Enterobacteriaceae Ail/OmpX/PagC/Lom family of outer membrane proteins (OMPs) are found in the genomes of all pathogenic Yersiniae. This review describes the current knowledge regarding the role of Ail in Y. pestis pathogenesis and virulence. The pronounced role of Ail in the following areas are discussed (1) inhibition of the bactericidal properties of complement, (2) attachment and Yersinia outer proteins (Yop) delivery to host tissue, (3) prevention of PMNL recruitment to the lymph nodes, and (4) inhibition of the inflammatory response. Finally, Ail homologs in Y. enterocolitica and Y. pseudotuberculosis are compared to illustrate differences that may have contributed to the drastic bacterial lifestyle change that shifted Y. pestis from an enteric to a vector-born systemic pathogen. PMID:22919692

  11. MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age.

    PubMed

    Ferree, Andrew W; Trudeau, Kyle; Zik, Eden; Benador, Ilan Y; Twig, Gilad; Gottlieb, Roberta A; Shirihai, Orian S

    2013-11-01

    To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport.

  12. Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein*

    PubMed Central

    Rahman, M. Azizur; Oomori, Tamotsu; Wörheide, Gert

    2011-01-01

    Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature. PMID:21768106

  13. Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis

    PubMed Central

    Trexler, Adam J.; Sochacki, Kem A.; Taraska, Justin W.

    2016-01-01

    How and when the dozens of molecules that control exocytosis assemble in living cells to regulate the fusion of a vesicle with the plasma membrane is unknown. Here we image with two-color total internal reflection fluorescence microscopy the local changes of 27 proteins at single dense-core vesicles undergoing calcium-triggered fusion. We identify two broad dynamic behaviors of exocytic molecules. First, proteins enriched at exocytic sites are associated with DCVs long before exocytosis, and near the time of membrane fusion, they diffuse away. These proteins include Rab3 and Rab27, rabphilin3a, munc18a, tomosyn, and CAPS. Second, we observe a group of classical endocytic proteins and lipids, including dynamins, amphiphysin, syndapin, endophilin, and PIP2, which are rapidly and transiently recruited to the exocytic site near the time of membrane fusion. Dynamin mutants unable to bind amphiphysin were not recruited, indicating that amphiphysin is involved in localizing dynamin to the fusion site. Expression of mutant dynamins and knockdown of endogenous dynamin altered the rate of cargo release from single vesicles. Our data reveal the dynamics of many key proteins involved in exocytosis and identify a rapidly recruited dynamin/PIP2/BAR assembly that regulates the exocytic fusion pore of dense-core vesicles in cultured endocrine beta cells. PMID:27307587

  14. Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis.

    PubMed

    Trexler, Adam J; Sochacki, Kem A; Taraska, Justin W

    2016-08-01

    How and when the dozens of molecules that control exocytosis assemble in living cells to regulate the fusion of a vesicle with the plasma membrane is unknown. Here we image with two-color total internal reflection fluorescence microscopy the local changes of 27 proteins at single dense-core vesicles undergoing calcium-triggered fusion. We identify two broad dynamic behaviors of exocytic molecules. First, proteins enriched at exocytic sites are associated with DCVs long before exocytosis, and near the time of membrane fusion, they diffuse away. These proteins include Rab3 and Rab27, rabphilin3a, munc18a, tomosyn, and CAPS. Second, we observe a group of classical endocytic proteins and lipids, including dynamins, amphiphysin, syndapin, endophilin, and PIP2, which are rapidly and transiently recruited to the exocytic site near the time of membrane fusion. Dynamin mutants unable to bind amphiphysin were not recruited, indicating that amphiphysin is involved in localizing dynamin to the fusion site. Expression of mutant dynamins and knockdown of endogenous dynamin altered the rate of cargo release from single vesicles. Our data reveal the dynamics of many key proteins involved in exocytosis and identify a rapidly recruited dynamin/PIP2/BAR assembly that regulates the exocytic fusion pore of dense-core vesicles in cultured endocrine beta cells. PMID:27307587

  15. High-Throughput Single-Molecule Studies of Protein-DNA Interactions

    PubMed Central

    Robison, Aaron D.; Finkelstein, Ilya J.

    2014-01-01

    Fluorescence and force-based single-molecule studies of protein-nucleic acid interactions continue to shed critical insights into many aspects of DNA and RNA processing. As single-molecule assays are inherently low-throughput, obtaining statistically relevant datasets remains a major challenge. Additionally, most fluorescence-based single-molecule particle-tracking assays are limited to observing fluorescent proteins that are in the low-nanomolar range, as spurious background signals predominate at higher fluorophore concentrations. These technical limitations have traditionally limited the types of questions that could be addressed via single-molecule methods. In this review, we describe new approaches for high-throughput and high-concentration single-molecule biochemical studies. We conclude with a discussion of outstanding challenges for the single-molecule biologist and how these challenges can be tackled to further approach the biochemical complexity of the cell. PMID:24859086

  16. Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

    SciTech Connect

    Hast, Michael A.; Nichols, Connie B.; Armstrong, Stephanie M.; Kelly, Shannon M.; Hellinga, Homme W.; Alspaugh, J. Andrew; Beese, Lorena S.

    2012-09-17

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

  17. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J.; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2012-10-01

    Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

  18. Sub-angstrom single-molecule measurements of motor proteins using a nanopore

    PubMed Central

    Derrington, Ian M; Craig, Jonathan M; Stava, Eric; Laszlo, Andrew H; Ross, Brian C; Brinkerhoff, Henry; Nova, Ian C; Doering, Kenji; Tickman, Benjamin I; Ronaghi, Mostafa; Mandell, Jeffrey G; Gunderson, Kevin L; Gundlach, Jens H

    2016-01-01

    Present techniques for measuring the motion of single motor proteins, such as FRET and optical tweezers, are limited to a resolution of ~300 pm. We use ion current modulation through the protein nanopore MspA to observe translocation of helicase Hel308 on DNA with up to ~40 picometer sensitivity. This approach should be applicable to any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and DNA repair enzymes. PMID:26414351

  19. Seeing is believing: Direct imaging of charge flow along pili proteins reveals new mechanism for bacterial electron transfer

    NASA Astrophysics Data System (ADS)

    Malvankar, Nikhil; Yalcin, Sibel Ebru; Adhikari, Ramesh; Tuominen, Mark; Lovley, Derek

    2015-03-01

    Visualization of charge flow on the nanoscale in proteins is crucial for a fundamental understanding of several life processes. Here, we report direct visualization of charge propagation along native pili of Geobacter sulfurreducens at nanometer resolution using electrostatic force microscopy. Surprisingly, charges injected at a single point into individual, untreated pili, still attached to cells, propagate over the entire filament. The charges propagate despite a lack of cytochromes on the pili, in contrast to the dominant biochemical model that proteins are electronically insulating and must incorporate redox-active cofactors in order to achieve electron transport functionality. The mobile charge density in pili is comparable to synthetic organic conductors, increasing with proton doping, and with temperature-dependence consistent with previously discovered metallic-like transport mechanism. Conductive pili enable syntrophic bacteria to share energy by directly exchanging electrons among each other. Measurements along individual pilus using nanoelectrodes showed ohmic behavior strongly dependent on the amino acid composition of pili. Electron transfer rate measurement revealed that the pili conductivity is the decisive factor in controlling the bacterial respiration rate. Funded by Office of Naval Research, DOE Genomic Sciences, NSF-NSEC CHM (CMMI-1025020) and Burroughs Wellcome Fund.

  20. Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells

    PubMed Central

    Brennecke, Philip; Reyes, Alejandro; Pinto, Sheena; Rattay, Kristin; Nguyen, Michelle; Küchler, Rita; Huber, Wolfgang; Kyewski, Bruno; Steinmetz, Lars M.

    2015-01-01

    Expression of tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for self-tolerance induction and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA-sequencing and provide evidence for numerous recurring TRA co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process, which might involve local re-modeling of chromatin and thus ensures a comprehensive representation of the immunological self. PMID:26237553

  1. DNA Replication Catalyzed by Herpes Simplex Virus Type 1 Proteins Reveals Trombone Loops at the Fork*♦

    PubMed Central

    Bermek, Oya; Willcox, Smaranda; Griffith, Jack D.

    2015-01-01

    Using purified replication factors encoded by herpes simplex virus type 1 and a 70-base minicircle template, we obtained robust DNA synthesis with leading strand products of >20,000 nucleotides and lagging strand fragments from 600 to 9,000 nucleotides as seen by alkaline gel electrophoresis. ICP8 was crucial for the synthesis on both strands. Visualization of the deproteinized products using electron microscopy revealed long, linear dsDNAs, and in 87%, one end, presumably the end with the 70-base circle, was single-stranded. The remaining 13% had multiple single-stranded segments separated by dsDNA segments 500 to 1,000 nucleotides in length located at one end. These features are diagnostic of the trombone mechanism of replication. Indeed, when the products were examined with the replication proteins bound, a dsDNA loop was frequently associated with the replication complex located at one end of the replicated DNA. Furthermore, the frequency of loops correlated with the fraction of DNA undergoing Okazaki fragment synthesis. PMID:25471368

  2. Effect of the microtubule-associated protein tau on dynamics of single-headed motor proteins KIF1A

    NASA Astrophysics Data System (ADS)

    Sparacino, J.; Farías, M. G.; Lamberti, P. W.

    2014-02-01

    Intracellular transport based on molecular motors and its regulation are crucial to the functioning of cells. Filamentary tracks of the cells are abundantly decorated with nonmotile microtubule-associated proteins, such as tau. Motivated by experiments on kinesin-tau interactions [Dixit et al., Science 319, 1086 (2008), 10.1126/science.1152993] we developed a stochastic model of interacting single-headed motor proteins KIF1A that also takes into account the interactions between motor proteins and tau molecules. Our model reproduces experimental observations and predicts significant effects of tau on bound time and run length which suggest an important role of tau in regulation of kinesin-based transport.

  3. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity.

    PubMed

    Brehme, Marc; Voisine, Cindy

    2016-08-01

    Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  4. Structure and dynamics of protein waters revealed by radiolysis and mass spectrometry

    PubMed Central

    Gupta, Sayan; D’Mello, Rhijuta; Chance, Mark R.

    2012-01-01

    Water is critical for the structure, stability, and functions of macromolecules. Diffraction and NMR studies have revealed structure and dynamics of bound waters at atomic resolution. However, localizing the sites and measuring the dynamics of bound waters, particularly on timescales relevant to catalysis and macromolecular assembly, is quite challenging. Here we demonstrate two techniques: first, temperature-dependent radiolytic hydroxyl radical labeling with a mass spectrometry (MS)-based readout to identify sites of bulk and bound water interactions with surface and internal residue side chains, and second, H218O radiolytic exchange coupled MS to measure the millisecond dynamics of bound water interactions with various internal residue side chains. Through an application of the methods to cytochrome c and ubiquitin, we identify sites of water binding and measure the millisecond dynamics of bound waters in protein crevices. As these MS-based techniques are very sensitive and not protein size limited, they promise to provide unique insights into protein–water interactions and water dynamics for both small and large proteins and their complexes. PMID:22927377

  5. Analysis of microdissected neurons by 18O mass spectrometry reveals altered protein expression in Alzheimer's disease

    PubMed Central

    Hashimoto, Masakazu; Bogdanovic, Nenad; Nakagawa, Hiroyuki; Volkmann, Inga; Aoki, Mikio; Winblad, Bengt; Sakai, Jun; Tjernberg, Lars O

    2012-01-01

    Abstract It is evident that the symptoms of Alzheimer's disease (AD) are derived from severe neuronal damage, and especially pyramidal neurons in the hippocampus are affected pathologically. Here, we analysed the proteome of hippocampal neurons, isolated from post-mortem brains by laser capture microdissection. By using 18O labelling and mass spectrometry, the relative expression levels of 150 proteins in AD and controls were estimated. Many of the identified proteins are involved in transcription and nucleotide binding, glycolysis, heat-shock response, microtubule stabilization, axonal transport or inflammation. The proteins showing the most altered expression in AD were selected for immunohistochemical analysis. These analyses confirmed the altered expression levels, and showed in many AD cases a pathological pattern. For comparison, we also analysed hippocampal sections by Western blot. The expression levels found by this method showed poor correlation with the neuron-specific analysis. Hence, we conclude that cell-specific proteome analysis reveals differences in the proteome that cannot be detected by bulk analysis. PMID:21883897

  6. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity

    PubMed Central

    2016-01-01

    ABSTRACT Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  7. Glycoproteomic Study Reveals Altered Plasma Proteins Associated with HIV Elite Suppressors

    PubMed Central

    Yang, Weiming; Laeyendecker, Oliver; Wendel, Sarah K.; Zhang, Bai; Sun, Shisheng; Zhou, Jian-Ying; Ao, Minghui; Moore, Richard D.; Jackson, J. Brooks; Zhang, Hui

    2014-01-01

    HIV elite suppressors (ES) or controllers are individuals achieving control of viremia by their natural immunological mechanisms without highly active antiretroviral therapy (HAART). Study of the mechanisms responsible for the immunological suppression of viremia in ES may lead to the detection of individuals with ES and the effective control of HIV infection. We hypothesize that plasma glycoproteins play essential roles in the immune system of ES since plasma proteins are critical and highly relevant in anti-viral immunity and most plasma proteins are glycoproteins. To examine glycoproteins associated with ES, plasma samples from ES individuals (n=20), and from individuals on HAART (n=20), with AIDS (n=20), and no HIV infection (n=10) were analyzed by quantitative glycoproteomics. We found that a number of glycoproteins changed between ES versus HAART, AIDS and HIV- individuals. In sharp contrast, the level of plasma glycoproteins in the HAART cohort showed fewer changes compared with AIDS and HIV- individuals. These results showed that although both ES and HAART effectively suppress viremia, ES appeared to profoundly affect immunologically relevant glycoproteins in plasma as consequence of or support for anti-viral immunity. Bioinformatic analysis revealed that altered proteins in ES plasma were mainly associated with inflammation. This analysis suggests that overlapping, while distinguishable, glycoprotein profiles for inflammation and immune activation appeared to be present between ES and non-ES (HAART+AIDS) cohorts, indicating different triggers for inflammation and immune activation between natural and treatment-related viral suppression. PMID:25285165

  8. Structure of the Yeast Polarity Protein Sro7 Reveals a SNARE Regulatory Mechanism

    SciTech Connect

    Hattendorf, D.A.; Andreeva, A.; Gangar, A.; Brennwald, P.J.; Weis, W.I.; /Stanford U., Med. School /North Carolina U.

    2007-07-09

    Polarized exocytosis requires coordination between the actin cytoskeleton and the exocytic machinery responsible for fusion of secretory vesicles at specific sites on the plasma membrane. Fusion requires formation of a complex between a vesicle-bound R-SNARE and plasma membrane Qa, Qb and Qc SNARE proteins. Proteins in the lethal giant larvae protein family, including lethal giant larvae and tomosyn in metazoans and Sro7 in yeast, interact with Q-SNAREs and are emerging as key regulators of polarized exocytosis. The crystal structure of Sro7 reveals two seven-bladed WD40 {beta}-propellers followed by a 60-residue-long 'tail', which is bound to the surface of the amino-terminal propeller. Deletion of the Sro7 tail enables binding to the Qbc SNARE region of Sec9 and this interaction inhibits SNARE complex assembly. The N-terminal domain of Sec9 provides a second, high-affinity Sro7 interaction that is unaffected by the tail. The results suggest that Sro7 acts as an allosteric regulator of exocytosis through interactions with factors that control the tail. Sequence alignments indicate that lethal giant larvae and tomosyn have a two-{beta}-propeller fold similar to that of Sro7, but only tomosyn appears to retain the regulatory tail.

  9. The single-stranded DNA-binding protein of Escherichia coli.

    PubMed Central

    Meyer, R R; Laine, P S

    1990-01-01

    The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake. PMID:2087220

  10. Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains.

    PubMed

    Boyer, Patrick D; Ganesh, Sairaam; Qin, Zhao; Holt, Brian D; Buehler, Markus J; Islam, Mohammad F; Dahl, Kris Noel

    2016-02-10

    Single-walled carbon nanotubes (SWCNTs) have great potential for cell-based therapies due to their unique intrinsic optical and physical characteristics. Consequently, broad classes of dispersants have been identified that individually suspend SWCNTs in water and cell media in addition to reducing nanotube toxicity to cells. Unambiguous control and verification of the localization and distribution of SWCNTs within cells, particularly to the nucleus, is needed to advance subcellular technologies utilizing nanotubes. Here we report delivery of SWCNTs to the nucleus by noncovalently attaching the tail domain of the nuclear protein lamin B1 (LB1), which we engineer from the full-length LMNB1 cDNA. More than half of this low molecular weight globular protein is intrinsically disordered but has an immunoglobulin-fold composed of a central hydrophobic core, which is highly suitable for associating with SWCNTs, stably suspending SWCNTs in water and cell media. In addition, LB1 has an exposed nuclear localization sequence to promote active nuclear import of SWCNTs. These SWCNTs-LB1 dispersions in water and cell media display near-infrared (NIR) absorption spectra with sharp van Hove peaks and an NIR fluorescence spectra, suggesting that LB1 individually disperses nanotubes. The dispersing capability of SWCNTs by LB1 is similar to that by albumin proteins. The SWCNTs-LB1 dispersions with concentrations ≥150 μg/mL (≥30 μg/mL) in water (cell media) remain stable for ≥75 days (≥3 days) at 4 °C (37 °C). Further, molecular dynamics modeling of association of LB1 with SWCNTs reveal that the exposure of the nuclear localization sequence is independent of LB1 binding conformation. Measurements from confocal Raman spectroscopy and microscopy, NIR fluorescence imaging of SWCNTs, and fluorescence lifetime imaging microscopy show that millions of these SWCNTs-LB1 complexes enter HeLa cells, localize to the nucleus of cells, and interact with DNA. We postulate that the

  11. Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains.

    PubMed

    Boyer, Patrick D; Ganesh, Sairaam; Qin, Zhao; Holt, Brian D; Buehler, Markus J; Islam, Mohammad F; Dahl, Kris Noel

    2016-02-10

    Single-walled carbon nanotubes (SWCNTs) have great potential for cell-based therapies due to their unique intrinsic optical and physical characteristics. Consequently, broad classes of dispersants have been identified that individually suspend SWCNTs in water and cell media in addition to reducing nanotube toxicity to cells. Unambiguous control and verification of the localization and distribution of SWCNTs within cells, particularly to the nucleus, is needed to advance subcellular technologies utilizing nanotubes. Here we report delivery of SWCNTs to the nucleus by noncovalently attaching the tail domain of the nuclear protein lamin B1 (LB1), which we engineer from the full-length LMNB1 cDNA. More than half of this low molecular weight globular protein is intrinsically disordered but has an immunoglobulin-fold composed of a central hydrophobic core, which is highly suitable for associating with SWCNTs, stably suspending SWCNTs in water and cell media. In addition, LB1 has an exposed nuclear localization sequence to promote active nuclear import of SWCNTs. These SWCNTs-LB1 dispersions in water and cell media display near-infrared (NIR) absorption spectra with sharp van Hove peaks and an NIR fluorescence spectra, suggesting that LB1 individually disperses nanotubes. The dispersing capability of SWCNTs by LB1 is similar to that by albumin proteins. The SWCNTs-LB1 dispersions with concentrations ≥150 μg/mL (≥30 μg/mL) in water (cell media) remain stable for ≥75 days (≥3 days) at 4 °C (37 °C). Further, molecular dynamics modeling of association of LB1 with SWCNTs reveal that the exposure of the nuclear localization sequence is independent of LB1 binding conformation. Measurements from confocal Raman spectroscopy and microscopy, NIR fluorescence imaging of SWCNTs, and fluorescence lifetime imaging microscopy show that millions of these SWCNTs-LB1 complexes enter HeLa cells, localize to the nucleus of cells, and interact with DNA. We postulate that the

  12. Structural and functional studies of the rat mitochondrial single strand DNA binding protein P16.

    PubMed

    Hoke, G D; Pavco, P A; Ledwith, B J; Van Tuyle, G C

    1990-10-01

    The rat mitochondrial single strand DNA binding protein (SSB) P16 was purified to apparent homogeneity by elution from single strand DNA agarose with ethidium bromide. Each monomer of P16 contains two tryptophan residues, and the intrinsic fluorescence from these residues is quenched upon binding to single strand polynucleotides. From fluorescence quench titrations of ligand to fixed amounts of DNA lattice, a binding site size of 8 or 9 nucleotides per P16 monomer was found. Measurement of the affinity of P16 for isolated sites by titration with either oligo(dT)8 or 5'-dephosphorylated oligo(dT)8 indicated values on the order of 10(7) M-1. P16 exhibited a binding preference for single strand DNA, poly(dT), and poly(dC) in comparison to double strand DNA, poly(U), or poly[d(A-T)]. Although it was not possible to show that P16 destabilizes double helical DNA or even poly[d(A-T)], binding of P16 does inhibit the process of renaturation as shown by inhibition of duplex formation between poly(dA) and poly(dT). The binding of saturating amounts of P16 to single strand poly(dT).oligo(dA)50 template-primers enhanced approximately 10-fold the activity of both the homologous mitochondrial DNA polymerase and the Escherichia coli DNA polymerase I Klenow fragment. However, the mitochondrial DNA primase was nearly completely inhibited by the saturation of the poly(dT) template with P16. Amino-terminal sequence analysis of P16 and a protease-insensitive, DNA binding domain (Mr approximately 6000) revealed that the DNA binding domain residues, at least in part, in the amino-terminal third of the P16 molecule. Furthermore, the amino-terminal sequence was found to be strikingly similar to that of the Xenopus laevis mtSSB-1 and to a lesser extent similar to E. coli SSB and E. coli F sex factor SSB.

  13. System-wide Analysis of SUMOylation Dynamics in Response to Replication Stress Reveals Novel Small Ubiquitin-like Modified Target Proteins and Acceptor Lysines Relevant for Genome Stability*

    PubMed Central

    Xiao, Zhenyu; Chang, Jer-Gung; Hendriks, Ivo A.; Sigurðsson, Jón Otti; Olsen, Jesper V.; Vertegaal, Alfred C.O.

    2015-01-01

    Genotoxic agents can cause replication fork stalling in dividing cells because of DNA lesions, eventually leading to replication fork collapse when the damage is not repaired. Small Ubiquitin-like Modifiers (SUMOs) are known to counteract replication stress, nevertheless, only a small number of relevant SUMO target proteins are known. To address this, we have purified and identified SUMO-2 target proteins regulated by replication stress in human cells. The developed methodology enabled single step purification of His10-SUMO-2 conjugates under denaturing conditions with high yield and high purity. Following statistical analysis on five biological replicates, a total of 566 SUMO-2 targets were identified. After 2 h of hydroxyurea treatment, 10 proteins were up-regulated for SUMOylation and two proteins were down-regulated for SUMOylation, whereas after 24 h, 35 proteins were up-regulated for SUMOylation, and 13 proteins were down-regulated for SUMOylation. A site-specific approach was used to map over 1000 SUMO-2 acceptor lysines in target proteins. The methodology is generic and is widely applicable in the ubiquitin field. A large subset of these identified proteins function in one network that consists of interacting replication factors, transcriptional regulators, DNA damage response factors including MDC1, ATR-interacting protein ATRIP, the Bloom syndrome protein and the BLM-binding partner RMI1, the crossover junction endonuclease EME1, BRCA1, and CHAF1A. Furthermore, centromeric proteins and signal transducers were dynamically regulated by SUMOylation upon replication stress. Our results uncover a comprehensive network of SUMO target proteins dealing with replication damage and provide a framework for detailed understanding of the role of SUMOylation to counteract replication stress. Ultimately, our study reveals how a post-translational modification is able to orchestrate a large variety of different proteins to integrate different nuclear processes with the

  14. SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell.

    PubMed

    Kim, Henry S; Martel, Anne; Girard, Eric; Moulin, Martine; Härtlein, Michael; Madern, Dominique; Blackledge, Martin; Franzetti, Bruno; Gabel, Frank

    2016-05-24

    Water molecules in the immediate vicinity of biomacromolecules, including proteins, constitute a hydration layer characterized by physicochemical properties different from those of bulk water and play a vital role in the activity and stability of these structures, as well as in intermolecular interactions. Previous studies using solution scattering, crystallography, and molecular dynamics simulations have provided valuable information about the properties of these hydration shells, including modifications in density and ionic concentration. Small-angle scattering of x-rays (SAXS) and neutrons (SANS) are particularly useful and complementary techniques to study biomacromolecular hydration shells due to their sensitivity to electronic and nuclear scattering-length density fluctuations, respectively. Although several sophisticated SAXS/SANS programs have been developed recently, the impact of physicochemical surface properties on the hydration layer remains controversial, and systematic experimental data from individual biomacromolecular systems are scarce. Here, we address the impact of physicochemical surface properties on the hydration shell by a systematic SAXS/SANS study using three mutants of a single protein, green fluorescent protein (GFP), with highly variable net charge (+36, -6, and -29). The combined analysis of our data shows that the hydration shell is locally denser in the vicinity of acidic surface residues, whereas basic and hydrophilic/hydrophobic residues only mildly modify its density. Moreover, the data demonstrate that the density modifications result from the combined effect of residue-specific recruitment of ions from the bulk in combination with water structural rearrangements in their vicinity. Finally, we find that the specific surface-charge distributions of the different GFP mutants modulate the conformational space of flexible parts of the protein. PMID:27224484

  15. Functional Mapping of Protein Kinase A Reveals Its Importance in Adult Schistosoma mansoni Motor Activity

    PubMed Central

    de Saram, Paulu S. R.; Ressurreição, Margarida; Davies, Angela J.; Rollinson, David; Emery, Aidan M.; Walker, Anthony J.

    2013-01-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and ‘smart’ anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology

  16. Functional mapping of protein kinase A reveals its importance in adult Schistosoma mansoni motor activity.

    PubMed

    de Saram, Paulu S R; Ressurreição, Margarida; Davies, Angela J; Rollinson, David; Emery, Aidan M; Walker, Anthony J

    2013-01-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and 'smart' anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology.

  17. Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas

    PubMed Central

    Hou, Yu; Guo, Huahu; Cao, Chen; Li, Xianlong; Hu, Boqiang; Zhu, Ping; Wu, Xinglong; Wen, Lu; Tang, Fuchou; Huang, Yanyi; Peng, Jirun

    2016-01-01

    Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells. PMID:26902283

  18. Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas.

    PubMed

    Hou, Yu; Guo, Huahu; Cao, Chen; Li, Xianlong; Hu, Boqiang; Zhu, Ping; Wu, Xinglong; Wen, Lu; Tang, Fuchou; Huang, Yanyi; Peng, Jirun

    2016-03-01

    Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells.

  19. Kinetics of small molecule interactions with membrane proteins in single cells measured with mechanical amplification

    PubMed Central

    Guan, Yan; Shan, Xiaonan; Zhang, Fenni; Wang, Shaopeng; Chen, Hong-Yuan; Tao, Nongjian

    2015-01-01

    Measuring small molecule interactions with membrane proteins in single cells is critical for understanding many cellular processes and for screening drugs. However, developing such a capability has been a difficult challenge. We show that molecular interactions with membrane proteins induce a mechanical deformation in the cellular membrane, and real-time monitoring of the deformation with subnanometer resolution allows quantitative analysis of small molecule–membrane protein interaction kinetics in single cells. This new strategy provides mechanical amplification of small binding signals, making it possible to detect small molecule interactions with membrane proteins. This capability, together with spatial resolution, also allows the study of the heterogeneous nature of cells by analyzing the interaction kinetics variability between different cells and between different regions of a single cell. PMID:26601298

  20. Single-Molecule Manipulation Studies of a Mechanically Activated Protein

    NASA Astrophysics Data System (ADS)

    Botello, Eric; Harris, Nolan; Choi, Huiwan; Bergeron, Angela; Dong, Jing-Fei; Kiang, Ching-Hwa

    2009-10-01

    Plasma von Willebrand factor (pVWF) is the largest multimeric adhesion ligand found in human blood and must be adhesively activated by exposure to shear stress, like at sites of vascular injury, to initiate blood clotting. Sheared pVWF (sVWF) will undergo a conformational change from a loose tangled coil to elongated strings forming adhesive fibers by binding with other sVWF. VWF's adhesion activity is also related to its length, with the ultra-large form of VWF (ULVWF) being hyper-actively adhesive without exposure to shear stress; it has also been shown to spontaneously form fibers. We used single molecule manipulation techniques with the AFM to stretch pVWF, sVWF and ULVWF and monitor the forces as a function of molecular extension. We showed a similar increase in resistance to unfolding for sVWF and ULVWF when compared to pVWF. This mechanical resistance to forced unfolding is reduced when other molecules known to disrupt their fibril formation are present. Our results show that sVWF and ULVWF domains unfold at higher forces than pVWF, which is consistent with the hypothesis that shear stress induces lateral association that alters adhesion activity of pVWF.

  1. IF-combined smRNA FISH reveals interaction of MCPIP1 protein with IER3 mRNA

    PubMed Central

    Kochan, Jakub; Wawro, Mateusz

    2016-01-01

    ABSTRACT MCPIP1 and IER3 are recently described proteins essential for maintenance of immune homeostasis. IER3 is involved in the regulation of apoptosis and differentiation and has been shown lately to protect activated T cells and macrophages from apoptosis. MCPIP1 is an RNase critical for controlling inflammation-related mRNAs. MCPIP1 interacts with and degrades a set of stem-loop-containing mRNAs (including IL-6). Our results demonstrate the involvement of MCPIP1 in the regulation of IER3 mRNA levels. A dual luciferase assay revealed that over-expression of MCPIP1 resulted in a decrease of luciferase activity in the samples co-transfected with constructs containing luciferase CDS attached to IER3 3′UTR. We identified a stem-loop structure similar to that described to be important for destabilization of the IL-6 mRNA by MCPIP1. Examination of IER3 3′UTR sequence, structure and evolutionary conservation revealed that the identified stem-loop is buried within a bigger element. Deletion of this fragment abolished the regulation of IER3 3′UTR-containing transcript by MCPIP1. Finally, using immunofluorescence-combined single-molecule RNA FISH we have shown that the MCPIP1 protein co-localizes with IER3 mRNA. By this method we also proved that the presence of the wild-type NYN/PIN-like domain of MCPIP1 correlated with the decreased level of IER3 mRNA. RNA immunoprecipitation further confirmed the interaction of MCPIP1 with IER3 transcripts in vivo. PMID:27256408

  2. Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

    PubMed Central

    Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

    2016-01-01

    Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exh