Distance measurements within a concatamer of the plasma membrane Cl?/HCO3? exchanger, AE1.
AuthorsBasu A, Mazor S, Casey JR,
JournalBiochemistry
PubMed ID20828148
'AE1, which exists in the erythrocyte plasma membrane as a noncovalent dimer, facilitates transmembrane Cl?/HCO3? exchange. Here a concatamer of AE1 (two AE1 monomers fused via a two-residue linker to form an intramolecular dimer) was designed to facilitate fluorescence resonance energy transfer (FRET) studies. The concatameric protein (AE1·AE1) was expressed ... More
Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis.
AuthorsNakase I, Tadokoro A, Kawabata N, Takeuchi T, Katoh H, Hiramoto K, Negishi M, Nomizu M, Sugiura Y, Futaki S
JournalBiochemistry
PubMed ID17209559
'Arginine-rich peptides, including octaarginine (R8), HIV-1 Tat, and branched-chain arginine-rich peptides, belong to one of the major classes of cell-permeable peptides which deliver various proteins and macromolecules to cells. The importance of the endocytic pathways has recently been demonstrated in the cellular uptake of these peptides. We have previously shown ... More
Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.
AuthorsSmith RM, Baibakov B, Ikebuchi Y, White BH, Lambert NA, Kaczmarek LK, Vogel SS
JournalJ Cell Biol
PubMed ID10684256
'Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of ... More
Actin binding to the central domain of WASP/Scar proteins plays a critical role in the activation of the Arp2/3 complex.
AuthorsKelly AE, Kranitz H, Dötsch V, Mullins RD
JournalJ Biol Chem
PubMed ID16403731
'The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind ... More
Tetanus toxin is transported in a novel neuronal compartment characterized by a specialized pH regulation.
AuthorsBohnert S, Schiavo G
JournalJ Biol Chem
PubMed ID16236708
'Tetanus toxin binds specifically to motor neurons at the neuromuscular junction. There, it is internalized into vesicular carriers undergoing fast retrograde transport to the spinal cord. Despite the importance of this axonal transport pathway in health and disease, its molecular and biophysical characterization is presently lacking. We sought to fill ... More
Coil-globule transition in the denatured state of a small protein.
AuthorsSherman E, Haran G
JournalProc Natl Acad Sci U S A
PubMed ID16857738
'Upon transfer from strongly denaturing to native conditions, proteins undergo a collapse that either precedes folding or occurs simultaneously with it. This collapse is similar to the well known coil-globule transition of polymers. Here we employ single-molecule fluorescence methods to fully characterize the equilibrium coil-globule transition in the denatured state ... More
Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy.
AuthorsSlaughter BD, Unruh JR, Price ES, Huynh JL, Bieber Urbauer RJ, Johnson CK
JournalJ Am Chem Soc
PubMed ID16117552
'We used single-pair fluorescence resonance energy transfer (spFRET) measurements to characterize denatured and partially denatured states of the multidomain calcium signaling protein calmodulin (CaM) in both its apo and Ca(2+)-bound forms. The results demonstrate the existence of an unfolding intermediate. A CaM mutant (CaM-T34C-T110C) was doubly labeled with fluorescent probes ... More
Electrokinetic molecular separation in nanoscale fluidic channels.
'This report presents a study of electrokinetic transport in a series of integrated macro- to nano-fluidic chips that allow for controlled injection of molecular mixtures into high-density arrays of nanochannels. The high-aspect-ratio nanochannels were fabricated on a Si wafer using interferometric lithography and standard semiconductor industry processes, and are capped ... More
Direct visualization of saposin remodelling of lipid bilayers.
AuthorsAlattia JR, Shaw JE, Yip CM, Privé GG
JournalJ Mol Biol
PubMed ID16949605
'Saposins A, B, C and D are soluble, non-enzymatic proteins that interact with lysosomal membranes to activate the breakdown and transfer of glycosphingolipids. The mechanisms of hydrolase activation and lipid transfer by saposins remain unknown. We have used in situ atomic force microscopy (AFM) with simultaneous confocal fluorescence microscopy to ... More
Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments.
AuthorsAllen MW, Urbauer RJ, Zaidi A, Williams TD, Urbauer JL, Johnson CK
JournalAnal Biochem
PubMed ID14751262
'We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring ... More
Optical properties of Alexa 488 and Cy5 immobilized on a glass surface.
AuthorsWang L, Gaigalas AK, Reipa V
JournalBiotechniques
PubMed ID15679095
'The absorption and emission spectra were measured for Cy5 and Alexa 488 fluorophores confined on a glass surface. The data were obtained using fluorometry and spectroscopic ellipsometry. Red shifts of the surface-immobilized fluorophore absorption spectra relative to the fluorophore spectra in aqueous solution were observed using both methods. We interpret ... More
Introduction of Tau Oligomers into Cortical Neurons Alters Action Potential Dynamics and Disrupts Synaptic Transmission and Plasticity.
AuthorsHill E, Karikari TK, Moffat KG, Richardson MJE, Wall MJ
Journal
PubMed ID31554666
'Tau is a highly soluble microtubule-associated protein that acts within neurons to modify microtubule stability. However, abnormally phosphorylated tau dissociates from microtubules to form oligomers and fibrils which associate in the somatodendritic compartment. Although tau can form neurofibrillary tangles (NFTs), it is the soluble oligomers that appear to be the ... More
FliW and FliS function independently to control cytoplasmic flagellin levels in Bacillus subtilis.
AuthorsMukherjee S, Babitzke P, Kearns DB,
JournalJ Bacteriol
PubMed ID23144244
'The cytoplasmic level of flagellin (called Hag) is homeostatically regulated in the Gram-positive bacterium Bacillus subtilis by a partner-switching mechanism between the protein FliW and either the Hag structural protein or CsrA, an RNA binding protein that represses hag translation. Here we show that FliW and the putative secretion chaperone ... More
Orientational changes of crossbridges during single turnover of ATP.
AuthorsBorejdo J, Akopova I
JournalBiophys J
PubMed ID12668452
'Muscle contraction results from rotation of actin-bound myosin crossbridges. Crossbridges consist of the globular N-terminal catalytic domain and the alpha-helical C-terminal regulatory domain containing the essential and regulatory light chains. The essential light chain exists in two isoforms, of which the larger one has a 41-amino acid extension piece added ... More
Assembly of the bacteriophage T4 primosome: single-molecule and ensemble studies.
AuthorsZhang Z, Spiering MM, Trakselis MA, Ishmael FT, Xi J, Benkovic SJ, Hammes GG
JournalProc Natl Acad Sci U S A
PubMed ID15728347
'Within replisomes for DNA replication, the primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with ... More
Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake.
AuthorsRichard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, Chernomordik LV, Lebleu B
JournalJ Biol Chem
PubMed ID12411431
'Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, ... More
Interaction of dihydrofolate reductase with methotrexate: ensemble and single-molecule kinetics.
'The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule ... More
Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots.
AuthorsBonasio R, Carman CV, Kim E, Sage PT, Love KR, Mempel TR, Springer TA, von Andrian UH
JournalProc Natl Acad Sci U S A
PubMed ID17785425
'The real-time observation of protein dynamics in living cells and organisms is of fundamental importance for understanding biological processes. Most approaches to labeling proteins exploit noncovalent interactions, unsuitable to long-term studies, or genetic fusion to naturally occurring fluorescent proteins that often have unsatisfactory optical properties. Here we used the fungal ... More
Zinc porphyrin: a fluorescent acceptor in studies of Zn-cytochrome c unfolding by fluorescence resonance energy transfer.
AuthorsEnsign AA, Jo I, Yildirim I, Krauss TD, Bren KL,
JournalProc Natl Acad Sci U S A
PubMed ID18669660
'FRET between the zinc porphyrin (ZnP) chromophore in zinc-substituted cytochrome c (Zn-cyt c) and an Alexa Fluor dye attached to specific surface sites was used to characterize Zn-cyt c unfolding. The use of ZnP as a fluorescent acceptor eliminates the need to doubly label the protein with exogenous dyes to ... More
Importin 13: a novel mediator of nuclear import and export.
AuthorsMingot JM, Kostka S, Kraft R, Hartmann E, Görlich D
JournalEMBO J
PubMed ID11447110
'Importin beta-related receptors mediate translocation through nuclear pore complexes. Co-operation with the RanGTPase system allows them to bind and subsequently release their substrates on opposite sides of the nuclear envelope, which in turn ensures a directed nucleocytoplasmic transport. Here we identify a novel family member from higher eukaryotes that functions ... More
Affibody Molecules for In vivo Characterization of HER2-Positive Tumors by Near-Infrared Imaging.
AuthorsLee SB, Hassan M, Fisher R, Chertov O, Chernomordik V, Kramer-Marek G, Gandjbakhche A, Capala J,
JournalClin Cancer Res
PubMed ID18559604
'PURPOSE: HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. We are developing molecular probes for in vivo quantitative imaging of HER2 receptors using near-infrared (NIR) optical imaging. The goal is to provide probes that will minimally interfere with the studied system, ... More
Dependence of fluorescence intensity on the spectral overlap between fluorophores and plasmon resonant single silver nanoparticles.
AuthorsChen Y, Munechika K, Ginger DS
JournalNano Lett
PubMed ID17315937
'We investigate the fluorescence from dyes coupled to individual DNA-functionalized metal nanoparticles. We use single-particle darkfield scattering and fluorescence microscopy to correlate the fluorescence intensity of the dyes with the localized surface plasmon resonance (LSPR) spectra of the individual metal nanoparticles to which they are attached. For each of three ... More
Insights into the action of the superfamily of cholesterol-dependent cytolysins from studies of intermedilysin.
'The cholesterol-dependent cytolysins (CDCs), a superfamily of pore-forming toxins, are characterized by a conserved undecapeptide motif that is believed to be critical for membrane recognition by means of cholesterol. Intermedilysin (ILY), an unusual member of the CDCs, exhibits specificity for human cells and contains nonconservative substitutions in the motif. We ... More
Fluorescence correlation spectroscopy shows that monomeric polyglutamine molecules form collapsed structures in aqueous solutions.
AuthorsCrick SL, Jayaraman M, Frieden C, Wetzel R, Pappu RV
JournalProc Natl Acad Sci U S A
PubMed ID17075061
'We have used fluorescence correlation spectroscopy measurements to quantify the hydrodynamic sizes of monomeric polyglutamine as a function of chain length (N) by measuring the scaling of translational diffusion times (tau(D)) for the peptide series (Gly)-(Gln)(N)-Cys-Lys(2) in aqueous solution. We find that tau(D) scales with N as tau(o)N(nu) and therefore ... More
Site-specific protein labeling by Sfp phosphopantetheinyl transferase.
AuthorsYin J, Lin AJ, Golan DE, Walsh CT,
JournalNat Protoc
PubMed ID17406245
'Sfp phosphopantetheinyl transferase covalently attaches small-molecule probes including biotin and various organic fluorophores to a specific serine residue in the peptidyl carrier protein (PCP) or a short 11-residue peptide tag ybbR through a phosphopantetheinyl linker. We describe here a protocol for site-specific protein labeling by Sfp-catalyzed protein post-translational modification that ... More
Fluorescence resonance energy transfer studies on anthrax lethal toxin.
AuthorsCroney JC, Cunningham KM, Collier RJ, Jameson DM
JournalFEBS Lett
PubMed ID12935906
'Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self-assemble on receptor-bearing eukaryotic cells to form toxic, non-covalent complexes. PA(63), a proteolytically activated form of PA, spontaneously oligomerizes to form ring-shaped heptamers that bind LF and translocate it into ... More
RNA conformation in catalytically active human telomerase.
AuthorsYeoman JA, Orte A, Ashbridge B, Klenerman D, Balasubramanian S,
JournalJ Am Chem Soc
PubMed ID20148555
'We have used single-molecule fluorescence microscopy to study the folded state of human telomerase RNA (hTR). Here we show that hTR adopts a new conformation on binding to human telomerase reverse transcriptase (hTERT) and reconstitution of an active ribonucleoprotein complex. Our data are consistent with the formation of an RNA ... More
Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking.
Authorsde Diesbach P, N'Kuli F, Berens C, Sonveaux E, Monsigny M, Roche AC, Courtoy PJ
JournalNucleic Acids Res
PubMed ID11917011
'Having identified an oligonucleotide (ON) receptor in the HepG2 cell line, we have re-examined here the kinetics of ON uptake, subcellular distribution and intracellular localisation in these cells, at concentrations relevant for the study of a receptor-dependent process. Kinetic parameters of ON endocytosis were comparable with those of the receptor-mediated ... More
A biomimetic motility assay provides insight into the mechanism of actin-based motility.
AuthorsWiesner S, Helfer E, Didry D, Ducouret G, Lafuma F, Carlier MF, Pantaloni D
JournalJ Cell Biol
PubMed ID12551957
'Abiomimetic motility assay is used to analyze the mechanism of force production by site-directed polymerization of actin. Polystyrene microspheres, functionalized in a controlled fashion by the N-WASP protein, the ubiquitous activator of Arp2/3 complex, undergo actin-based propulsion in a medium that consists of five pure proteins. We have analyzed the ... More
Site-directed fluorescence studies of a prokaryotic ClC antiporter.
AuthorsBell SP, Curran PK, Choi S, Mindell JA
JournalBiochemistry
PubMed ID16734414
'Channels and transporters of the ClC family serve a variety of physiological functions. Understanding of their gating and transport mechanisms remains incomplete, with disagreement over the extent of protein conformational change involved. Using site-directed fluorescence labeling, we probe ClC-ec1, a prokaryotic ClC, for transport-related structural rearrangements. We specifically label cysteines ... More
Dynamics of equilibrium structural fluctuations of apomyoglobin measured by fluorescence correlation spectroscopy.
AuthorsChen H, Rhoades E, Butler JS, Loh SN, Webb WW
JournalProc Natl Acad Sci U S A
PubMed ID17556539
'The spectra of equilibrium chain conformation fluctuations of apomyoglobin (apoMb) as a function of folding, from the acid-denatured state at pH 2.6 through the stable molten globule state pH approximately 4.1 to the folded state at pH 6.3, are reported, as measured by fluorescence correlation spectroscopy. The conformational fluctuations, which ... More
Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy.
AuthorsSchuler B, Lipman EA, Eaton WA
JournalNature
PubMed ID12384704
'Protein folding is inherently a heterogeneous process because of the very large number of microscopic pathways that connect the myriad unfolded conformations to the unique conformation of the native structure. In a first step towards the long-range goal of describing the distribution of pathways experimentally, Förster resonance energy transfer (FRET) ... More
Multicolor single-molecule FRET to explore protein folding and binding.
AuthorsGambin Y, Deniz AA,
JournalMol Biosyst
PubMed ID20601974
'Proper protein function in cells, tissues and organisms depends critically on correct protein folding or interaction with partners. Over the last decade, single-molecule FRET (smFRET) has emerged as a powerful tool to probe complex distributions, dynamics, pathways and landscapes in protein folding and binding reactions, leveraging its ability to avoid ... More
Influence of hydrophobic mismatch and amino acid composition on the lateral diffusion of transmembrane peptides.
'We investigated the effect of amino acid composition and hydrophobic length of alpha-helical transmembrane peptides and the role of electrostatic interactions on the lateral diffusion of the peptides in lipid membranes. Model peptides of varying length and composition, and either tryptophans or lysines as flanking residues, were synthesized. The peptides ... More
Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient.
AuthorsKrantz BA, Finkelstein A, Collier RJ
JournalJ Mol Biol
PubMed ID16343527
'Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF ... More
On the stiffness of the natural actin filament decorated with alexa fluor tropomyosin.
AuthorsAdami R, Cintio O, Trombetta G, Choquet D, Grazi E
JournalBiophys Chem
PubMed ID12878314
Natural, phalloidin-free, actin filaments were decorated with tropomyosin made fluorescent by reaction with alexa fluor (R) 488 C(5) maleimide. The elastic modulus by stretching of these filaments was then determined and found to span between 38.2 MPa and 61.48 MPa. We tried also to determine the yield strength of the ... More
Reconstitution of DNA segregation driven by assembly of a prokaryotic actin homolog.
Multiple unrelated polymer systems have evolved to partition DNA molecules between daughter cells at division. To better understand polymer-driven DNA segregation, we reconstituted the three-component segregation system of the R1 plasmid from purified components. We found that the ParR/parC complex can construct a simple bipolar spindle by binding the ends ... More
Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.
AuthorsDeRocco V, Anderson T, Piehler J, Erie DA, Weninger K,
JournalBiotechniques
PubMed ID21091445
To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to ... More
Identification of two novel RanGTP-binding proteins belonging to the importin beta superfamily.
AuthorsKutay U, Hartmann E, Treichel N, Calado A, Carmo-Fonseca M, Prehn S, Kraft R, Gorlich D, Bischoff FR
JournalJ Biol Chem
PubMed ID11024021
Nucleo-cytoplasmic transport comprises a large number of distinct pathways, many of which are defined by members of the importin beta superfamily of nuclear transport receptors. These transport receptors all directly interact with RanGTP to modulate the compartment-specific binding of their transport substrates. To identify new members of the importin beta ... More
Protein-protein interactions as a tool for site-specific labeling of proteins.
AuthorsJäger M, Michalet X, Weiss S
JournalProtein Sci
PubMed ID15987886
Probing structures and dynamics within biomolecules using ensemble and single-molecule fluorescence resonance energy transfer requires the conjugation of fluorophores to proteins in a site-specific and thermodynamically nonperturbative fashion. Using single-molecule fluorescence-aided molecular sorting and the chymotrypsin inhibitor 2-subtilisin BPN' complex as an example, we demonstrate that protein-protein interactions can be ... More
Organization of the archaeal MCM complex on DNA and implications for the helicase mechanism.
AuthorsMcGeoch AT, Trakselis MA, Laskey RA, Bell SD
JournalNat Struct Mol Biol
PubMed ID16116441
The homomultimeric archaeal mini-chromosome maintenance (MCM) complex serves as a simple model for the analogous heterohexameric eukaryotic complex. Here we investigate the organization and orientation of the MCM complex of the hyperthermophilic archaeon Sulfolobus solfataricus (Sso) on model DNA substrates. Sso MCM binds as a hexamer and slides on the ... More
Fluorescence of alexa fluor dye tracks protein folding.
Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly ... More
Multiplexed flow cytometry: high-throughput screening of single-chain antibodies.
AuthorsAyriss J, Valero R, Bradbury AR, Pavlik P,
JournalMethods Mol Biol
PubMed ID19252858
The development of high-throughput screening (HTS) technologies has become essential for initial characterization of recombinant antibodies and alternative affinity reagents, selected from large combinatorial libraries. Such binding ligands are routinely selected against a single antigen and screened for desired binding specificities. Recent progress with genome sequencing projects has led to ... More
Simulation of fluorescence anisotropy experiments: probing protein dynamics.
AuthorsSchröder GF, Alexiev U, Grubmüller H,
JournalBiophys J
PubMed ID16169987
Time-resolved fluorescence anisotropy decay experiments on a protein-attached dye can probe local protein dynamics and steric restrictions, but are difficult to interpret at the structural level. Aiming at an atomistic description, we have carried out molecular dynamics simulations of such experiments. Our simulations describe an Alexa488 fluorescent dye maleimide derivative ... More
Specificity of the anaphase-promoting complex: a single-molecule study.
AuthorsLu Y, Wang W, Kirschner MW,
Journal
PubMed ID25859049
Biological processes require specific enzymatic reactions, paradoxically involving short recognition sequences. As an example, cell-cycle timing depends on a sequence of ubiquitylation events mediated by the anaphase-promoting complex (APC) based on short redundant motifs. To understand the origin of specificity, we designed single-molecule fluorescence assays that capture transient ubiquitylation reactions. ... More
Site-specific, thiol-mediated conjugation of fluorescent probes to cysteine-modified diabodies targeting CD20 or HER2.
AuthorsSirk SJ, Olafsen T, Barat B, Bauer KB, Wu AM,
JournalBioconjug Chem
PubMed ID19053310
Small, engineered antibody fragments such as diabodies (50 kDa noncovalent dimers of single-chain Fv fragments) are useful alternatives to their larger antibody counterparts. However, due to their size, they are more susceptible to disruption of their antigen binding sites when modified using random conjugation techniques. Previous work has demonstrated the ... More
Strategy for efficient site-specific FRET-dye labeling of ubiquitin.
AuthorsKao MW, Yang LL, Lin JC, Lim TS, Fann W, Chen RP,
JournalBioconjug Chem
PubMed ID18507427
To study conformational changes within a single protein molecule, sp-FRET (single pair fluorescence resonance energy transfer) is an important technique to provide distance information. However, incorporating donor and acceptor dyes into the same protein molecule is not an easy task. Here, we report a strategy for the efficient double-labeling of ... More
Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs.
Cysteines with reactive thiol groups are attractive tools for site-specific labeling of proteins. Engineering a reactive cysteine residue into proteins with multiple disulfide bonds is often a challenging task as it may interfere with structural and functional properties of the protein. Here we developed a phage display-based biochemical assay, PHESELECTOR ... More
Single-molecule FRET reveals sugar-induced conformational dynamics in LacY.
AuthorsMajumdar DS, Smirnova I, Kasho V, Nir E, Kong X, Weiss S, Kaback HR,
JournalProc Natl Acad Sci U S A
PubMed ID17502603
The N- and C-terminal six-helix bundles of lactose permease (LacY) form a large internal cavity open on the cytoplasmic side and closed on the periplasmic side with a single sugar-binding site at the apex of the cavity near the middle of the molecule. During sugar/H(+) symport, an outward-facing cavity is ... More
High-throughput screening of single-chain antibodies using multiplexed flow cytometry.
AuthorsAyriss J, Woods T, Bradbury A, Pavlik P,
JournalJ Proteome Res
PubMed ID17330944
We have developed a screening method that has the potential to streamline the high-throughput analysis of affinity reagents for proteomic projects. By using multiplexed flow cytometry, we can simultaneously determine the relative expression levels, the identification of nonspecific binding, and the discrimination of fine specificities to generate a complete functional ... More
Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling.
AuthorsYin J, Lin AJ, Buckett PD, Wessling-Resnick M, Golan DE, Walsh CT,
JournalChem Biol
PubMed ID16183024
Fluorescence imaging of living cells depends on an efficient and specific method for labeling the target cellular protein with fluorophores. Here we show that Sfp phosphopantetheinyl transferase-catalyzed protein labeling is suitable for fluorescence imaging of membrane proteins that spend at least part of their membrane trafficking cycle at the cell ... More
Effect of flexibility and cis residues in single-molecule FRET studies of polyproline.
AuthorsBest RB, Merchant KA, Gopich IV, Schuler B, Bax A, Eaton WA,
JournalProc Natl Acad Sci U S A
PubMed ID18029448
Polyproline has recently been used as a spacer between donor and acceptor chromophores to help establish the accuracy of distances determined from single-molecule Förster resonance energy transfer (FRET) measurements. This work showed that the FRET efficiency in water is higher than expected for a rigid spacer and was attributed to ... More
Novel system to achieve one-pot modification of cargo molecules with oligoarginine vectors for intracellular delivery.
AuthorsTakayama K, Tadokoro A, Pujals S, Nakase I, Giralt E, Futaki S,
JournalBioconjug Chem
PubMed ID19161253
There is a growing number of reports showing the usefulness of cell-penetrating peptides (CPPs) including oligoarginines for intracellular delivery of macromolecules. Although the covalent attachment of the CPP segments to the cargo molecules is usually required to ensure effective delivery, conventional methods of conjugation need several manipulation steps that are ... More
Efficient site-specific labeling of proteins via cysteines.
AuthorsKim Y, Ho SO, Gassman NR, Korlann Y, Landorf EV, Collart FR, Weiss S,
JournalBioconjug Chem
PubMed ID18275130
Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using ... More
Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens.
AuthorsStowell SR, Arthur CM, Mehta P, Slanina KA, Blixt O, Leffler H, Smith DF, Cummings RD,
JournalJ Biol Chem
PubMed ID18216021
Human galectins have functionally divergent roles, although most of the members of the galectin family bind weakly to the simple disaccharide lactose (Galbeta1-4Glc). To assess the specificity of galectin-glycan interactions in more detail, we explored the binding of several important galectins (Gal-1, Gal-2, and Gal-3) using a dose-response approach toward ... More
Photobleaching pathways in single-molecule FRET experiments.
AuthorsKong X, Nir E, Hamadani K, Weiss S,
JournalJ Am Chem Soc
PubMed ID17375921
To acquire accurate structural and dynamical information on complex biomolecular machines using single-molecule fluorescence resonance energy transfer (sm-FRET), a large flux of donor and acceptor photons is needed. To achieve such fluxes, one may use higher laser excitation intensity; however, this induces increased rates of photobleaching. Anti-oxidant additives have been ... More
Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors.
Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT ... More
Time-resolved fluorescence anisotropy studies show domain-specific interactions of calmodulin with IQ target sequences of myosin V.
AuthorsBayley P, Martin S, Browne P, Royer C
JournalEur Biophys J
PubMed ID12734700
Single cysteine mutants of calmodulin, Cam(S38C) and Cam(N111C), have been specifically labelled with Alexa488 maleimide to study the effects of calcium on the structural dynamics of calmodulin complexed with IQ3, IQ4 and IQ34 target peptide motifs of mouse unconventional myosin-V. Using phase fluorometry, the time-resolved anisotropy shows well-separated global and ... More
A robust method for production of MHC tetramers with small molecule fluorophores.
AuthorsRamachandiran V, Grigoriev V, Lan L, Ravkov E, Mertens SA, Altman JD
JournalJ Immunol Methods
PubMed ID17187819
Tetramers of major histocompatibility complex molecules (MHC) are now well-established reagents for the detection of antigen-specific T cells by flow cytometry. MHC tetramers are prepared by mixing enzymatically biotinylated MHC molecules with commercial preparations of streptavidin, usually conjugated to a fluorescent phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC). While ... More
Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases.
AuthorsZhou Z, Cironi P, Lin AJ, Xu Y, Hrvatin S, Golan DE, Silver PA, Walsh CT, Yin J
JournalACS Chem Biol
PubMed ID17465518
Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: ... More
Arp2/3 ATP hydrolysis-catalysed branch dissociation is critical for endocytic force generation.
AuthorsMartin AC, Welch MD, Drubin DG
JournalNat Cell Biol
PubMed ID16862144
The Arp2/3 complex, which is crucial for actin-based motility, nucleates actin filaments and organizes them into y-branched networks. The Arp2 subunit has been shown to hydrolyse ATP, but the functional importance of Arp2/3 ATP hydrolysis is not known. Here, we analysed an Arp2 mutant in Saccharomyces cerevisiae that is defective ... More
Noble metals strip peptides from class II MHC proteins.
Class II major histocompatibility complex (MHC) proteins are essential for normal immune system function but also drive many autoimmune responses. They bind peptide antigens in endosomes and present them on the cell surface for recognition by CD4(+) T cells. A small molecule could potentially block an autoimmune response by disrupting ... More
Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions.
AuthorsRusinova E, Tretyachenko-Ladokhina V, Vele OE, Senear DF, Alexander Ross JB
JournalAnal Biochem
PubMed ID12234459
The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and ... More
Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse.
AuthorsAoyama K, Suh SW, Hamby AM, Liu J, Chan WY, Chen Y, Swanson RA
JournalNat Neurosci
PubMed ID16311588
Uptake of the neurotransmitter glutamate is effected primarily by transporters expressed on astrocytes, and downregulation of these transporters leads to seizures and neuronal death. Neurons also express a glutamate transporter, termed excitatory amino acid carrier-1 (EAAC1), but the physiological function of this transporter remains uncertain. Here we report that genetically ... More
Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification.
AuthorsJäger M, Nir E, Weiss S
JournalProtein Sci
PubMed ID16452617
An often limiting factor for studying protein folding by single-molecule fluorescence resonance energy transfer (FRET) is the ability to site-specifically introduce a photostable organic FRET donor (D) and a complementary acceptor (A) into a polypeptide chain. Using alternating-laser excitation and chymotrypsin inhibitor 2 as a model, we show that chemical ... More
An effective and rapid method for functional characterization of immunoadsorbents using POROS beads and flow cytometry.
AuthorsAnderson NL, Haines LR, Pearson TW
JournalJ Proteome Res
PubMed ID15113098
To facilitate the construction, functional characterization, and use of immunoadsorbents, we have developed a flow cytometry method that allows rapid assessment of large numbers of particle-bound antibodies. Protein G derivitized POROS beads were used to bind affinity-purified antibodies specific for synthetic peptides designed from human plasma proteins. The antibodies were ... More
Homologous versus heterologous interactions in the bicomponent staphylococcal gamma-haemolysin pore.
AuthorsViero G, Cunaccia R, Prévost G, Werner S, Monteil H, Keller D, Joubert O, Menestrina G, Dalla Serra M
JournalBiochem J
PubMed ID16241903
Staphylococcal gamma-haemolysin HlgA-HlgB forms a beta-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues - placed near the right and ... More
Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence.
AuthorsSchuler B, Lipman EA, Steinbach PJ, Kumke M, Eaton WA
JournalProc Natl Acad Sci U S A
PubMed ID15699337
To determine whether Forster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those ... More
Voltage-clamp fluorometry in the local environment of the C255-C511 disulfide bridge of the Na+/glucose cotransporter.
AuthorsGagnon DG, Frindel C, Lapointe JY
JournalBiophys J
PubMed ID17208964
We recently identified a functionally important disulfide bridge between C255 and C511 of the human Na+/glucose cotransporter SGLT1. In this study, voltage-clamp fluorometry was used to characterize the fluorescence of four different dyes attached to C255 and C511 under various ionic and substrate/inhibitor conditions. State-dependent fluorescence changes (DeltaF) were observed ... More
Submicron streptavidin patterns for protein assembly.
AuthorsChristman KL, Requa MV, Enriquez-Rios VD, Ward SC, Bradley KA, Turner KL, Maynard HD
JournalLangmuir
PubMed ID16893251
Micron and submicron-scale features of aldehyde functionality were fabricated in polymer films by photolithography to develop a platform for protein immobilization and assembly at a biologically relevant scale. Films containing the pH-reactive polymer poly(3,3'-diethoxypropyl methacrylate) and a photoacid generator (PAG) were patterned from 500 nm to 40 mum by exposure ... More
Correlation spectroscopy of minor fluorescent species: signal purification and distribution analysis.
AuthorsLaurence TA, Kwon Y, Yin E, Hollars CW, Camarero JA, Barsky D
JournalBiophys J
PubMed ID17189306
We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable ... More
On the mechanism of nucleosome assembly by histone chaperone NAP1.
AuthorsMazurkiewicz J, Kepert JF, Rippe K
JournalJ Biol Chem
PubMed ID16531623
The process of mononucleosome assembly mediated by histone chaperone NAP1 was investigated using DNA fragments 146 and 207 bp in length containing the Lytechinus variegatus 5 S rDNA nucleosome positioning sequence. A quantitative description was derived using gel electrophoresis and fluorescent anisotropy data. First, NAP1-bound H3.H4 was released forming a ... More
Real-time enzyme dynamics illustrated with fluorescence spectroscopy of p-hydroxybenzoate hydroxylase.
AuthorsWestphal AH, Matorin A, Hink MA, Borst JW, van Berkel WJ, Visser AJ
JournalJ Biol Chem
PubMed ID16492664
We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair Förster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels ... More
Site-specific dimensions across a highly denatured protein; a single molecule study.
AuthorsMcCarney ER, Werner JH, Bernstein SL, Ruczinski I, Makarov DE, Goodwin PM, Plaxco KW
JournalJ Mol Biol
PubMed ID16095607
Do highly denatured proteins adopt random coil configurations? Here, we address this question by measuring residue-to-residue separations across the denatured FynSH3 domain. Using single-molecule Forster resonance energy transfer techniques, we have collected transfer efficiency probability distributions for dye-labeled, denatured protein. Applying maximum likelihood analysis to the interpretation of these distributions, ... More
In vitro characterization of the interaction between HIV-1 Gag and human lysyl-tRNA synthetase.
AuthorsKovaleski BJ, Kennedy R, Hong MK, Datta SA, Kleiman L, Rein A, Musier-Forsyth K
JournalJ Biol Chem
PubMed ID16702215
Human immunodeficiency virus type 1 (HIV-1) viral assembly is mediated by multiple protein-protein and protein-nucleic acid interactions. Human tRNA(Lys3) is used as the primer for HIV reverse transcription, and HIV Gag and GagPol are required for packaging of the tRNA into virions. Human lysyl-tRNA synthetase (LysRS) is also specifically packaged ... More
Use of a fluorescent maleimide to probe structure-function relationships in stalk segments 4 and 5 of the yeast plasma-membrane H+-ATPase.
AuthorsSlayman CW, Miranda M, Pardo JP, Allen KE
JournalAnn N Y Acad Sci
PubMed ID12763792
In the yeast plasma-membrane H(+)-ATPase and other P-type ATPases, conformational changes are transmitted between cytoplasmic and membrane-embedded domains via a stalk region composed of cytoplasmic extensions of membrane segments 2, 3, 4, and 5. The present study has used a fluorescent maleimide (Alexa-488) to probe Cys residues introduced into stalk ... More
Stalk segment 5 of the yeast plasma membrane H(+)-ATPase. Labeling with a fluorescent maleimide reveals a conformational change during glucose activation.
AuthorsMiranda M, Pardo JP, Allen KE, Slayman CW
JournalJ Biol Chem
PubMed ID12169695
Glucose is well known to cause a rapid, reversible activation of the yeast plasma membrane H(+)-ATPase, very likely mediated by phosphorylation of two or more Ser/Thr residues near the C terminus. Recent mutagenesis studies have shown that glucose-dependent activation can be mimicked constitutively by amino acid substitutions in stalk segment ... More
Protein-protein association in polymer solutions: from dilute to semidilute to concentrated.
AuthorsKozer N, Kuttner YY, Haran G, Schreiber G
JournalBiophys J
PubMed ID17189316
In a typical cell, proteins function in the crowded cytoplasmic environment where 30% of the space is occupied by macromolecules of varying size and nature. This environment may be simulated in vitro using synthetic polymers. Here, we followed the association and diffusion rates of TEM1-beta-lactamase (TEM) and the beta-lactamase inhibitor ... More
Binding of the b-subunit in the ATP synthase from Escherichia coli.
AuthorsDiez M, Börsch M, Zimmermann B, Turina P, Dunn SD, Gräber P
JournalBiochemistry
PubMed ID14744151
The rotary mechanism of ATP synthase requires a strong binding within stator subunits. In this work we studied the binding affinity of the b-subunit to F(1)-ATPase of Escherichia coli. The dimerization of the truncated b-subunit without amino acids 1-33, b(34-156)T62C, was investigated by analytical ultracentrifugation, resulting in a dissociation constant ... More
Arrangement of apolipoprotein A-I in reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer experiments.
AuthorsTricerri MA, Behling Agree AK, Sanchez SA, Bronski J, Jonas A
JournalBiochemistry
PubMed ID11305923
The folding and organization of apolipoprotein A-I (apoA-I) in discoidal, high-density lipoprotein (HDL) complexes with phospholipids are not yet completely resolved. For about 20 years, it was generally accepted that the amphipathic helices of apoA-I lie parallel to the acyl chains of the phospholipids ("picket fence" model). However, based on ... More
Lymphocyte surface thiol levels.
AuthorsSahaf B, Heydari K, Herzenberg LA, Herzenberg LA
JournalProc Natl Acad Sci U S A
PubMed ID12642656
Recent studies have implicated reduced thiols (cysteine -SH) in the function of individual cell surface proteins. Studies presented here demonstrate that the overall level of reduced thiols on cell surface molecules differs on individual subsets of peripheral blood mononuclear cells and that these levels can be manipulated in vitro by ... More
Trafficking of spontaneously endocytosed MHC proteins.
AuthorsChiu I, Davis DM, Strominger JL
JournalProc Natl Acad Sci U S A
PubMed ID10570178
Class I MHC protein primarily presents endogenous antigen but also may present exogenous antigen. Here, we investigated the intracellular pathway of spontaneously internalized class I MHC protein by confocal microscopy. beta(2)-microglobulin (beta(2)m), labeled with a single fluorophore, was exchanged at the surface of B cell transfectants to specifically mark cell ... More
Unfolding energetics of G-alpha-actin: a discrete intermediate can be re-folded to the native state by CCT.
AuthorsAltschuler GM, Klug DR, Willison KR
JournalJ Mol Biol
PubMed ID16171816
Nascent actin requires interactions with the highly conserved and essential eukaryotic chaperonin-containing TCP-1 (CCT) for its correct folding to the native state in vivo. Biochemical and structural analysis of the interaction between actin and CCT has been studied extensively but the underlying energetics and kinetics of the CCT-dependent actin folding ... More
Binding and internalization of fluorescent opioid peptide conjugates in living cells.
AuthorsArttamangkul S, Alvarez-Maubecin V, Thomas G, Williams JT, Grandy DK
JournalMol Pharmacol
PubMed ID11093798
The dynamics of agonist-stimulated opioid receptor internalization and trafficking have been difficult to study in living cells in part because the available probes were inadequate. To overcome this obstacle, six new fluorescent opioid peptides were developed. Dermorphin (DERM), deltorphin (DELT), TIPP, and endomorphin were conjugated to BODIPY TR or Alexa ... More
Synthesis, characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels.
AuthorsPragl B, Koschak A, Trieb M, Obermair G, Kaufmann WA, Gerster U, Blanc E, Hahn C, Prinz H, Schütz G, Darbon H, Gruber HJ, Knaus HG
JournalBioconjug Chem
PubMed ID12009929
Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In ... More
A highly sensitive method for quantification of myosin light chain phosphorylation by capillary isoelectric focusing with laser-induced fluorescence detection.
AuthorsShiraishi M, Loutzenhiser RD, Walsh MP
JournalElectrophoresis
PubMed ID15690429
Activation of myosin II by phosphorylation of the 20 kDa regulatory light chains (LC20) has been implicated in numerous contractile and motile events, e.g., smooth muscle contraction, cytokinesis, and cell migration. The ability to analyze LC20 phosphorylation in minute samples is critical to determine the importance of LC20 phosphorylation in ... More
Conformational substates of calmodulin revealed by single-pair fluorescence resonance energy transfer: influence of solution conditions and oxidative modification.
AuthorsSlaughter BD, Unruh JR, Allen MW, Bieber Urbauer RJ, Johnson CK
JournalBiochemistry
PubMed ID15751946
A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains ... More
Quantitative regulation of the dynamic steady state of actin networks.
AuthorsManhart A, Icheva TA, Guerin C, Klar T, Boujemaa-Paterski R, Thery M, Blanchoin L, Mogilner A
JournalElife
PubMed ID30869077
'Principles of regulation of actin network dimensions are fundamentally important for cell functions, yet remain unclear. Using both in vitro and in silico approaches, we studied the effect of key parameters, such as actin density, ADF/Cofilin concentration and network width on the network length. In the presence of ADF/Cofilin, networks ... More
Mechanisms of PDZ domain scaffold assembly illuminated by use of supported cell membrane sheets.
AuthorsErlendsson S, Thorsen TS, Vauquelin G, Ammendrup-Johnsen I, Wirth V, Martinez KL, Teilum K, Gether U, Madsen KL
JournalElife
PubMed ID30605082
'PDZ domain scaffold proteins are molecular modules orchestrating cellular signalling in space and time. Here, we investigate assembly of PDZ scaffolds using supported cell membrane sheets, a unique experimental setup enabling direct access to the intracellular face of the cell membrane. Our data demonstrate how multivalent protein-protein and protein-lipid interactions ... More
Preparation of stable tau oligomers for cellular and biochemical studies.
AuthorsKarikari TK, Nagel DA, Grainger A, Clarke-Bland C, Hill EJ, Moffat KG
JournalAnal Biochem
PubMed ID30315761
'Increasing evidence suggests that small oligomers are the principal neurotoxic species of tau in Alzheimer''s disease and other tauopathies. However, mechanisms of tau oligomer-mediated neurodegeneration are poorly understood. The transience of oligomers due to aggregation can compromise the stability of oligomers prepared in vitro. Consequently, we sought to develop an ... More
NRF2 negatively regulates primary ciliogenesis and hedgehog signaling.
AuthorsLiu P, Dodson M, Fang D, Chapman E, Zhang DD
JournalPLoS Biol
PubMed ID32053600
'Primary cilia are lost during cancer development, but the mechanism regulating cilia degeneration is not determined. While transcription factor nuclear factor-erythroid 2-like 2 (NRF2) protects cells from oxidative, proteotoxic, and metabolic stress in normal cells, hyperactivation of NRF2 is oncogenic, although the detailed molecular mechanisms by which uncontrolled NRF2 activation ... More
Deamidation disrupts native and transient contacts to weaken the interaction between UBC13 and RING-finger E3 ligases.
AuthorsMohanty P, Agrata R, Habibullah BI, G S A, Das R
JournalElife
PubMed ID31638574
The deamidase OspI from enteric bacteria
The type IV pilus protein PilU functions as a PilT-dependent retraction ATPase.
AuthorsAdams DW, Pereira JM, Stoudmann C, Stutzmann S, Blokesch M
JournalPLoS Genet
PubMed ID31525185
Type IV pili are dynamic cell surface appendages found throughout the bacteria. The ability of these structures to undergo repetitive cycles of extension and retraction underpins their crucial roles in adhesion, motility and natural competence for transformation. In the best-studied systems a dedicated retraction ATPase PilT powers pilus retraction. Curiously, ... More
Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13.
AuthorsLiu Z, Dong X, Yi HW, Yang J, Gong Z, Wang Y, Liu K, Zhang WP, Tang C
JournalCell Discov
PubMed ID30962947
The interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13 ... More
Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling.
AuthorsTischer DK, Weiner OD
JournalElife
PubMed ID30947808
T cells are thought to discriminate self from foreign peptides by converting small differences in ligand binding half-life into large changes in cell signaling. Such a kinetic proofreading model has been difficult to test directly, as existing methods of altering ligand binding half-life also change other potentially important biophysical parameters, ... More
Structural insights into DNA recognition by AimR of the arbitrium communication system in the SPbeta phage.
AuthorsGuan Z, Pei K, Wang J, Cui Y, Zhu X, Su X, Zhou Y, Zhang D, Tang C, Yin P, Liu Z, Zou T
JournalCell Discov
PubMed ID31149347
A newly identified arbitrium communication system regulates the lysis-to-lysogeny decision in a
Distinct Conformations, Aggregation and Cellular Internalization of Different Tau Strains.
AuthorsKarikari TK, Nagel DA, Grainger A, Clarke-Bland C, Crowe J, Hill EJ, Moffat KG
JournalFront Cell Neurosci
PubMed ID31338022
The inter-cellular propagation of tau aggregates in several neurodegenerative diseases involves, in part, recurring cycles of extracellular tau uptake, initiation of endogenous tau aggregation, and extracellular release of at least part of this protein complex. However, human brain tau extracts from diverse tauopathies exhibit variant or "strain" specificity in inducing ... More
GalNAc Conjugation Attenuates the Cytotoxicity of Antisense Oligonucleotide Drugs in Renal Tubular Cells.
AuthorsSewing S, Gubler M, Gérard R, Avignon B, Mueller Y, Braendli-Baiocco A, Odin M, Moisan A
JournalMol Ther Nucleic Acids
PubMed ID30583097
Targeted delivery of antisense oligonucleotide (AON) drugs is a promising strategy to increase their concentration in the desired tissues and cell types while reducing access to other organs. Conjugation of AONs to N-acetylgalactosamine (GalNAc) has been shown to efficiently shift their biodistribution toward the liver via high-affinity binding to the ... More
Annexin B12 Trimer Formation is Governed by a Network of Protein-Protein and Protein-Lipid Interactions.
AuthorsTao M, Isas JM, Langen R
JournalSci Rep
PubMed ID32210350
Membrane protein oligomerization mediates a wide range of biological events including signal transduction, viral infection and membrane curvature induction. However, the relative contributions of protein-protein and protein-membrane interactions to protein oligomerization remain poorly understood. Here, we used the Ca ... More