Membrane protein stoichiometry determined from the step-wise photobleaching of dye-labelled subunits.
AuthorsDas SK, Darshi M, Cheley S, Wallace MI, Bayley H
JournalChembiochem
PubMed ID17503420
Reversible transition between the surface trimer and membrane-inserted monomer of annexin 12.
AuthorsLadokhin AS, Haigler HT
JournalBiochemistry
PubMed ID15736950
'Under mildly acidic conditions, annexin 12 (ANX) inserts into lipid membranes to form a transbilayer pore [Langen, R., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 14060]. In this study, we have addressed the question of the oligomeric state of ANX in this transbilayer conformation by means of Forster-type ... More
Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex.
AuthorsBowen ME, Weninger K, Ernst J, Chu S, Brunger AT
JournalBiophys J
PubMed ID15821166
'The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in ... More
Chaperoning of insertion of membrane proteins into lipid bilayers by hemifluorinated surfactants: application to diphtheria toxin.
'Hemifluorinated compounds, such as HF-TAC, make up a novel class of nondetergent surfactants designed to keep membrane proteins soluble under nondissociating conditions [Breyton, C., et al. (2004) FEBS Lett. 564, 312]. Because fluorinated and hydrogenated chains do not mix well, supramicellar concentrations of these surfactants can coexist with intact lipid ... 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
An improved cell-penetrating, caspase-activatable, near-infrared fluorescent peptide for apoptosis imaging.
AuthorsMaxwell D, Chang Q, Zhang X, Barnett EM, Piwnica-Worms D,
JournalBioconjug Chem
PubMed ID19331388
'Apoptosis is required for normal cellular homeostasis, and deregulation of the apoptotic process is implicated in various diseases. Previously, we developed a cell-penetrating near-infrared fluorescence (NIRF) probe based on an activatable strategy to detect apoptosis-associated caspase activity in vivo. This probe consisted of a cell-penetrating Tat peptide conjugated to an ... More
Single-molecule tracking of mRNA exiting from RNA polymerase II.
AuthorsAndrecka J, Lewis R, Brückner F, Lehmann E, Cramer P, Michaelis J,
JournalProc Natl Acad Sci U S A
PubMed ID18162559
'Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5'' end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active ... More
The conserved core domains of annexins A1, A2, A5, and B12 can be divided into two groups with different Ca2+-dependent membrane-binding properties.
AuthorsPatel DR, Isas JM, Ladokhin AS, Jao CC, Kim YE, Kirsch T, Langen R, Haigler HT
JournalBiochemistry
PubMed ID15723527
'The hallmark of the annexin super family of proteins is Ca(2+)-dependent binding to phospholipid bilayers, a property that resides in the conserved core domain of these proteins. Despite the structural similarity between the core domains, studies reported herein showed that annexins A1, A2, A5, and B12 could be divided into ... 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
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
Evidence of an intermediate and parallel pathways in protein unfolding from single-molecule fluorescence.
AuthorsOrte A, Craggs TD, White SS, Jackson SE, Klenerman D,
JournalJ Am Chem Soc
PubMed ID18507381
Determining how proteins fold into their native structures is a subject of great importance, since ultimately it will allow protein structure and function to be predicted from primary sequence data. In addition, there is now a clear link between protein unfolding and misfolding events and many disease states. However, since ... 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
Optimizing methods to recover absolute FRET efficiency from immobilized single molecules.
Microscopy-based fluorescence resonance energy transfer (FRET) experiments measure donor and acceptor intensities by isolating these signals with a series of optical elements. Because this filtering discards portions of the spectrum, the observed FRET efficiency is dependent on the set of filters in use. Similarly, observed FRET efficiency is also affected ... 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
Comparison of the C2A domain of synaptotagmin-I and annexin-V as probes for detecting cell death.
AuthorsAlam IS, Neves AA, Witney TH, Boren J, Brindle KM,
JournalBioconjug Chem
PubMed ID20402461
The induction of apoptosis is frequently accompanied by the exposure of phosphatidylserine (PS) on the cell surface, which has been detected using radionuclide and fluorescently labeled derivatives of the PS-binding protein, Annexin V. The fluorescently labeled protein has been used extensively in vitro as a diagnostic reagent for detecting cell ... 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
Resolving cadherin interactions and binding cooperativity at the single-molecule level.
AuthorsZhang Y, Sivasankar S, Nelson WJ, Chu S,
JournalProc Natl Acad Sci U S A
PubMed ID19114658
The cadherin family of Ca(2+)-dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions between cadherins that are at the core of cell-cell adhesion are a matter of considerable debate. A widely-accepted model is that cadherins adhere in 3 ... 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
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
Nuclear import time and transport efficiency depend on importin beta concentration.
AuthorsYang W, Musser SM
JournalJ Cell Biol
PubMed ID16982803
Although many components and reaction steps necessary for bidirectional transport across the nuclear envelope (NE) have been characterized, the mechanism and control of cargo migration through nuclear pore complexes (NPCs) remain poorly understood. Single-molecule fluorescence microscopy was used to track the movement of cargos before, during, and after their interactions ... More
Prion recognition elements govern nucleation, strain specificity and species barriers.
AuthorsTessier PM, Lindquist S
JournalNature
PubMed ID17495929
Prions are proteins that can switch to self-perpetuating, infectious conformations. The abilities of prions to replicate, form structurally distinct strains, and establish and overcome transmission barriers between species are poorly understood. We exploit surface-bound peptides to overcome complexities of investigating such problems in solution. For the yeast prion Sup35, we ... More
Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations.
AuthorsWeninger K, Bowen ME, Chu S, Brunger AT
JournalProc Natl Acad Sci U S A
PubMed ID14657376
Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, ... More
Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis.
AuthorsWheelock AM, Morin D, Bartosiewicz M, Buckpitt AR
JournalProteomics
PubMed ID16429456
2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) ... More
Clustering and coupled gating modulate the activity in KcsA, a potassium channel model.
Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into planar lipid bilayers, ... More
Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions.
AuthorsKuzmenkina EV, Heyes CD, Nienhaus GU
JournalProc Natl Acad Sci U S A
PubMed ID16221762
Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of ... More
Dynamic instability of clathrin assembly provides proofreading control for endocytosis.
AuthorsChen Y, Yong J, Martínez-Sánchez A, Yang Y, Wu Y, De Camilli P, Fernández-Busnadiego R, Wu M
JournalJ Cell Biol
PubMed ID31451612
'Clathrin-mediated endocytosis depends on the formation of functional clathrin-coated pits that recruit cargos and mediate the uptake of those cargos into the cell. However, it remains unclear whether the cargos in the growing clathrin-coated pits are actively monitored by the coat assembly machinery. Using a cell-free reconstitution system, we report ... More
Influenza A virus surface proteins are organized to help penetrate host mucus.
AuthorsVahey MD, Fletcher DA
JournalElife
PubMed ID31084711
'Influenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance ... More
Reconceptualizing Fluorescence Correlation Spectroscopy for Monitoring and Analyzing Periodically Passing Objects.
AuthorsZamir E, Frey C, Weiss M, Antona S, Frohnmayer JP, Janiesch JW, Platzman I, Spatz JP
JournalAnal Chem
PubMed ID28985462
'Fluorescence correlation spectroscopy (FCS) is a sensitive technique commonly applied for studying the dynamics of nanoscale-labeled objects in solution. Current analysis of FCS data is largely based on the assumption that the labeled objects are stochastically displaced due to Brownian motion. However, this assumption is often invalid for microscale objects, ... More
Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET.
AuthorsChung HS, Meng F, Kim JY, McHale K, Gopich IV, Louis JM
JournalProc Natl Acad Sci U S A
PubMed ID28760960
'We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency-lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at ... More
Binding of NF?B Appears to Twist the Ankyrin Repeat Domain of I?Ba.
AuthorsTrelle MB, Ramsey KM, Lee TC, Zheng W, Lamboy J, Wolynes PG, Deniz A, Komives EA
JournalBiophys J
PubMed ID26910425
Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of I?Ba, the temporally regulated inhibitor of canonical NF?B signaling. Under native conditions, most of the I?Ba molecules showed stable, high FRET signals consistent with distances between the fluorophores ... More
Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis.
AuthorsChandrasekaran R, Kenworthy AK, Lacy DB
JournalPLoS Pathog
PubMed ID27942025
Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. ... More
Protein Delivery into Plant Cells: Toward
AuthorsCedeño C, Pauwels K, Tompa P
JournalFront Plant Sci
PubMed ID28469623
Understanding the biologically relevant structural and functional behavior of proteins inside living plant cells is only possible through the combination of structural biology and cell biology. The state-of-the-art structural biology techniques are typically applied to molecules that are isolated from their native context. Although most experimental conditions can be easily ... More
Interdependence between EGFR and Phosphatases Spatially Established by Vesicular Dynamics Generates a Growth Factor Sensing and Responding Network.
AuthorsStanoev A, Mhamane A, Schuermann KC, Grecco HE, Stallaert W, Baumdick M, Brüggemann Y, Joshi MS, Roda-Navarro P, Fengler S, Stockert R, Roßmannek L, Luig J, Koseska A, Bastiaens PIH
JournalCell Syst
PubMed ID30145116
The proto-oncogenic epidermal growth factor receptor (EGFR) is a tyrosine kinase whose sensitivity to growth factors and signal duration determines cellular behavior. We resolve how EGFR's response to epidermal growth factor (EGF) originates from dynamically established recursive interactions with spatially organized protein tyrosine phosphatases (PTPs). Reciprocal genetic PTP perturbations enabled ... More
Reconstitution of a 26-Subunit Human Kinetochore Reveals Cooperative Microtubule Binding by CENP-OPQUR and NDC80.
AuthorsPesenti ME, Prumbaum D, Auckland P, Smith CM, Faesen AC, Petrovic A, Erent M, Maffini S, Pentakota S, Weir JR, Lin YC, Raunser S, McAinsh AD, Musacchio A
JournalMol Cell
PubMed ID30174292
The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we ... More
Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage.
AuthorsWendorff TJ, Berger JM
JournalElife
PubMed ID29595473
Type II topoisomerases manage DNA supercoiling and aid chromosome segregation using a complex, ATP-dependent duplex strand passage mechanism. Type IIB topoisomerases and their homologs support both archaeal/plant viability and meiotic recombination. Topo VI, a prototypical type IIB topoisomerase, comprises two Top6A and two Top6B protomers; how these subunits cooperate to ... More
Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules.
AuthorsScarborough EA, Davis TN, Asbury CL
JournalElife
PubMed ID31045495
Regulation of the outer kinetochore complex Ndc80 is essential to ensure correct kinetochore-microtubule attachments during mitosis. Here, we present a novel mechanism of regulation that is intrinsic to its structure; tight bending of the Ndc80 complex inhibits its microtubule binding. Using single molecule Förster resonance energy transfer (FRET), we show ... More
Functional analysis of the secondary HIV-1 capsid binding site in the host protein cyclophilin A.
AuthorsPeng W, Shi J, Márquez CL, Lau D, Walsh J, Faysal KMR, Byeon CH, Byeon IL, Aiken C, Böcking T
JournalRetrovirology
PubMed ID30947724
Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme's active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular ... More
PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins.
AuthorsDuan Y, Du A, Gu J, Duan G, Wang C, Gui X, Ma Z, Qian B, Deng X, Zhang K, Sun L, Tian K, Zhang Y, Jiang H, Liu C, Fang Y
JournalCell Res
PubMed ID30728452
Mutations in RNA-binding proteins (RBPs) localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregation, which is a pathological hallmark of various neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. In ... More
Actin assembly ruptures the nuclear envelope by prying the lamina away from nuclear pores and nuclear membranes in starfish oocytes.
AuthorsWesolowska N, Avilov I, Machado P, Geiss C, Kondo H, Mori M, Lenart P
JournalElife
PubMed ID31989921
The nucleus of oocytes (germinal vesicle) is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) that are stockpiled for embryonic development. We showed that breakdown of this specialized NE is mediated by an Arp2/3-nucleated F-actin 'shell' in starfish oocytes, in contrast to microtubule-driven ... More