Shimon Weiss

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Name: Weiss, Shimon

Organization: University of California at Los Angeles , USA

Department: Department of Chemistry and Biochemistry

Title: (PhD)

TOPICS

Analytical Chemistry

Chemicals
References

Design Rules for Membrane-Embedded Voltage-Sensing Nanoparticles

Co-reporter:Kyoungwon Park, Shimon Weiss

Biophysical Journal 2017 Volume 112, Issue 4(Volume 112, Issue 4) pp:

Publication Date(Web):28 February 2017

DOI:10.1016/j.bpj.2016.12.047

Voltage-sensing dyes and voltage-sensing fluorescence proteins have been continually improved and as a result have provided a wealth of insights into neuronal circuits. Further improvements in voltage-sensing dyes and voltage-sensing fluorescence proteins are needed, however, for routine detection of single action potentials across a large number of individual neurons in a large field-of-view of a live mammalian brain. On the other hand, recent experiments and calculations suggest that semiconducting nanoparticles could act as efficient voltage sensors, suitable for the above-mentioned task. This study presents quantum mechanical calculations, including Auger recombination rates, of the quantum-confined Stark effect in membrane-embedded semiconducting nanoparticles, examines their possible utility as membrane voltage sensors, and provide design rules for their structure and composition.

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Co-reporter:Jookyung Lee;Eitan Lerner;Shijia W. Lu;Shuang Wang;SangYoon Chung;Benjamin L. Allen;Logan W. Grimaud;Xavier Michalet;Sergei Borukhov;Antonino Ingargiola;Yazan Alhadid;Dylan J. Taatjes;Terence R. Strick

PNAS 2016 Volume 113 (Issue 43 ) pp:E6562-E6571

Publication Date(Web):2016-10-25

DOI:10.1073/pnas.1605038113

Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP–GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused–backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.

Far-Field Super-resolution Detection of Plasmonic Near-Fields

Co-reporter:Robert Charles Boutelle, Daniel Neuhauser, and Shimon Weiss

ACS Nano 2016 Volume 10(Issue 8) pp:7955

Publication Date(Web):August 8, 2016

DOI:10.1021/acsnano.6b03873

We demonstrate a far-field single molecule super-resolution method that maps plasmonic near-fields. The method is largely invariant to fluorescence quenching (arising from probe proximity to a metal), has reduced point-spread-function distortion compared to fluorescent dyes (arising from strong coupling to nanoscopic metallic features), and has a large dynamic range (of 2 orders of magnitude) allowing mapping of plasmonic field-enhancements regions. The method takes advantage of the sensitivity of quantum dot (QD) stochastic blinking to plasmonic near-fields. The modulation of the blinking characteristics thus provides an indirect measure of the local field strength. Since QD blinking can be monitored in the far-field, the method can measure localized plasmonic near-fields at high throughput using a simple far-field optical setup. Using this method, propagation lengths and penetration depths were mapped-out for silver nanowires of different diameters and for different dielectric environments, with a spatial accuracy of ∼15 nm. We initially use sparse sampling to ensure single molecule localization for accurate characterization of the plasmonic near-field with plans to increase density of emitters in further studies. The measured propagation lengths and penetration depths values agree well with Maxwell finite-difference time-domain calculations and with published literature values. This method offers advantages such as low cost, high throughput, and superresolved mapping of localized plasmonic fields at high sensitivity and fidelity.Keywords: blinking; far-field; near-field; plasmonics; quantum dot; super-resolution

A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement–Förster-Type Resonance Energy Transfer (PIFE-FRET)

Co-reporter:Eitan Lerner, Evelyn Ploetz, Johannes Hohlbein, Thorben Cordes, and Shimon Weiss

The Journal of Physical Chemistry B 2016 Volume 120(Issue 26) pp:6401-6410

Publication Date(Web):May 17, 2016

DOI:10.1021/acs.jpcb.6b03692

Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein–DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging

Co-reporter:Lydia Kisley, Rachel Brunetti, Lawrence J. Tauzin, Bo Shuang, Xiyu Yi, Alec W. Kirkeminde, Daniel A. Higgins, Shimon Weiss, and Christy F. Landes

ACS Nano 2015 Volume 9(Issue 9) pp:9158

Publication Date(Web):August 3, 2015

DOI:10.1021/acsnano.5b03430

Porous materials such as cellular cytosol, hydrogels, and block copolymers have nanoscale features that determine macroscale properties. Characterizing the structure of nanopores is difficult with current techniques due to imaging, sample preparation, and computational challenges. We produce a super-resolution optical image that simultaneously characterizes the nanometer dimensions of and diffusion dynamics within porous structures by correlating stochastic fluctuations from diffusing fluorescent probes in the pores of the sample, dubbed here as “fluorescence correlation spectroscopy super-resolution optical fluctuation imaging” or “fcsSOFI”. Simulations demonstrate that structural features and diffusion properties can be accurately obtained at sub-diffraction-limited resolution. We apply our technique to image agarose hydrogels and aqueous lyotropic liquid crystal gels. The heterogeneous pore resolution is improved by up to a factor of 2, and diffusion coefficients are accurately obtained through our method compared to diffraction-limited fluorescence imaging and single-particle tracking. Moreover, fcsSOFI allows for rapid and high-throughput characterization of porous materials. fcsSOFI could be applied to soft porous environments such hydrogels, polymers, and membranes in addition to hard materials such as zeolites and mesoporous silica.Keywords: correlation; diffusion; fluorescence microscopy; hydrogel; liquid crystal; super-resolution;

Toward Single-Molecule Optical Mapping of the Epigenome

Co-reporter:Michal Levy-Sakin, Assaf Grunwald, Soohong Kim, Natalie R. Gassman, Anna Gottfried, Josh Antelman, Younggyu Kim, Sam O. Ho, Robin Samuel, Xavier Michalet, Ron R. Lin, Thomas Dertinger, Andrew S. Kim, Sangyoon Chung, Ryan A. Colyer, Elmar Weinhold, Shimon Weiss, and Yuval Ebenstein

ACS Nano 2014 Volume 8(Issue 1) pp:14

Publication Date(Web):December 11, 2013

DOI:10.1021/nn4050694

The past decade has seen an explosive growth in the utilization of single-molecule techniques for the study of complex systems. The ability to resolve phenomena otherwise masked by ensemble averaging has made these approaches especially attractive for the study of biological systems, where stochastic events lead to inherent inhomogeneity at the population level. The complex composition of the genome has made it an ideal system to study at the single-molecule level, and methods aimed at resolving genetic information from long, individual, genomic DNA molecules have been in use for the last 30 years. These methods, and particularly optical-based mapping of DNA, have been instrumental in highlighting genomic variation and contributed significantly to the assembly of many genomes including the human genome. Nanotechnology and nanoscopy have been a strong driving force for advancing genomic mapping approaches, allowing both better manipulation of DNA on the nanoscale and enhanced optical resolving power for analysis of genomic information. During the past few years, these developments have been adopted also for epigenetic studies. The common principle for these studies is the use of advanced optical microscopy for the detection of fluorescently labeled epigenetic marks on long, extended DNA molecules. Here we will discuss recent single-molecule studies for the mapping of chromatin composition and epigenetic DNA modifications, such as DNA methylation.Keywords: chromatin; epigenetics; fluorescence microscopy; methylation; nanoscopy; nanotechnology; optical mapping; single molecule

Processing of microRNA primary transcripts requires heme in mammalian cells

Co-reporter:Sara H. Weitz;Ming Gong;Ian Barr;Feng Guo

PNAS 2014 Volume 111 (Issue 5 ) pp:1861-1866

Publication Date(Web):2014-02-04

DOI:10.1073/pnas.1309915111

DiGeorge syndrome critical region gene 8 (DGCR8) is the RNA-binding partner protein of the nuclease Drosha. DGCR8 and Drosha recognize and cleave primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical microRNAs (miRNAs) in animals. We previously reported that human, frog, and starfish DGCR8 bind heme when expressed in Escherichia coli and that Fe(III) heme activates apoDGCR8 in reconstituted pri-miRNA processing assays. However, the physiological relevance of heme in miRNA maturation has not been clear. Here, we present a live-cell pri-miRNA processing assay that produces robust signals and faithfully indicates DGCR8 and Drosha activities. We demonstrate that all known heme-binding–deficient DGCR8 mutants are defective in pri-miRNA processing in HeLa cells. DGCR8 contains a previously uncharacterized heme-binding motif, “IPCL,” that is also required for its activity. Heme availability and biosynthesis in HeLa cells positively affect pri-miRNA processing and production of mature miRNA. These results establish an essential function for heme in pri-miRNA processing in mammalian cells. Our study suggests that abnormal heme biosynthesis and degradation may contribute to diseases via miRNA-mediated gene regulation networks.

Resolving the spatial relationship between intracellular components by dual color super resolution optical fluctuations imaging (SOFI)

Co-reporter:Maria Elena Gallina;Jianmin Xu;Thomas Dertinger;Adva Aizer

Optical Nanoscopy 2013 Volume 2( Issue 1) pp:

Publication Date(Web):2013 December

DOI:10.1186/2192-2853-2-2

Multi-color super-resolution (SR) imaging microscopy techniques can resolve ultrastructural relationships between- and provide co-localization information of- different proteins inside the cell or even within organelles at a higher resolution than afforded by conventional diffraction-limited imaging. While still very challenging, important SR colocalization results have been reported in recent years using STED, PALM and STORM techniques.In this work, we demonstrate dual-color Super Resolution Optical Fluctuations Imaging (SOFI) using a standard far-field fluorescence microscope and different color blinking quantum dots. We define the spatial relationship between hDcp1a, a processing body (P-body, PB) protein, and the tubulin cytoskeletal network. Our finding could open up new perspectives on the role of the cytoskeleton in PB formation and assembly. Further insights into PB internal organization are also reported and discussed.Our results demonstrate the suitability and facile use of multi-color SOFI for the investigation of intracellular ultrastructures.

Labeling Cytosolic Targets in Live Cells with Blinking Probes

Co-reporter:Jianmin Xu, Jason Chang, Qi Yan, Thomas Dertinger, Marcel P. Bruchez, and Shimon Weiss

The Journal of Physical Chemistry Letters 2013 Volume 4(Issue 13) pp:2138-2146

Publication Date(Web):June 13, 2013

DOI:10.1021/jz400682m

With the advent of superresolution imaging methods, fast dynamic imaging of biological processes in live cells remains a challenge. A subset of these methods requires the cellular targets to be labeled with spontaneously blinking probes. The delivery and specific targeting of cytosolic targets and the control of the probes’ blinking properties are reviewed for three types of blinking probes: quantum dots, synthetic dyes, and fluorescent proteins.

Slow Unfolded-State Structuring in Acyl-CoA Binding Protein Folding Revealed by Simulation and Experiment

Co-reporter:Vincent A. Voelz ; Marcus Jäger ; Shuhuai Yao □; Yujie Chen ; Li Zhu △; Steven A. Waldauer ; Gregory R. Bowman ; Mark Friedrichs ▽; Olgica Bakajin ▲; Lisa J. Lapidus ; Shimon Weiss ▼;Vijay S. Pande ■

Journal of the American Chemical Society 2012 Volume 134(Issue 30) pp:12565-12577

Publication Date(Web):July 2, 2012

DOI:10.1021/ja302528z

Protein folding is a fundamental process in biology, key to understanding many human diseases. Experimentally, proteins often appear to fold via simple two- or three-state mechanisms involving mainly native-state interactions, yet recent network models built from atomistic simulations of small proteins suggest the existence of many possible metastable states and folding pathways. We reconcile these two pictures in a combined experimental and simulation study of acyl-coenzyme A binding protein (ACBP), a two-state folder (folding time ∼10 ms) exhibiting residual unfolded-state structure, and a putative early folding intermediate. Using single-molecule FRET in conjunction with side-chain mutagenesis, we first demonstrate that the denatured state of ACBP at near-zero denaturant is unusually compact and enriched in long-range structure that can be perturbed by discrete hydrophobic core mutations. We then employ ultrafast laminar-flow mixing experiments to study the folding kinetics of ACBP on the microsecond time scale. These studies, along with Trp-Cys quenching measurements of unfolded-state dynamics, suggest that unfolded-state structure forms on a surprisingly slow (∼100 μs) time scale, and that sequence mutations strikingly perturb both time-resolved and equilibrium smFRET measurements in a similar way. A Markov state model (MSM) of the ACBP folding reaction, constructed from over 30 ms of molecular dynamics trajectory data, predicts a complex network of metastable stables, residual unfolded-state structure, and kinetics consistent with experiment but no well-defined intermediate preceding the main folding barrier. Taken together, these experimental and simulation results suggest that the previously characterized fast kinetic phase is not due to formation of a barrier-limited intermediate but rather to a more heterogeneous and slow acquisition of unfolded-state structure.

Stable, Compact, Bright Biofunctional Quantum Dots with Improved Peptide Coating

Co-reporter:Jianmin Xu, Piotr Ruchala, Yuval Ebenstain, J. Jack Li, and Shimon Weiss

The Journal of Physical Chemistry B 2012 Volume 116(Issue 36) pp:11370-11378

Publication Date(Web):August 17, 2012

DOI:10.1021/jp306453y

We developed a new peptide, natural phytochelatin (PC), which tightly binds to CdSe/ZnS quantum dots’ (QDs) surfaces and renders them water-soluble. Coating QDs with this flexible and all-hydrophilic peptide offers high colloidal stability, adds only 0.8–0.9 nm to the radius of the particles (as compared to their original inorganic radius), preserves very high quantum yield (QY) in water, and affords facile bioconjugation with various functional groups. We demonstrate specific targeting (with minimal nonspecific binding) of such fluorescein-conjugated QDs to ScFv-fused mouse prion protein expressed in live N2A cells. We also demonstrated homogeneous in vivo biodistribution with no significant toxicity in live zebrafish.

Single Molecule Quantum-Confined Stark Effect Measurements of Semiconductor Nanoparticles at Room Temperature

Co-reporter:KyoungWon Park, Zvicka Deutsch, J. Jack Li, Dan Oron, and Shimon Weiss

ACS Nano 2012 Volume 6(Issue 11) pp:10013

Publication Date(Web):October 17, 2012

DOI:10.1021/nn303719m

We measured the quantum-confined Stark effect (QCSE) of several types of fluorescent colloidal semiconductor quantum dots and nanorods at the single molecule level at room temperature. These measurements demonstrate the possible utility of these nanoparticles for local electric field (voltage) sensing on the nanoscale. Here we show that charge separation across one (or more) heterostructure interface(s) with type-II band alignment (and the associated induced dipole) is crucial for an enhanced QCSE. To further gain insight into the experimental results, we numerically solved the Schrödinger and Poisson equations under self-consistent field approximation, including dielectric inhomogeneities. Both calculations and experiments suggest that the degree of initial charge separation (and the associated exciton binding energy) determines the magnitude of the QCSE in these structures.Keywords: nanorod; quantum dot; quantum-confined Stark effect; type-I band alignment; type-II band alignment; voltage sensing; wave function engineering

Back Cover: Enzymatically Incorporated Genomic Tags for Optical Mapping of DNA-Binding Proteins (Angew. Chem. Int. Ed. 15/2012)

Co-reporter:Soohong Kim;Anna Gottfried;Ron R. Lin;Dr. Thomas Dertinger;Andrew S. Kim;Sangyoon Chung;Dr. Ryan A. Colyer;Dr. Elmar Weinhold; Shimon Weiss;Dr. Yuval Ebenstein

Angewandte Chemie International Edition 2012 Volume 51( Issue 15) pp:

Publication Date(Web):

DOI:10.1002/anie.201200628

Enzymatically Incorporated Genomic Tags for Optical Mapping of DNA-Binding Proteins

Co-reporter:Soohong Kim;Anna Gottfried;Ron R. Lin;Dr. Thomas Dertinger;Andrew S. Kim;Sangyoon Chung;Dr. Ryan A. Colyer;Dr. Elmar Weinhold; Shimon Weiss;Dr. Yuval Ebenstein

Angewandte Chemie International Edition 2012 Volume 51( Issue 15) pp:3578-3581

Publication Date(Web):

DOI:10.1002/anie.201107714

SOFI-based 3D superresolution sectioning with a widefield microscope

Co-reporter:Thomas Dertinger;Jianmin Xu;Omeed Foroutan Naini;Robert Vogel

Optical Nanoscopy 2012 Volume 1( Issue 1) pp:

Publication Date(Web):2012 December

DOI:10.1186/2192-2853-1-2

Fluorescence-based biological imaging has been revolutionized by the recent introduction of superresolution microscopy methods. 3D superresolution microscopy, however, remains a challenge as its implementation by existing superresolution methods is non-trivial.Here we demonstrate a facile and straightforward 3D superresolution imaging and sectioning of the cytoskeletal network of a fixed cell using superresolution optical fluctuation imaging (SOFI) performed on a conventional lamp-based widefield microscope.SOFI’s inherent sectioning capability effectively transforms a conventional widefield microscope into a superresolution ‘confocal widefield’ microscope.

Aromatic Aldehyde and Hydrazine Activated Peptide Coated Quantum Dots for Easy Bioconjugation and Live Cell Imaging

Co-reporter:Gopal Iyer, Fabien Pinaud, Jianmin Xu, Yuval Ebenstein, Jack Li, Jessica Chang, Maxime Dahan, and Shimon Weiss

Bioconjugate Chemistry 2011 Volume 22(Issue 6) pp:1006

Publication Date(Web):May 10, 2011

DOI:10.1021/bc100593m

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody–QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.

Superresolution Optical Fluctuation Imaging with Organic Dyes

Co-reporter:Dr. Thomas Dertinger;Dr. Mike Heilemann;Robert Vogel; Markus Sauer; Shimon Weiss

Angewandte Chemie 2010 Volume 122( Issue 49) pp:9631-9633

Publication Date(Web):

DOI:10.1002/ange.201004138

Superresolution Optical Fluctuation Imaging with Organic Dyes

Co-reporter:Dr. Thomas Dertinger;Dr. Mike Heilemann;Robert Vogel; Markus Sauer; Shimon Weiss

Angewandte Chemie International Edition 2010 Volume 49( Issue 49) pp:9441-9443

Publication Date(Web):

DOI:10.1002/anie.201004138

Lighting Up Individual DNA Binding Proteins with Quantum Dots

Co-reporter:Yuval Ebenstein, Natalie Gassman, Soohong Kim, Josh Antelman, Younggyu Kim, Sam Ho, Robin Samuel, Xavier Michalet and Shimon Weiss

Nano Letters 2009 Volume 9(Issue 4) pp:1598-1603

Publication Date(Web):March 16, 2009

DOI:10.1021/nl803820b

The ability to determine the precise loci and occupancy of DNA-binding proteins is instrumental to our understanding of cellular processes like gene expression and regulation. We propose a single-molecule approach for the direct visualization of proteins bound to their template DNA. Fluorescent quantum dots (QD) are used to label proteins bound to DNA, allowing multicolor, nanometer-resolution localization. Protein−DNA complexes are linearly extended and imaged to determine the precise location of the protein binding sites. The method is demonstrated by detecting individual QD-labeled T7-RNA polymerases on the T7 bacteriophage genome. This work demonstrates the potential of this approach to precisely read protein binding position or, alternatively, “write” such information on extended DNA with QDs via sequence-specific molecular recognition.

Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled proteinDNA complexes

Co-reporter:Yuval Ebenstein;Natalie Gassman;Soohong Kim

Journal of Molecular Recognition 2009 Volume 22( Issue 5) pp:397-402

Publication Date(Web):

DOI:10.1002/jmr.956

Abstract

Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA. Copyright © 2009 John Wiley & Sons, Ltd.

Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)

Co-reporter:T. Dertinger;R. Colyer;G. Iyer;S. Weiss;J. Enderlein

PNAS 2009 Volume 106 (Issue 52 ) pp:22287-22292

Publication Date(Web):2009-12-29

DOI:10.1073/pnas.0907866106

Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.

Suppression of Quantum Dot Blinking in DTT-Doped Polymer Films

Co-reporter:Josh Antelman, Yuval Ebenstein, Thomas Dertinger, Xavier Michalet and Shimon Weiss

The Journal of Physical Chemistry C 2009 Volume 113(Issue 27) pp:11541-11545

Publication Date(Web):March 31, 2009

DOI:10.1021/jp811078e

In this report we evaluate the emission properties of single quantum dots embedded in a thin, thiol-containing polymer film. We report the suppression of quantum dot blinking leading to a continuous photon flux from both organic and water soluble quantum dots and demonstrate their application as robust fluorescent point sources for ultrahigh resolution localization. In addition, we apply the polymer coating to cell samples immunostained with antibody conjugated QDs and show that fluorescence intensity from the polymer embedded cells shows no sign of degradation after 67 h of continuous excitation. The reported thin polymer film coating may prove advantageous for immuno-cyto/histo-chemistry as well as for the fabrication of quantum dot containing devices requiring a reliable and stable photon source (including a single photon source) or stable charge characteristics while maintaining intimate contact between the quantum dot and the surrounding matrix.

High Affinity scFv−Hapten Pair as a Tool for Quantum Dot Labeling and Tracking of Single Proteins in Live Cells

Co-reporter:Gopal Iyer, Xavier Michalet, Yun-Pei Chang, Fabien F. Pinaud, Stephanie E. Matyas, Gregory Payne and Shimon Weiss

Nano Letters 2008 Volume 8(Issue 12) pp:4618-4623

Publication Date(Web):November 20, 2008

DOI:10.1021/nl8032284

We describe a general approach to label cell surface proteins using quantum dots (QD) for single-molecule tracking. QDs coated with small-hapten modified peptides are targeted to cell surface fusion proteins containing the corresponding single-chain fragment antibody (scFv). The approach is illustrated with the small hapten fluorescein (FL) and a high-affinity anti-FL scFv fused to two different proteins in yeast and murine neuronal cell line N2a.

Efficient Site-Specific Labeling of Proteins via Cysteines

Co-reporter:Younggyu Kim, Sam O. Ho, Natalie R. Gassman, You Korlann, Elizabeth V. Landorf, Frank R. Collart and Shimon Weiss

Bioconjugate Chemistry 2008 Volume 19(Issue 3) pp:786

Publication Date(Web):February 15, 2008

DOI:10.1021/bc7002499

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 thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein’s amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein’s function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70−90%, and specificities are better than ∼95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

Co-reporter:Devdoot S. Majumdar;Xiangxu Kong;Irina Smirnova;Eyal Nir;H. Ronald Kaback;Vladimir Kasho

PNAS 2007 Volume 104 (Issue 31 ) pp:12640-12645

Publication Date(Web):2007-07-31

DOI:10.1073/pnas.0700969104

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 thought to open with closing of the inward-facing cavity so that the sugar-binding site is alternately accessible to either face of the membrane. In this communication, single-molecule fluorescence (Förster) resonance energy transfer is used to test this model with wild-type LacY and a conformationally restricted mutant. Pairs of Cys residues at the ends of two helices on the cytoplasmic or periplasmic sides of wild-type LacY and the mutant were labeled with appropriate donor and acceptor fluorophores, single-molecule fluorescence resonance energy transfer was determined in the absence and presence of sugar, and distance changes were calculated. With wild-type LacY, binding of a galactopyranoside, but not a glucopyranoside, results in a decrease in distance on the cytoplasmic side and an increase in distance on the periplasmic side. In contrast, with the mutant, a more pronounced decrease in distance and in distance distribution is observed on the cytoplasmic side, but there is no change on the periplasmic side. The results are consistent with the alternating access model and indicate that the defect in the mutant is due to impaired ligand-induced flexibility on the periplasmic side.

Initial Transcription by RNA Polymerase Proceeds Through a DNA-Scrunching Mechanism

Co-reporter:Achillefs N. Kapanidis;Emmanuel Margeat;Sam On Ho;Ekaterine Kortkhonjia;Richard H. Ebright

Science 2006 Vol 314(5802) pp:1144-1147

Publication Date(Web):17 Nov 2006

DOI:10.1126/science.1131399

Abstract

Using fluorescence resonance energy transfer to monitor distances within single molecules of abortively initiating transcription initiation complexes, we show that initial transcription proceeds through a “scrunching” mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA into itself and past its active center. We show further that putative alternative mechanisms for RNAP active-center translocation in initial transcription, involving “transient excursions” of RNAP relative to DNA or “inchworming” of RNAP relative to DNA, do not occur. The results support a model in which a stressed intermediate, with DNA-unwinding stress and DNA-compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of interactions between RNAP and promoter DNA and between RNAP and initiation factors during promoter escape.

Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics

Co-reporter:X. Michalet;F. F. Pinaud;L. A. Bentolila;J. M. Tsay;S. Doose;J. J. Li;G. Sundaresan;A. M. Wu;S. S. Gambhir;S. Weiss

Science 2005 Vol 307(5709) pp:538-544

Publication Date(Web):28 Jan 2005

DOI:10.1126/science.1104274

Abstract

Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.

Wavefunction engineering: From quantum wells to near-infrared type-II colloidal quantum dots synthesized by layer-by-layer colloidal epitaxy

Co-reporter:J. Jack Li, James M. Tsay, Xavier Michalet, Shimon Weiss

Chemical Physics 2005 Volume 318(1–2) pp:82-90

Publication Date(Web):15 November 2005

DOI:10.1016/j.chemphys.2005.04.029

Abstract

We review the concept and the evolution of bandgap and wavefunction engineering, the seminal contributions of Dr. Chemla to the understanding of the rich phenomena displayed in epitaxially grown quantum confined systems, and demonstrate the application of these concepts to the colloidal synthesis of high quality type-II CdTe/CdSe quantum dots using successive ion layer adsorption and reaction chemistry. Transmission electron microscopy reveals that CdTe/CdSe can be synthesized layer by layer, yielding particles of narrow size distribution. Photoluminescence emission and excitation spectra reveal discrete type-II transitions, which correspond to energy lower than the type-I bandgap. The increase in the spatial separation between photoexcited electrons and holes as a function of successive addition of CdSe monolayers was monitored by photoluminescence lifetime measurements. Systematic increase in lifetimes demonstrates the high level of wavefunction engineering and control in these systems.

Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins

Co-reporter:Xiangxu Kong;Ted A. Laurence;Marcus Jäger

PNAS 2005 Volume 102 (Issue 48 ) pp:17348-17353

Publication Date(Web):2005-11-29

DOI:10.1073/pnas.0508584102

We study protein and nucleic acid structure and dynamics using single-molecule FRET and alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on single-molecule signals detected within 100 μs to 1 ms. Distance distributions caused by fluctuations faster than 100 μs are studied within these subpopulations by using time-correlated single-photon counting. Measured distance distributions for dsDNA can be accounted for by considering fluorophore linkers and fluorophore rotational diffusion, except that we find smaller fluctuations for internally labeled dsDNA than DNA with one of the fluorophores positioned at a terminal site. We find that the electrostatic portion of the persistence length of short single-stranded poly(dT) varies approximately as the ionic strength (I) to the –1/2 power (I –1/2), and that the average contribution to the contour length per base is 0.40–0.45 nm. We study unfolded chymotrypsin inhibitor 2 (CI2) and unfolded acyl-CoA binding protein (ACBP) even under conditions where folded and unfolded subpopulations coexist (contributions from folded proteins are excluded by using alternating-laser excitation). At lower denaturant concentrations, unfolded CI2 and ACBP are more compact and display larger fluctuations than at higher denaturant concentrations where only unfolded proteins are present. The experimentally measured fluctuations are larger than the fluctuations predicted from a Gaussian chain model or a wormlike chain model. We propose that the larger fluctuations may indicate transient residual structure in the unfolded state.

Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules

Co-reporter:Achillefs N. Kapanidis;Nam Ki Lee;Ted A. Laurence;Sören Doose;Emmanuel Margeat

PNAS 2004 Volume 101 (Issue 24 ) pp:8936-8941

Publication Date(Web):2004-06-15

DOI:10.1073/pnas.0401690101

We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of biomolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor–acceptor pairs for Förster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics.

Nonequilibrium Single Molecule Protein Folding in a Coaxial Mixer

Co-reporter:Kambiz M. Hamadani, Shimon Weiss

Biophysical Journal (1 July 2008) Volume 95(Issue 1) pp:

Publication Date(Web):1 July 2008

DOI:10.1529/biophysj.107.127431

We have developed a continuous-flow mixing device suitable for monitoring bioconformational reactions at the single-molecule level with a response time of ∼10 ms under single-molecule flow conditions. Its coaxial geometry allows three-dimensional hydrodynamic focusing of sample fluids to diffraction-limited dimensions where diffusional mixing is rapid and efficient. The capillary-based design enables rapid in-lab construction of mixers without the need for expensive lithography-based microfabrication facilities. In-line filtering of sample fluids using granulated silica particles virtually eliminates clogging and extends the lifetime of each device to many months. In this article, to determine both the distance-to-time transfer function and the instrument response function of the device we characterize its fluid flow and mixing properties using both fluorescence cross-correlation spectroscopy velocimetry and finite element fluid dynamics simulations. We then apply the mixer to single molecule FRET protein folding studies of Chymotrypsin Inhibitor protein 2. By transiently populating the unfolded state of Chymotrypsin Inhibitor Protein 2 (CI2) under nonequilibrium in vitro refolding conditions, we spatially and temporally resolve the denaturant-dependent nonspecific collapse of the unfolded state from the barrier-limited folding transition of CI2. Our results are consistent with previous CI2 mixing results that found evidence for a heterogeneous unfolded state consisting of cis- and trans-proline conformers.

Three-Color Alternating-Laser Excitation of Single Molecules: Monitoring Multiple Interactions and Distances

Co-reporter:Nam Ki Lee, Achillefs N. Kapanidis, Hye Ran Koh, You Korlann, Sam On Ho, Younggyu Kim, Natalie Gassman, Seong Keun Kim, Shimon Weiss

Biophysical Journal (1 January 2007) Volume 92(Issue 1) pp:

Publication Date(Web):1 January 2007

DOI:10.1529/biophysj.106.093211

We introduce three-color alternating-laser excitation (3c-ALEX), a fluorescence resonance energy transfer (FRET) method that measures up to three intramolecular distances and complex interaction stoichiometries of single molecules in solution. This tool extends substantially the capabilities of two-color ALEX, which employs two alternating lasers to study molecular interactions (through probe stoichiometry S) and intramolecular distances (through FRET efficiency E), and sorts fluorescent molecules in multi-dimensional probe-stoichiometry and FRET-efficiency histograms. Probe-stoichiometry histograms allowed analytical sorting, identification, and selection of diffusing species; selected molecules were subsequently represented in FRET-efficiency histograms, generating up to three intramolecular distances. Using triply labeled DNAs, we established that 3c-ALEX enables 1), FRET-independent analysis of three-component interactions; 2), observation and sorting of singly, doubly, and triply labeled molecules simultaneously present in solution; 3), measurements of three intramolecular distances within single molecules from a single measurement; and 4), dissection of conformational heterogeneity with improved resolution compared to conventional single-molecule FRET. We also used 3c-ALEX to study large biomolecules such as RNA polymerase-DNA transcription complexes, and monitor the downstream translocation of RNA polymerase on DNA from two perspectives within the complex. This study paves the way for advanced single-molecule analysis of complex mixtures and biomolecular machinery.