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
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
Spatial dynamics of receptor-mediated endocytic trafficking in budding yeast revealed by using fluorescent alpha-factor derivatives.
AuthorsToshima JY, Toshima J, Kaksonen M, Martin AC, King DS, Drubin DG
JournalProc Natl Acad Sci U S A
PubMed ID16574772
'Much progress defining the order and timing of endocytic internalization events has come as a result of real-time, live-cell fluorescence microscopy. Although the availability of numerous endocytic mutants makes yeast an especially valuable organism for functional analysis of endocytic dynamics, a serious limitation has been the lack of a fluorescent ... More
Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface.
AuthorsImamoto Y, Kataoka M, Tokunaga F, Palczewski K
JournalBiochemistry
PubMed ID11106502
'A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that ... More
Self-assembling light-harvesting systems from synthetically modified tobacco mosaic virus coat proteins.
AuthorsMiller RA, Presley AD, Francis MB
JournalJ Am Chem Soc
PubMed ID17319656
'A new protein-based approach has been developed for the construction of light-harvesting systems through self-assembly. The building blocks were prepared by attaching fluorescent chromophores to cysteine residues introduced on tobacco mosaic virus coat protein monomers. When placed under the appropriate buffer conditions, these conjugates could be assembled into stacks of ... More
A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer.
AuthorsBrustad EM, Lemke EA, Schultz PG, Deniz AA,
JournalJ Am Chem Soc
PubMed ID19108697
'A general strategy for the site-specific dual-labeling of proteins for single-molecule fluorescence resonance energy transfer is presented. A genetically encoded unnatural ketone amino acid was labeled with a hydroxylamine-containing fluorophore with high yield (>95%) and specificity. This methodology was used to construct dual-labeled T4 lysozyme variants, allowing the study of ... More
Requirements for recruitment of a G protein-coupled receptor to clathrin-coated pits in budding yeast.
'Endocytic internalization of G protein-coupled receptors (GPCRs) plays a critical role in down-regulation of GPCR signaling. The yeast mating pheromone receptor Ste2p has been used as a model to investigate mechanisms of signal transduction, modification, and endocytic internalization of GPCRs. We previously used a fluorescently labeled mating pheromone derivative to ... More
Homo- and heterooligomeric SNARE complexes studied by site-directed spin labeling.
AuthorsMargittai M, Fasshauer D, Pabst S, Jahn R, Langen R
JournalJ Biol Chem
PubMed ID11278719
'SNARE (soluble NSF acceptor protein receptor) proteins are thought to mediate membrane fusion by assembling into heterooligomeric complexes that connect the fusing membranes and initiate the fusion reaction. Here we used site-directed spin labeling to map conformational changes that occur upon homo- and heterooligomeric complex formation of neuronal SNARE proteins. ... 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
The human natural killer cell immune synapse.
AuthorsDavis DM, Chiu I, Fassett M, Cohen GB, Mandelboim O, Strominger JL
JournalProc Natl Acad Sci U S A
PubMed ID10611338
'Inhibitory killer Ig-like receptors (KIR) at the surface of natural killer (NK) cells induced clustering of HLA-C at the contacting surface of target cells. In this manner, inhibitory immune synapses were formed as human NK cells surveyed target cells. At target/NK cell synapses, HLA-C/KIR distributed into rings around central patches ... More
A processive single-headed motor: kinesin superfamily protein KIF1A.
AuthorsOkada Y, Hirokawa N
JournalScience
PubMed ID10024239
A single kinesin molecule can move "processively" along a microtubule for more than 1 micrometer before detaching from it. The prevailing explanation for this processive movement is the "walking model," which envisions that each of two motor domains (heads) of the kinesin molecule binds coordinately to the microtubule. This implies ... 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
Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes.
AuthorsMarras SA, Kramer FR, Tyagi S
JournalNucleic Acids Res
PubMed ID12409481
An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy transfer for a large number of combinations of commonly used fluorophores and quenchers. We ... More
Engineering a terbium-binding site into an integral membrane protein for luminescence energy transfer.
AuthorsVázquez-Ibar JL, Weinglass AB, Kaback HR
JournalProc Natl Acad Sci U S A
PubMed ID11891311
Luminescence resonance energy transfer with a lanthanide like Tb(3+) as donor is a useful technique for estimating intra- and intermolecular distances in macromolecules. However, the technique usually requires the use of a bulky chelator with a flexible linker attached to a Cys residue to bind Tb(3+) and, for intramolecular studies, ... 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
Selective cell uptake of modified Tat peptide-fluorophore conjugates in rat retina in ex vivo and in vivo models.
AuthorsBarnett EM, Elangovan B, Bullok KE, Piwnica-Worms D
JournalInvest Ophthalmol Vis Sci
PubMed ID16723475
PURPOSE: To determine the pattern of retinal uptake of modified Tat peptide-fluorophore conjugates in the rat after ex vivo application and intravitreal injection. METHODS: Modified Tat peptide (RKKRRORRRGC) was conjugated at the C terminus to Alexa Fluor 594 to enable visualization of uptake. In the ex vivo model, posterior segments ... More
A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity.
AuthorsBosch M, Benito A, Ribó M, Puig T, Beaumelle B, Vilanova M
JournalBiochemistry
PubMed ID14979713
Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all ... More
Mapping ATP-dependent activation at a sigma54 promoter.
AuthorsLeach RN, Gell C, Wigneshweraraj S, Buck M, Smith A, Stockley PG
JournalJ Biol Chem
PubMed ID16926155
The sigma(54) promoter specificity factor is distinct from other bacterial RNA polymerase (RNAP) sigma factors in that it forms a transcriptionally silent closed complex upon promoter binding. Transcriptional activation occurs through a nucleotide-dependent isomerization of sigma(54), mediated via its interactions with an enhancer-binding activator protein that utilizes the energy released ... 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
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
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