Dynamic fluorescent imaging of human immunodeficiency virus type 1 gag in live cells by biarsenical labeling.
AuthorsRudner L, Nydegger S, Coren LV, Nagashima K, Thali M, Ott DE,
JournalJ Virol
PubMed ID15767407
'Human immunodeficiency virus type 1 (HIV-1) Gag is the primary structural protein of the virus and is sufficient for particle formation. We utilized the recently developed biarsenical-labeling method to dynamically observe HIV-1 Gag within live cells by adding a tetracysteine tag (C-C-P-G-C-C) to the C terminus of Gag in both ... More
Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase.
AuthorsZhang C, Cai Z, Kim YC, Kumar R, Yuan F, Shi PY, Kao C, Luo G,
JournalJ Virol
PubMed ID15994762
'Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other ... More
Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.
AuthorsIgnatova Z, Gierasch LM,
JournalProc Natl Acad Sci U S A
PubMed ID14701904
'In vivo fluorescent labeling of an expressed protein has enabled the observation of its stability and aggregation directly in bacterial cells. Mammalian cellular retinoic acid-binding protein I (CRABP I) was mutated to incorporate in a surface-exposed omega loop the sequence Cys-Cys-Gly-Pro-Cys-Cys, which binds specifically to a biarsenical fluorescein dye (FlAsH). ... More
Multicolor and electron microscopic imaging of connexin trafficking.
Authors Gaietta Guido; Deerinck Thomas J; Adams Stephen R; Bouwer James; Tour Oded; Laird Dale W; Sosinsky Gina E; Tsien Roger Y; Ellisman Mark H;
JournalScience
PubMed ID11964472
'Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in ... More
Visualization of mRNA translation in living cells.
The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of beta-actin mRNA. Constructs coding for ... More
The Salmonella enterica serovar typhimurium-encoded type III secretion systems can translocate Chlamydia trachomatis proteins into the cytosol of host cells.
AuthorsHo TD, Starnbach MN,
JournalInfect Immun
PubMed ID15664932
Chlamydia trachomatis is an obligate, intracellular pathogen that is a major cause of preventable blindness and infertility worldwide. Although the published genome sequence suggests that C. trachomatis encodes a type III secretion system, the lack of genetic tools for studying Chlamydia has hindered the examination of this potentially important class ... More
AuthorsTour O, Meijer RM, Zacharias DA, Adams SR, Tsien RY,
JournalNat Biotechnol
PubMed ID14625562
Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject ... More
Association of ebola virus matrix protein VP40 with microtubules.
AuthorsRuthel G, Demmin GL, Kallstrom G, Javid MP, Badie SS, Will AB, Nelle T, Schokman R, Nguyen TL, Carra JH, Bavari S, Aman MJ,
JournalJ Virol
PubMed ID15795257
Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or ... More
Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics.
AuthorsSosinsky GE, Gaietta GM, Hand G, Deerinck TJ, Han A, Mackey M, Adams SR, Bouwer J, Tsien RY, Ellisman MH,
JournalCell Commun Adhes
PubMed ID14681013
Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine ... More
A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells.
Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein-coupled receptor (GPCR) activation ... More