Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer.
AuthorsPosson DJ, Ge P, Miller C, Bezanilla F, Selvin PR
JournalNature
PubMed ID16094368
'Voltage-gated ion channels open and close in response to voltage changes across electrically excitable cell membranes. Voltage-gated potassium (Kv) channels are homotetramers with each subunit constructed from six transmembrane segments, S1-S6 (ref. 2). The voltage-sensing domain (segments S1-S4) contains charged arginine residues on S4 that move across the membrane electric ... More
Quantitation of microparticles released from coated-platelets.
AuthorsDale GL, Remenyi G, Friese P,
JournalJ Thromb Haemost
PubMed ID16102115
'Dual agonist stimulation of platelets with thrombin and convulxin results in generation of coated-platelets, a sub-population of cells known formerly as COAT-platelets (collagen and thrombin). Coated-platelets retain several procoagulant proteins on their surface and express phosphatidylserine (PS). In this report, we utilize a new methodology to demonstrate that coated-platelets also ... More
Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging.
AuthorsYang J, Chen H, Vlahov IR, Cheng JX, Low PS
JournalProc Natl Acad Sci U S A
PubMed ID16950881
'Despite functional evidence for disulfide bond-reducing activity in endosomal compartments, the mechanistic details pertaining to such process (e.g., kinetics and sites of disulfide reduction) remain largely controversial. To address these questions directly, we have synthesized a previously uncharacterized fluorescent folate conjugate, folate-(BODIPY FL)-SS-rhodamine (folate-FRET), that changes fluorescence from red to ... More
An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly.
AuthorsStray SJ, Johnson JM, Kopek BG, Zlotnick A
JournalNat Biotechnol
PubMed ID16474383
'Virus assembly has not been routinely targeted in the development of antiviral drugs, in part because of the lack of tractable methods for screening in vitro. We have developed an in vitro assay of hepatitis B virus (HBV) capsid assembly, based on fluorescence quenching of dye-labeled capsid protein, for testing ... More
The achondroplasia mutation does not alter the dimerization energetics of the fibroblast growth factor receptor 3 transmembrane domain.
AuthorsYou M, Li E, Hristova K
JournalBiochemistry
PubMed ID16634636
The Gly380 --> Arg mutation in the TM domain of fibroblast growth factor receptor 3 (FGFR3) of the RTK family is linked to achondroplasia, the most common form of human dwarfism. The molecular mechanism of pathology induction is under debate, and two different mechanisms have been proposed to contribute to ... More
Lactadherin and clearance of platelet-derived microvesicles.
AuthorsDasgupta SK, Abdel-Monem H, Niravath P, Le A, Bellera RV, Langlois K, Nagata S, Rumbaut RE, Thiagarajan P,
JournalBlood
PubMed ID19023116
The transbilayer movement of phosphatidylserine from the inner to the outer leaflet of the membrane bilayer during platelet activation is associated with the release of procoagulant phosphatidylserine-rich small membrane vesicles called platelet-derived microvesicles. We tested the effect of lactadherin, which promotes the phagocytosis of phosphatidylserine-expressing lymphocytes and red blood cells, ... More
Detecting changes in the thiol redox state of proteins following a decrease in oxygen concentration using a dual labeling technique.
AuthorsLui JK, Lipscombe R, Arthur PG,
JournalJ Proteome Res
PubMed ID19894774
Cells are routinely exposed to hyperoxic conditions when cultured in the presence of 95% air and 5% carbon dioxide. Hyperoxic conditions can increase the generation of reactive oxygen species and cause oxidative stress. Oxidative stress has been proposed to cause cells in culture to behave differently from cells in vivo. ... More
Role of peroxiredoxin 1 and peroxiredoxin 4 in protection of respiratory syncytial virus-induced cysteinyl oxidation of nuclear cytoskeletal proteins.
AuthorsJamaluddin M, Wiktorowicz JE, Soman KV, Boldogh I, Forbus JD, Spratt H, Garofalo RP, Brasier AR,
JournalJ Virol
PubMed ID20610706
The respiratory epithelium plays a central role in innate immunity by secreting networks of inflammatory mediators in response to respiratory syncytial virus (RSV) infection. Previous proteomic studies focusing on the host cellular response to RSV indicated the existence of a nuclear heat shock response and cytoplasmic depletion of antioxidant proteins ... More
Conformations of the signal recognition particle protein Ffh from Escherichia coli as determined by FRET.
AuthorsBuskiewicz I, Peske F, Wieden HJ, Gryczynski I, Rodnina MV, Wintermeyer W,
JournalJ Mol Biol
PubMed ID16005894
The signal recognition particle (SRP) initiates the co-translational targeting of proteins to the plasma membrane in bacteria by binding to the N-terminal signal sequence emerging from the translating ribosome. SRP in Escherichia coli is composed of one protein, Ffh, and 4.5S RNA. In the present work, we probe the structure ... More
Fluorescence applications in molecular neurobiology.
AuthorsTaraska JW, Zagotta WN,
JournalNeuron
PubMed ID20434995
Macromolecules drive the complex behavior of neurons. For example, channels and transporters control the movements of ions across membranes, SNAREs direct the fusion of vesicles at the synapse, and motors move cargo throughout the cell. Understanding the structure, assembly, and conformational movements of these and other neuronal proteins is essential ... More
Manipulation of carrier proteins in antibiotic biosynthesis.
Engineering biosynthetic pathways into suitable host organisms has become an attractive venue for the design, evaluation, and production of small molecule therapeutics. Polyketide (PK) and nonribosomal peptide (NRP) synthases have been of particular interest due to their modular structure, yet routine cloning and expression of these enzymes remains challenging. Here ... More
Comprehensive two-dimensional separation system by coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection.
AuthorsMao Y, Zhang X
JournalElectrophoresis
PubMed ID14518059
A comprehensive two-dimensional (2-D) separation system, coupling capillary reverse-phase liquid chromatography (cRPLC) to capillary isoelectric focusing (CIEF), is described for protein and peptide mapping. cRPLC, the first dimension, provided high-resolution separations for salt-free proteins. CIEF, the second dimension with an orthogonal mechanism to cRPLC afforded excellent resolution capability for proteins ... More
Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.
AuthorsBoonacker E, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID11724895
Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative ... More
Inhibition of human ether à go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-termini.
AuthorsZiechner U, Schönherr R, Born AK, Gavrilova-Ruch O, Glaser RW, Malesevic M, Küllertz G, Heinemann SH
JournalFEBS J
PubMed ID16478480
Human ether à go-go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca2+/calmodulin (CaM), presumably binding to the C-terminal domain of the channel subunits. Deletion of the cytosolic N-terminal domain resulted in complete abolition of Ca2+/CaM sensitivity suggesting the existence of further CaM binding ... 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
Chemoenzymatic approaches for streamlined detection of active site modifications on thiotemplate assembly lines using mass spectrometry.
AuthorsMcLoughlin SM, Mazur MT, Miller LM, Yin J, Liu F, Walsh CT, Kelleher NL
JournalBiochemistry
PubMed ID16245932
For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, ... More