Intracellular calmodulin availability accessed with two-photon cross-correlation.
AuthorsKim SA, Heinze KG, Waxham MN, Schwille P
JournalProc Natl Acad Sci U S A
PubMed ID14695888
'The availability and interactions of signaling proteins are tightly regulated in time and space to produce specific and localized effects. For calmodulin (CaM), a key transducer of intracellular Ca(2+) signaling, binding to its variety of targets initiates signaling cascades and regulates its subcellular localization, thereby making it unavailable for subsequent ... More
Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots.
AuthorsBonasio R, Carman CV, Kim E, Sage PT, Love KR, Mempel TR, Springer TA, von Andrian UH
JournalProc Natl Acad Sci U S A
PubMed ID17785425
'The real-time observation of protein dynamics in living cells and organisms is of fundamental importance for understanding biological processes. Most approaches to labeling proteins exploit noncovalent interactions, unsuitable to long-term studies, or genetic fusion to naturally occurring fluorescent proteins that often have unsatisfactory optical properties. Here we used the fungal ... More
Real-time determination of picomolar free Cu(II) in seawater using a fluorescence-based fiber optic biosensor.
We report real-time, in situ determination of free copper ion at picomolar levels in seawater using a fluorescence-based fiber optic biosensor. The sensor transducer is a protein molecule, site-specifically labeled with a fluorophore that is attached to the distal end of an optical fiber, which binds free Cu(II) with high ... More
An eight residue fragment of an acyl carrier protein suffices for post-translational introduction of fluorescent pantetheinyl arms in protein modification in vitro and in vivo.
AuthorsZhou Z, Koglin A, Wang Y, McMahon AP, Walsh CT,
JournalJ Am Chem Soc
PubMed ID18593165
Genetically encoded tags for tracking a given protein continue to be of great interest in a multitude of in vitro and in vivo contexts. Acyl carrier proteins, both free-standing and as embedded 80-100 residue domains, contain a specific serine side chain that undergoes post-translational pantetheinylation from CoASH as donor substrate. ... More
Modulation of cell surface protein free thiols: a potential novel mechanism of action of the sesquiterpene lactone parthenolide.
AuthorsSkalska J, Brookes PS, Nadtochiy SM, Hilchey SP, Jordan CT, Guzman ML, Maggirwar SB, Briehl MM, Bernstein SH,
JournalPLoS One
PubMed ID19956548
BACKGROUND: There has been much interest in targeting intracellular redox pathways as a therapeutic approach for cancer. Given recent data to suggest that the redox status of extracellular protein thiol groups (i.e. exofacial thiols) effects cell behavior, we hypothesized that redox active anti-cancer agents would modulate exofacial protein thiols. METHODOLOGY/PRINCIPAL ... More
Dual-color fluorescence-burst analysis to probe protein efflux through the mechanosensitive channel MscL.
Authorsvan den Bogaart G, Krasnikov V, Poolman B
JournalBiophys J
PubMed ID17142294
The mechanosensitive channel protein of large conductance, MscL, from Escherichia coli has been implicated in protein efflux, but the passage of proteins through the channel has never been demonstrated. We used dual-color fluorescence-burst analysis to evaluate the efflux of fluorescent labeled compounds through MscL. The method correlates the fluctuations in ... 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
Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles.
AuthorsDe Keyzer J, Van Der Does C, Driessen AJ
JournalJ Biol Chem
PubMed ID12226104
Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, ... More
T cell surface redox levels determine T cell reactivity and arthritis susceptibility.
AuthorsGelderman KA, Hultqvist M, Holmberg J, Olofsson P, Holmdahl R
JournalProc Natl Acad Sci U S A
PubMed ID16908843
Rats and mice with a lower capacity to produce reactive oxygen species (ROS) because of allelic polymorphisms in the Ncf1 gene (which encodes neutrophil cytosolic factor 1) are more susceptible to develop severe arthritis. These data suggest that ROS are involved in regulating the immune response. We now show that ... More
On the mechanism of nucleosome assembly by histone chaperone NAP1.
AuthorsMazurkiewicz J, Kepert JF, Rippe K
JournalJ Biol Chem
PubMed ID16531623
The process of mononucleosome assembly mediated by histone chaperone NAP1 was investigated using DNA fragments 146 and 207 bp in length containing the Lytechinus variegatus 5 S rDNA nucleosome positioning sequence. A quantitative description was derived using gel electrophoresis and fluorescent anisotropy data. First, NAP1-bound H3.H4 was released forming a ... 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