'As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. ... More
Spectral fluctuation of a single fluorophore conjugated to a protein molecule.
AuthorsWazawa T, Ishii Y, Funatsu T, Yanagida T
JournalBiophys J
PubMed ID10692340
'We have measured the fluorescence spectra of a single fluorophore attached to a single protein molecule in aqueous solution using a total internal reflection fluorescence microscope. The most reactive cysteine residue of myosin subfragment-1 (S1) was labeled with tetramethylrhodamine. The spectral shift induced by a change in solvent from aqueous ... More
Differential association of CD45 isoforms with CD4 and CD8 regulates the actions of specific pools of p56lck tyrosine kinase in T cell antigen receptor signal transduction.
AuthorsDornan S, Sebestyen Z, Gamble J, Nagy P, Bodnar A, Alldridge L, Doe S, Holmes N, Goff LK, Beverley P, Szollosi J, Alexander DR
JournalJ Biol Chem
PubMed ID11694532
'An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4(+)CD8(+) HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was ... More
A fluorescence polarization assay for screening inhibitors against the ribonuclease H activity of HIV-1 reverse transcriptase.
AuthorsNakayama GR, Bingham P, Tan D, Maegley KA
JournalAnal Biochem
PubMed ID16527235
A fluorescence polarization (FP) microplate assay suitable for screening compounds against the ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase has been developed. This homogeneous assay uses a hybrid 18-mer DNA/RNA duplex substrate composed of an RNA oligonucleotide labeled with 6-carboxytetramethyl rhodamine at the 3' end that is annealed ... More
Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy.
AuthorsMoore KJ, Turconi S, Ashman S, Ruediger M, Haupts U, Emerick V, Pope AJ
JournalJ Biomol Screen
PubMed ID10838431
Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule ... More
Quantitative hybridization kinetics of DNA probes to RNA in solution followed by diffusional fluorescence correlation analysis.
AuthorsSchwille P, Oehlenschläger F, Walter NG
JournalBiochemistry
PubMed ID8756483
Binding kinetics in solution of six N,N,N',N'-tetramethyl-5-carboxyrhodamine-labeled oligodeoxyribonucleotide probes to a 101mer target RNA comprising the primer binding site for HIV-1 reverse transcriptase were characterized using fluorescence correlation spectroscopy (FCS). FCS allows a sensitive, non-radioactive real time observation of hybridization of probes to the RNA target in the buffer of ... More
Kinetic mechanism of the inhibition of cathepsin G by alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor.
AuthorsDuranton J, Adam C, Bieth JG
JournalBiochemistry
PubMed ID9698370
Uncontrolled proteolysis due to cathepsin G (cat G) may cause severe pathological disorders. Cat G is inhibited by alpha 1-antichymotrypsin (ACT) and alpha 1-proteinase inhibitor (alpha 1PI), two members of the serpin superfamily of proteins. To see whether these two inhibitors play a physiological proteolysis-preventing function, we have made a ... More