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
Functional Effects of Nemaline Myopathy Mutations on Human Skeletal {alpha}-Actin.
AuthorsMiller BM, Trybus KM,
JournalJ Biol Chem
PubMed ID18477565
Mutations in human alpha-skeletal actin have been implicated in causing congenital nemaline myopathy, a disease characterized histopathologically by nemaline bodies in skeletal muscle and manifested in the patient as skeletal muscle weakness. Here we investigate the functional effects of three severe nemaline myopathy mutations (V43F, A138P, and R183G) in human ... More
Pathway of actin filament branch formation by Arp2/3 complex.
AuthorsBeltzner CC, Pollard TD,
JournalJ Biol Chem
PubMed ID18165685
A spectroscopic assay using pyrene-labeled fission yeast Arp2/3 complex revealed that the complex binds to and dissociates from actin filaments extremely slowly with or without the nucleation-promoting factor fission yeast Wsp1-VCA. Wsp1-VCA binds both Arp2/3 complex and actin monomers with high affinity. These two ligands have only modest impacts on ... More
Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions.
AuthorsRusinova E, Tretyachenko-Ladokhina V, Vele OE, Senear DF, Alexander Ross JB
JournalAnal Biochem
PubMed ID12234459
The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and ... More
Protein disulfide isomerase and sulfhydryl-dependent pathways in platelet activation.
AuthorsEssex DW, Li M, Miller A, Feinman RD
JournalBiochemistry
PubMed ID11352743
The inhibition of blood platelet aggregation and secretion was studied using covalent thiol reagents, maleimides, or mercuribenzoates, or using inhibitors of protein disulfide isomerase (PDI), bacitracin or antibodies to PDI. As expected, both types of inhibitors were effective against stimulation by normal physiologic stimuli. On the other hand, when stimulation ... More
Streptokinase binds to human plasmin with high affinity, perturbs the plasmin active site, and induces expression of a substrate recognition exosite for plasminogen.
AuthorsBoxrud PD, Fay WP, Bock PE
JournalJ Biol Chem
PubMed ID10799544
Binding of streptokinase (SK) to plasminogen (Pg) conformationally activates the zymogen and converts both Pg and plasmin (Pm) into specific Pg activators. The interaction of SK with Pm and its relationship to the mechanism of Pg activation were evaluated in equilibrium binding studies with active site-labeled fluorescent Pm derivatives and ... More
The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin.
AuthorsKovar DR, Kuhn JR, Tichy AL, Pollard TD,
JournalJ Cell Biol
PubMed ID12796476
Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 ... More
Linkage between fructose 1,6-bisphosphate binding and the dimer-tetramer equilibrium of Escherichia coli glycerol kinase: critical behavior arising from change of ligand stoichiometry.
AuthorsYu P, Pettigrew DW
JournalBiochemistry
PubMed ID12680779
Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) is inhibited allosterically by fructose 1,6-bisphosphate (FBP), and this inhibition is a primary mechanism by which glucose controls glycerol utilization in vivo. Earlier work indicates that glycerol kinase displays a dimer-tetramer equilibrium in solution, FBP shifts the equilibrium toward the tetramer, and ... More