Properties of spin and fluorescent labels at a receptor-ligand interface.
AuthorsOwenius R, Osterlund M, Lindgren M, Svensson M, Olsen OH, Persson E, Freskgård PO, Carlsson U
JournalBiophys J
PubMed ID10512843
'Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently ... More
Interaction of Escherichia coli hemolysin with biological membranes. A study using cysteine scanning mutagenesis.
AuthorsSchindel C, Zitzer A, Schulte B, Gerhards A, Stanley P, Hughes C, Koronakis V, Bhakdi S, Palmer M
JournalEur J Biochem
PubMed ID11168421
'Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca2(+) to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were ... More
Crystal structures of the yeast prion Ure2p functional region in complex with glutathione and related compounds.
AuthorsBousset L, Belrhali H, Melki R, Morera S
JournalBiochemistry
PubMed ID11695904
'The [URE3] phenotype in yeast Saccharomyces cerevisiae is due to an altered prion form of Ure2p, a protein involved in nitrogen catabolism. To understand possible conformational changes at the origin of prion propagation, we previously solved the crystal structure of the Ure2p functional region [Bousset et al. (2001) Structure 9, ... More
Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids.
'Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No ... More
Dynamics of trigger factor interaction with translating ribosomes.
AuthorsRutkowska A, Mayer MP, Hoffmann A, Merz F, Zachmann-Brand B, Schaffitzel C, Ban N, Deuerling E, Bukau B,
JournalJ Biol Chem
PubMed ID18045873
'In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of ... More
Fluorescent peptide probes for high-throughput measurement of protein phosphatases.
AuthorsNoble JE, Ganju P, Cass AE
JournalAnal Chem
PubMed ID12720338
'A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore ... More
Engineering a polarity-sensitive biosensor for time-lapse imaging of apoptotic processes and degeneration.
AuthorsKim YE, Chen J, Chan JR, Langen R,
JournalNat Methods
PubMed ID19966809
Apoptosis is of central importance to many areas of biological research, but there is a lack of methods that permit continuous monitoring of apoptosis or cell viability in a nontoxic and noninvasive manner. Here we report the development of a tool applicable to live-cell imaging that facilitates the visualization of ... More
Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells.
AuthorsSugimoto K, Nishida M, Otsuka M, Makino K, Ohkubo K, Mori Y, Morii T
JournalChem Biol
PubMed ID15123242
Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP(3)) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) delta. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine ... More
Real-time observation of trigger factor function on translating ribosomes.
AuthorsKaiser CM, Chang HC, Agashe VR, Lakshmipathy SK, Etchells SA, Hayer-Hartl M, Hartl FU, Barral JM
JournalNature
PubMed ID17051157
The contribution of co-translational chaperone functions to protein folding is poorly understood. Ribosome-associated trigger factor (TF) is the first molecular chaperone encountered by nascent polypeptides in bacteria. Here we show, using fluorescence spectroscopy to monitor TF function and structural rearrangements in real time, that TF interacts with ribosomes and translating ... More
Role of the Met3534-Ala4271 region of the ryanodine receptor in the regulation of Ca2+ release induced by calmodulin binding domain peptide.
AuthorsGangopadhyay JP, Ikemoto N
JournalBiophys J
PubMed ID16387763
CaMBP, a peptide corresponding to the 3614-3643 calmodulin (CaM) binding region of the ryanodine receptor (RyR1), is known to activate RyR1 Ca2+ channel. To analyze the mechanism of channel regulation by the CaMBP-RyR1 interaction, we investigated a), CaMBP binding to RyR1, b), induced local conformational changes in the CaMBP binding ... More
Synthesis and characterization of 6-chloroacetyl-2-dimethylaminonaphthalene as a fluorogenic substrate and a mechanistic probe for glutathione transferases.
AuthorsSvensson R, Grenö C, Johansson AS, Mannervik B, Morgenstern R
JournalAnal Biochem
PubMed ID12470677
Here we demonstrate that the thiol-reactive, environmentally sensitive fluorogenic molecules 6-bromoacetyl-2-dimethylaminonaphthalene and 6-acryloyl-2-dimethylaminonaphthalene are substrates for glutathione transferases (GSTs). Product formation can be measured by strong increase in fluorescence of the glutathione conjugate. As these substances display a high nonenzymatic background reaction rate, we have synthesized and characterized 6-chloroacetyl-2-dimethylaminonaphthalene, which ... More
Micro method for determination of nonesterified fatty acid in whole blood obtained by fingertip puncture.
AuthorsHansen JS, Villadsen JK, Gaster M, Faergeman NJ, Knudsen J
JournalAnal Biochem
PubMed ID16814738
Diagnostic tools for early identification of subjects at high risk for type 2 diabetes and other obesity-related disorders are important in prevention of these diseases. Nonesterified fatty acids (NEFAs) have been suggested to serve as a prediagnostic marker of diabetes and obesity-related disorders. In the current study, we developed a ... More
ATP-induced opposite changes in the local environments around Cys(697) (SH2) and Cys(707) (SH1) of the myosin motor domain revealed by the prodan fluorescence.
AuthorsHiratsuka T
JournalJ Biol Chem
PubMed ID10506171
To obtain a consistent view of the nucleotide-induced conformational changes around Cys(697) (SH2) and Cys(707) (SH1) in skeletal myosin subfragment-1 (S-1), the two thiols were labeled with the same environmentally sensitive fluorophore, 6-acyl-2-dimethylaminonaphthalene group, using 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AD) and 6-bromoacetyl-2-dimethylaminonaphthalene (BD), respectively. The resultant fluorescent derivatives, AD-S-1 and BD-S-1, have ... 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
Methane monooxygenase hydroxylase and B component interactions.
The interaction of the soluble methane monooxygenase regulatory component (MMOB) and the active site-bearing hydroxylase component (MMOH) is investigated using spin and fluorescent probes. MMOB from Methylosinus trichosporium OB3b is devoid of cysteine. Consequently, site-directed mutagenesis was used to incorporate single cysteine residues, allowing specific placement of the probe molecules. ... More
Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe.
AuthorsTsalkova TN, Davydova NY, Halpert JR, Davydov DR
JournalBiochemistry
PubMed ID17198380
Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled ... More
High-resolution probing of local conformational changes in proteins by the use of multiple labeling: unfolding and self-assembly of human carbonic anhydrase II monitored by spin, fluorescent, and chemical reactivity probes.
AuthorsHammarström P, Owenius R, Mårtensson LG, Carlsson U, Lindgren M
JournalBiophys J
PubMed ID11371460
Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)-ethyl)amino)naphtalene-1-sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation. HCA II unfolds in a multi-step manner with ... More