'A 14 nt DNA sequence 5''-AGAATGTGGCAAAG-3'' from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar-phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) ... More
New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling.
'Multicolour in situ hybridisation (MFISH) is increasingly applied to karyotyping and detection of chromosomal abnormalities. So far 27 colour analyses have been described using fluorescently labelled chromosome painting probes in a so-called combinatorial approach. In this paper a new strategy is presented to use efficiently the currently available number of ... More
Cooperative formation of a substrate binding pocket by alpha- and beta-subunits of mitochondrial processing peptidase.
AuthorsKojima K, Kitada S, Shimokata K, Ogishima T, Ito A
JournalJ Biol Chem
PubMed ID9829989
'Mitochondrial processing peptidase (MPP) specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme is a metalloprotease and forms a heterodimer consisting of structurally related alpha- and beta-subunits. To investigate the responsibility of MPP subunits for substrate recognition, we monitored interaction of the ... More
Binding of an N-terminal rhodanese peptide to DnaJ and to ribosomes.
AuthorsKudlicki W, Odom OW, Kramer G, Hardesty B
JournalJ Biol Chem
PubMed ID8940114
A peptide corresponding to the N-terminal 17 amino acids of bovine rhodanese was fluorescently labeled with a coumarin derivative at its primary amino group(s) and then purified by high performance liquid chromatography. This peptide interacted with the molecular chaperone DnaJ in the absence of other chaperones and ATP. In the ... More
A newly designed microspectrofluorometer for kinetic studies on protein crystals in combination with x-ray diffraction.
AuthorsKlink BU, Goody RS, Scheidig AJ
JournalBiophys J
PubMed ID16698776
We present a new design for a fluorescence microspectrophotometer for use in kinetic crystallography in combination with x-ray diffraction experiments. The FLUMIX device (Fluorescence spectroscopy to monitor intermediates in x-ray crystallography) is built for 0 degrees fluorescence detection, which has several advantages in comparison to a conventional fluorometer with 90 ... More
A real-time fluorescence method to monitor the melting of duplex DNA during transcription initiation by RNA polymerase.
AuthorsMatlock DL, Heyduk T
JournalAnal Biochem
PubMed ID10328775
The melting of duplex DNA in the vicinity of the transcription start site is an essential step of transcription initiation. Here we describe a fluorescent promoter technique which allows the melting of promoter DNA to be observed in a real-time manner with high sensitivity. We have constructed a 114-bp lacUV5 ... More
Fluorescent coumarin-labeled nucleotides to measure ADP release from actomyosin.
AuthorsWebb MR, Corrie JE
JournalBiophys J
PubMed ID11509369
Several coumarin-labeled nucleotides have been synthesized, based on 2'(3')-O-(2-aminoethyl)carbamoyl-ATP (edaATP). The fluorescent coumarins coupled with the free amino group are 7-diethylaminocoumarin-3-carboxylic acid (to give deac-edaATP), coumarin 343 (but-edaATP) and 7-ethylamino-8-bromocoumarin-3-carboxylic acid (mbc-edaATP). The carbamoyl linkage of these nucleotide analogs undergoes interconversion between 2'- and 3'-hydroxyl attachment very slowly, so that ... More
A function for the psi subunit in loading the Escherichia coli DNA polymerase sliding clamp.
AuthorsAnderson SG, Williams CR, O'donnell M, Bloom LB
JournalJ Biol Chem
PubMed ID17210572
Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, chi and psi, which are not present in the crystal structures, were determined by ... More
Structural and functional heterogeneity among the zinc fingers of human MRE-binding transcription factor-1.
AuthorsChen X, Agarwal A, Giedroc DP
JournalBiochemistry
PubMed ID9698361
MRE-binding transcription factor-1 (MTF-1) activates the expression of metallothionein (MT) genes in mouse and human cells upon binding to one or more tandem metal-response elements (MREs; 5'-ctnTGCRCnCgGCCc) in the MT promoter. MTF-1 contains six Cys2-His2 zinc finger sequences. Previous work suggests that the zinc finger domain itself may function as ... More
Glutamate residues required for substrate binding and cleavage activity in mitochondrial processing peptidase.
AuthorsKitada S, Kojima K, Shimokata K, Ogishima T, Ito A
JournalJ Biol Chem
PubMed ID9829990
Mitochondrial processing peptidase, a metalloendopeptidase consisting of alpha- and beta-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular ... More
Quantitation of myoglobin saturation in the perfused heart using myoglobin as an optical inner filter.
AuthorsLeisey JR, Scott DA, Grotyohann LW, Scaduto RC
JournalAm J Physiol
PubMed ID8067420
Quantitation of metabolic parameters using the technique of cardiac surface fluorescence is complicated by motion and changes in tissue absorption. Because ratio fluorescence methodology can be applied to eliminate motion-induced errors, in the current study, we used a ratio fluorescence technique to evaluate myoglobin saturation in the perfused rat heart, ... More
In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force.
AuthorsSteinberg BE, Touret N, Vargas-Caballero M, Grinstein S
JournalProc Natl Acad Sci U S A
PubMed ID17517624
Phagosomes employ lytic enzymes, cationic peptides, and reactive oxygen intermediates to eliminate invading microorganisms. The effectiveness of these microbicidal mechanisms is potentiated by the acidic pH created by H(+)-pumping vacuolar-type ATPases (V-ATPases) on the phagosomal membrane. The degree of phagosomal acidification varies greatly among neutrophils, macrophages, and dendritic cells and ... More
Native and multimeric vitronectin exhibit similar affinity for heparin. Differences in heparin binding properties induced upon denaturation are due to self-association into a multivalent form.
AuthorsZhuang P, Chen AI, Peterson CB
JournalJ Biol Chem
PubMed ID9054371
For many years, the concept that the heparin-binding sequence is sequestered within vitronectin and exposed upon denaturation of the protein has guided experimental design and interpretation of related structure-function studies on the protein. To evaluate binding of heparin to both native and denatured/renatured vitronectin, methods for monitoring binding in solution ... More