Protein stability induced by ligand binding correlates with changes in protein flexibility.
AuthorsCelej MS, Montich GG, Fidelio GD
JournalProtein Sci
PubMed ID12824495
'The interaction between ligands and proteins usually induces changes in protein thermal stability with modifications in the midpoint denaturation temperature, enthalpy of unfolding, and heat capacity. These modifications are due to the coupling of unfolding with binding equilibrium. Furthermore, they can be attained by changes in protein structure and conformational ... More
High-density miniaturized thermal shift assays as a general strategy for drug discovery.
AuthorsPantoliano MW, Petrella EC, Kwasnoski JD, Lobanov VS, Myslik J, Graf E, Carver T, Asel E, Springer BA, Lane P, Salemme FR
JournalJ Biomol Screen
PubMed ID11788061
'More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins ... More
A fluorometric assay for the biotin-avidin interaction based on displacement of the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid.
AuthorsMock DM, Langford G, Dubois D, Criscimagna N, Horowitz P
JournalAnal Biochem
PubMed ID4091277
'Avidin and biotin can be sensitively and accurately quantitated using the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS). In the presence of avidin, the fluorescence of 2,6-ANS is blue shifted with a large increase in quantum yield. Biotin binding causes complete displacement of the bound fluorophore with concomitant quenching of the fluorescence. ... More
Fluorescence probes for structure.
AuthorsBrand L, Gohlke JR
JournalAnnu Rev Biochem
PubMed ID4563443
Interaction of aromatic dyes with the coenzyme A binding site of choline acetyltransferase.
AuthorsMautner HG, Merrill RE, Currier SF, Harvey G
JournalJ Med Chem
PubMed ID7310833
The interaction of a series of aromatic dyes with the coenzyme A binding site of choline acetyltransferase was studied. Several of the dyes were very potent inhibitors of the enzyme. With few exceptions, inhibition was competitive with respect to acetylcoenzyme A and noncompetitive with respect to choline. It appears likely ... More
Biotin-fluorophore conjugates with poly(ethylene glycol) spacers retain intense fluorescence after binding to avidin and streptavidin.
AuthorsGruber HJ, Marek M, Schindler H, Kaiser K
JournalBioconjug Chem
PubMed ID9258455
Conventional biotin-fluorophore conjugates with approximately 14 atom spacers lose most of their fluorescence when binding to avidin or streptavidin, as is demonstrated in the present study. This explains the unusual fact that only biotinylated marker enzymes, but not fluorescent biotins, are regularly used in bioanalytic assays. Novel biotin-spacer-fluorophore conjugates are ... More
Accurate titration of avidin and streptavidin with biotin-fluorophore conjugates in complex, colored biofluids.
AuthorsGruber HJ, Kada G, Marek M, Kaiser K
JournalBiochim Biophys Acta
PubMed ID9685643
A new fluorimetric assay is presented for the specific and reliable quantitation of >/=2 nM avidin and streptavidin. The assay is based on pronounced changes in the fluorescence properties of commercial fluorescein-biotin, or of a newly synthesized biotin-poly(ethylene glycol)-pyrene conjugate, which occur upon binding to avidin and streptavidin. Accurate measurement ... More
The effects of the anilinonaphthalenesulfonates on the alkylation of tubulin: correlation between the appearance of sulfhydryl groups and apolar binding sites.
AuthorsLuduena RF, Roach MC, Horowitz P
JournalBiochim Biophys Acta
PubMed ID3509976
We have previously found that the sulfhydryl groups of tubulin are sensitive reporters of the effects of ligands on the tubulin molecule. In this study, we examined the effects of three anilinonaphthalenesulfonates on the interaction of tubulin with iodo[14C]acetamide and N, N'-ethylenebis(iodoacetamide). We found that 1,8-anilinonapthalensulfonate (1,8-ANS) and 2,6-anilinonaphthalenesulfonate (2,6-ANS) ... More
Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid.
AuthorsSecnik J, Wang Q, Chang CM, Jentoft JE
JournalBiochemistry
PubMed ID2261456
The structural and functional properties of the nucleocapsid (NC) protein of the avian myeloblastosis virus were examined by steady-state fluorescence and fluorescence anisotropy measurements of the complex between the NC and the extrinsic fluorophore 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid (bis-ANS). The intrinsic fluorescence of bis-ANS is enhanced many fold upon forming a complex ... More
Biotin-pyrene conjugates with poly(ethylene glycol) spacers are convenient fluorescent probes for avidin and streptavidin.
AuthorsMarek M, Kaiser K, Gruber HJ
JournalBioconjug Chem
PubMed ID9258456
Conventional biotin-fluorophore conjugates with approximately 14 atom spacers are strongly quenched when bound to avidin or streptavidin, whereas fluorescence becomes insensitive to receptor binding if typical fluorophores are linked to biotin via poly(ethylene glycol) (PEG) chains (Gruber et al., see the second of three papers in this issue). In the ... More
A regulatory hydrophobic area in the flexible joint region of plasminogen activator inhibitor-1, defined with fluorescent activity-neutralizing ligands. Ligand-induced serpin polymerization.
AuthorsEgelund R, Einholm AP, Pedersen KE, Nielsen RW, Christensen A, Deinum J, Andreasen PA
JournalJ Biol Chem
PubMed ID11278457
We have characterized the neutralization of the inhibitory activity of the serpin plasminogen activator inhibitor-1 (PAI-1) by a number of structurally distinct organochemicals, including compounds with environment-sensitive spectroscopic properties. In contrast to latent and reactive center-cleaved PAI-1 and PAI-1 in complex with urokinase-type plasminogen activator (uPA), active PAI-1 strongly increased ... More
Polyion complex micelles with protein-modified corona for receptor-mediated delivery of oligonucleotides into cells.
AuthorsVinogradov S, Batrakova E, Li S, Kabanov A
JournalBioconjug Chem
PubMed ID10502353
Graft-copolymers, containing poly(ethylene glycol) (PEG) and polyethyleneimine (PEI) chains have been proposed as carriers for delivery of phosphorothioate oligonucleotides (SODNs). Complexes of such copolymers with SODN self-assemble into particles having a core of neutralized PEI and SODN and a corona of PEG. Transferrin molecules are attached to the PEG corona ... More
A study of the interaction of avidin with 2-anilinonaphthalene-6-sulfonic acid as a probe of the biotin binding site.
AuthorsMock DM, Lankford G, Horowitz P
JournalBiochim Biophys Acta
PubMed ID3408737
The environment of the biotin binding site on avidin was investigated by determining the fluorescence enhancement of a series of fluorescent probes that are anilinonaphthalene sulfonic acid derivatives. Of the compounds tested, 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) exhibited the greatest enhancement under the conditions used (which would reflect both molar fluorescence enhancement ... More