Show simple item record Layton, CJ Hellinga, HW
dc.coverage.spatial United States 2011-06-21T17:22:10Z 2010-12-28
dc.identifier.citation Biochemistry, 2010, 49 (51), pp. 10831 - 10841
dc.description.abstract The quantification of protein-ligand interactions is essential for systems biology, drug discovery, and bioengineering. Ligand-induced changes in protein thermal stability provide a general, quantifiable signature of binding and may be monitored with dyes such as Sypro Orange (SO), which increase their fluorescence emission intensities upon interaction with the unfolded protein. This method is an experimentally straightforward, economical, and high-throughput approach for observing thermal melts using commonly available real-time polymerase chain reaction instrumentation. However, quantitative analysis requires careful consideration of the dye-mediated reporting mechanism and the underlying thermodynamic model. We determine affinity constants by analysis of ligand-mediated shifts in melting-temperature midpoint values. Ligand affinity is determined in a ligand titration series from shifts in free energies of stability at a common reference temperature. Thermodynamic parameters are obtained by fitting the inverse first derivative of the experimental signal reporting on thermal denaturation with equations that incorporate linear or nonlinear baseline models. We apply these methods to fit protein melts monitored with SO that exhibit prominent nonlinear post-transition baselines. SO can perturb the equilibria on which it is reporting. We analyze cases in which the ligand binds to both the native and denatured state or to the native state only and cases in which protein:ligand stoichiometry needs to treated explicitly.
dc.format.extent 10831 - 10841
dc.language ENG
dc.language.iso en_US en_US
dc.relation.ispartof Biochemistry
dc.relation.isversionof 10.1021/bi101414z
dc.subject Escherichia coli
dc.subject Escherichia coli Proteins
dc.subject Fluorescent Dyes
dc.subject Ligands
dc.subject Maltose-Binding Proteins
dc.subject Micrococcal Nuclease
dc.subject Periplasmic Binding Proteins
dc.subject Protein Binding
dc.subject Protein Stability
dc.subject Protein Unfolding
dc.subject Proteins
dc.subject Thermodynamics
dc.title Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes.
dc.title.alternative en_US
dc.type Journal Article
dc.description.version Version of Record en_US 2010-12-28 en_US
duke.description.endpage 10841 en_US
duke.description.issue 51 en_US
duke.description.startpage 10831 en_US
duke.description.volume 49 en_US
dc.relation.journal Biochemistry en_US
pubs.issue 51
pubs.organisational-group /Duke
pubs.organisational-group /Duke/School of Medicine
pubs.organisational-group /Duke/School of Medicine/Basic Science Departments
pubs.organisational-group /Duke/School of Medicine/Basic Science Departments/Biochemistry
pubs.publication-status Published
pubs.volume 49
dc.identifier.eissn 1520-4995

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