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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
5
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pubmed:dateCreated |
2009-12-1
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pubmed:abstractText |
We report the quantitative, label-free analysis of protein-protein interactions in free solution within picoliter volumes using backscatter interferometry (BSI). Changes in the refractive index are measured for solutions introduced on a PDMS microchip allowing determination of forward and reverse rate constants for two-mode binding. Time-dependent BSI traces are directly fit using a global analysis approach to characterize the interaction of the small heat-shock protein alpha-Crystallin with two substrates: destabilized mutants of T4 lysozyme and the in vivo target betaB1-Crystallin. The results recapitulate the selectivity of alphaB-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an alphaA-Crystallin mutant linked to hereditary cataract has activated binding to betaB1-Crystallin. Binding isotherms obtained from steady-state values of the BSI signal yielded meaningful dissociation constants and establishes BSI as a novel tool for the rapid identification of molecular partners using exceedingly small sample quantities under physiological conditions. This work demonstrates that BSI can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.
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pubmed:grant |
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/19178288-10037719,
http://linkedlifedata.com/resource/pubmed/commentcorrection/19178288-10217480,
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Mar
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pubmed:issn |
1520-6882
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pubmed:author |
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pubmed:issnType |
Electronic
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pubmed:day |
1
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pubmed:volume |
81
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
1865-71
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pubmed:dateRevised |
2011-8-1
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pubmed:meshHeading |
pubmed-meshheading:19178288-Crystallins,
pubmed-meshheading:19178288-Drug Interactions,
pubmed-meshheading:19178288-Interferometry,
pubmed-meshheading:19178288-Molecular Chaperones,
pubmed-meshheading:19178288-Mutagenesis, Site-Directed,
pubmed-meshheading:19178288-Protein Conformation,
pubmed-meshheading:19178288-Protein Folding,
pubmed-meshheading:19178288-Staining and Labeling,
pubmed-meshheading:19178288-Structure-Activity Relationship,
pubmed-meshheading:19178288-alpha-Crystallins
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pubmed:year |
2009
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pubmed:articleTitle |
Free-solution label-free detection of alpha-crystallin chaperone interactions by back-scattering interferometry.
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pubmed:affiliation |
Department of Chemistry and The Vanderbilt Institute for Chemical Biology, Vanderbilt University, VU Station B 351822, Nashville, Tennessee 37235-1822, USA.
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pubmed:publicationType |
Journal Article,
Research Support, N.I.H., Extramural
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