Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes.
Repository Usage Stats
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.
Escherichia coli Proteins
Periplasmic Binding Proteins
Published Version (Please cite this version)10.1021/bi101414z
Publication InfoLayton, Curtis J; & Hellinga, Homme W (2010). Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes. Biochemistry, 49(51). pp. 10831-10841. 10.1021/bi101414z. Retrieved from https://hdl.handle.net/10161/4015.
This is constructed from limited available data and may be imprecise. To cite this article, please review & use the official citation provided by the journal.
More InfoShow full item record
James B. Duke Distinguished Professor of Medicine
The work in this laboratory takes a combined theoretical and experimental approach to problems in structural biophysics. Computer simulations play an increasingly important role in our understanding of protein folding, stability, activity, and the specificity of protein-ligand interactions. Design methods are being developed which can be used to rationally modify the structure and function of a protein. This design methodology allows us to ask very specific question firmly based on a theore
Showing items related by title, author, creator, and subject.
LKB1 Loss induces characteristic patterns of gene expression in human tumors associated with NRF2 activation and attenuation of PI3K-AKT. Kaufman, Jacob M; Amann, Joseph M; Park, Kyungho; Arasada, Rajeswara Rao; Li, Haotian; Shyr, Yu; Carbone, David P (Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer, 2014-06)Inactivation of serine/threonine kinase 11 (STK11 or LKB1) is common in lung cancer, and understanding the pathways and phenotypes altered as a consequence will aid the development of targeted therapeutic strategies. Gene ...
Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro. Kuehn, MJ; Schekman, R; Ljungdahl, PO (J Cell Biol, 1996-11)In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the ...
Touhara, K; Hawes, BE; van Biesen, T; Lefkowitz, RJ (Proc Natl Acad Sci U S A, 1995-09-26)The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, ...