High throughput methods of assessing protein stability and aggregation

Senisterra, Guillermo; Finerty, P.J.

doi:10.1039/b814377c

Cited by 105 publications

(95 citation statements)

References 36 publications

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“…It should be noted that T m shifts observed at ramp rates that are too fast to allow equilibration are still useful for screening ligand binding and provide semiquantitative rank ordering of affinities. We also note that it is possible to design experiments that rely on isothermal relaxation kinetics to extract stabilities and binding constants (12). At temperatures near the T m , protein-dependent irreversible processes are at work and may preclude accurate analysis with equilibrium models for some proteins (43)(44)(45).…”

Section: Discussionmentioning

confidence: 99%

“…The measurement of protein stability typically also has required relatively large amounts of protein and low-throughput instrumentation and, consequently, has not been widely used as a tool to assess function. However, recently a number of techniques that allow protein stabilities to be determined with small amounts of material in a high-throughput manner have been developed (8)(9)(10)(11)(12). One such method is based on extrinsic fluorescent dyes that monitor protein (un)folding (2,3,13).…”

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Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes

2010

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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.The interaction of proteins with ligands is a fundamental aspect of biomolecular function. The quantification of such interactions is essential for systems biology, drug discovery, and bioengineering. Traditional, generalizable methods for measuring protein-ligand affinity such as equilibrium dialysis or isothermal titration calorimetry require large amounts of material or radiolabeled ligands. Ligand-induced changes in protein stability also provide a general, quantifiable signature of protein-ligand interactions (1-6), and hence of biological function (7). The measurement of protein stability typically also has required relatively large amounts of protein and low-throughput instrumentation and, consequently, has not been widely used as a tool to assess function. However, recently a number of techniques that allow protein stabilities to be determined with small amounts of material in a high-throughput manner have been developed (8-12). One such method is based on extrinsic fluorescent dyes that monitor protein (un)folding (2, 3, 13). This technique uses the relatively inexpensive fluorescent dye Sypro Orange (SO) 1 (14) in combination with readily available real-time polymerase chain reaction (RT-PCR) instrumentation (3, 13, 15) and is being adopted as a straightforward, economical, high-throughput screening tool for ligand discovery in the pharmaceutical industry (4, 15), structural genomics efforts (16,17), and high-throughput protein engineering (18). Here we present insights into the prop...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes

2010

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“…Binding of SE-1 to purified VirF was tested by comparing the VirF melting temperature (T m ) in the absence of SE-1 to that in its presence using a thermal shift assay with the fluorescent dye Sypro Orange (69, 70) (Molecular Probes). This method is also known as differential scanning fluorimetry (DSF) (71). We used the protocol of Ericsson et al (70) with the following minor modifications.…”

Section: Methodsmentioning

confidence: 99%

Small-Molecule Inhibitor of the Shigella flexneri Master Virulence Regulator VirF

et al. 2013

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VirF is an AraC family transcriptional activator that is required for the expression of virulence genes associated with invasion and cell-to-cell spread by Shigella flexneri, including multiple components of the type three secretion system (T3SS) machinery and effectors. We tested a small-molecule compound, SE-1 (formerly designated OSSL_051168), which we had identified as an effective inhibitor of the AraC family proteins RhaS and RhaR, for its ability to inhibit VirF. Cell-based reporter gene assays with Escherichia coli and Shigella, as well as in vitro DNA binding assays with purified VirF, demonstrated that SE-1 inhibited DNA binding and transcription activation (likely by blocking DNA binding) by VirF. Analysis of mRNA levels using real-time quantitative reverse transcription-PCR (qRT-PCR) further demonstrated that SE-1 reduced the expression of the VirF-dependent virulence genes icsA, virB, icsB, and ipaB in Shigella. We also performed eukaryotic cell invasion assays and found that SE-1 reduced invasion by Shigella. The effect of SE-1 on invasion required preincubation of Shigella with SE-1, in agreement with the hypothesis that SE-1 inhibited the expression of VirF-activated genes required for the formation of the T3SS apparatus and invasion. We found that the same concentrations of SE-1 had no detectable effects on the growth or metabolism of the bacterial cells or the eukaryotic host cells, respectively, indicating that the inhibition of invasion was not due to general toxicity. Overall, SE-1 appears to inhibit transcription activation by VirF, exhibits selectivity toward AraC family proteins, and has the potential to be developed into a novel antibacterial agent.

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“…5 and supplemental Table 1). The rationale underlying the isothermal denaturation assay is that binding of a small molecule to the p53-binding pocket of MDMX may stabilize the protein, and in the presence of the SYPRO orange hydrophobic dye, the temperature for denaturation and dye binding would shift (30,31). Indeed, GST-MDMX-(1-185) showed a melting point shift in the presence of p53 peptide from 46.9 Ϯ 0.6°C for native GST-MDMX-(1-185) protein to 50.8 Ϯ 0.6°C for GST-MDMX-(1-185) protein bound to the p53 peptide (supplemental Fig.…”

Section: Journal Of Biological Chemistrymentioning

confidence: 99%

Identification and Characterization of the First Small Molecule Inhibitor of MDMX

Reed

Shen

Shelat

et al. 2010

Journal of Biological Chemistry

180

162

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The p53 pathway is disrupted in virtually every human tumor. In ϳ50% of human cancers, the p53 gene is mutated, and in the remaining cancers, the pathway is dysregulated by genetic lesions in other genes that modulate the p53 pathway. One common mechanism for inactivation of the p53 pathway in tumors that express wild-type p53 is increased expression of MDM2 or MDMX. MDM2 and MDMX bind p53 and inhibit its function by distinct nonredundant mechanisms. Small molecule inhibitors and small peptides have been developed that bind MDM2 in the p53-binding pocket and displace the p53 protein, leading to p53-mediated cell cycle exit and apoptosis. Tumorigenesis is a multistep process that involves dysregulation of several pathways that are crucial for cell growth and survival (1). The p53 pathway regulates cell survival in response to cellular stress (e.g. DNA damage) or oncogenic stress (e.g. Rb pathway dysregulation) (2, 3) and is suppressed in virtually every human cancer by genetic lesions in the p53 gene or other components of the pathway (4). Approximately half of all cancers express wild-type p53, and considerable research over the past decade has focused on inducing p53-mediated cell death in these tumors (4, 5). Most efforts to date have focused on inhibiting MDM2, a negative regulator of p53 (6 -14).Another key regulator of the p53 pathway is a protein related to MDM2 called MDMX (15-17). MDM2 and MDMX share homology in their p53-binding domains, but MDMX is believed to regulate p53 through distinct mechanisms. Specifically, MDM2 primarily regulates p53 stability and subcellular localization, whereas MDMX may directly regulate p53 transcription (17-21). MDMX is genetically amplified in 19% of breast carcinomas, 19% of colon carcinomas, 18% of lung carcinomas, and a smaller percentage of gliomas (17). One of the best characterized tumors with an MDMX amplification is retinoblastoma. Approximately 65% of human retinoblastomas have increased MDMX copy number, which correlates with increased MDMX mRNA and protein (22). Previous studies have demonstrated that the MDMX amplification suppresses p53-mediated cell death in Rb pathway-deficient retinoblasts (22).A general consensus is emerging that to efficiently induce a p53 response in tumor cells that express wild-type p53, it may be necessary to inactivate both MDM2 and MDMX (18,23,24). To date, no screens to identify small molecule inhibitors of MDMX have been reported, and MDM2 inhibitors probably do not bind as efficiently to MDMX because of structural differences in the p53-binding pockets of the two proteins (25-27). Consistent with this theory, nutlin-3a binds MDMX with at least a 40-fold weaker equilibrium binding constant than for MDM2 (22). Therefore, to identify small molecules that bind MDMX and prevent its interaction with p53, we developed biochemical and cell-based assays suitable for high throughput screening (HTS)

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High throughput methods of assessing protein stability and aggregation

Cited by 105 publications

References 36 publications

Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes

Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes

Small-Molecule Inhibitor of the Shigella flexneri Master Virulence Regulator VirF

Identification and Characterization of the First Small Molecule Inhibitor of MDMX

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