Article pubs.acs.org/ac
Fast Screening of Ligand-Protein Interactions based on LigandInduced Protein Stabilization of Gold Nanoparticles Siu Yee New,†,⊥ Khin Moh Moh Aung,†,⊥ Gek Liang Lim,‡ Shuzhen Hong,‡ Si Kee Tan,‡ Yi Lu,†,§ Edwin Cheung,*,‡ and Xiaodi Su*,† †
Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), 3 Research Link, 117602 Singapore ‡ Cancer Biology and Pharmacology, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, 138672 Singapore § Department of Chemistry, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States S Supporting Information *
ABSTRACT: High throughput screening of small molecular weight (LMW) ligands for protein and sensitive determination of ligand-induced protein stabilization is an important task in drug discovery and in protein structural and functional genomics studies. In this study, gold nanoparticles (AuNPs) and their aggregation property are used to develop a rapid and less equipment intensive assay for screening the interactions between LMW ligands and transcription factors (TFs) and human serum albumin. The assay is based on the fact that the aggregation/discpersion status of AuNPs is very sensitive to the conformation of surrounding proteins, and when a LMW ligand binds to the proteins, it can enhance proteins’ salt and thermal stability, and therefore the protective effect on AuNPs from aggregation. Two TFs, i.e. FoxA1 (Forkhead box A1) and AP-2γ (activating enhancer binding protein 2 gamma), and 14 compounds from an NCI compounds library and human serum albumin (HSA) and three known ligands (ibuprofen, warfarin, and phenytoin) are involved to demonstrate the concept and to prove its generality and robustness. With this AuNP method, two strong LMW binders are identified for FoxA1 and AP-2γ; ligand induced protein stabilization is determined. The results have been verified using surface plasmon resonance spectroscopy (SPR) and differential static light scattering (DSLS) techniques. Tryptophan fluorescent measurement is also conducted to provide further information on protein conformational change upon LMW ligand loading as can be observed from AuNPs’ UV− vis spectra. FoxA1 and AP-2γ are pivotal in regulating the transcriptional activity of estrogen receptor alpha and controlling the expression of estrogen-responsive breast cancer cells. Identification of drug candidates targeting these two transcription factors could be an alternative in treating breast cancer, in particular those that have become endocrine resistance. are unsuitable for proteins with a high fluorescence background and proteins that do not aggregate upon unfolding, respectively.12 In this regard, Schaeffer and co-workers have developed a new method, i.e., green fluorescent protein-based stability assay (GFP-Basta), to study the thermal stability of the fused GFP-protein of interest.13 This method depends on GFP stability, thus it is not applicable for proteins that are more stable than GFP. Furthermore, GFP suffers from fluorescence signal loss and restricted storage conditions, although it is a considerably stable protein. Due to the recent advancement of nanofabrication and biofunctionalization techniques, nanomaterials (particularly inorganic nanoparticles and nanostructures) have been extensively used in biomedical reserach, for sensing, diagnostics,
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t is known that most low molecular weight (LMW) ligands, upon binding, would increase the Gibbs free energy of protein unfolding and stabilize the protein against denaturants.1,2 Such ligand-induced protein stabilization is pivotal to proteomic research, as it improves the protein purification, simplifies the drug discovery process, and facilitates protein structural elucidation, especially in crystallization.2 Because of the importance, a plethora of biophysical methods have been developed to study protein−ligand interaction, including X-ray crystallography,3 NMR,4 differential scanning fluorimetry (DSF),5 isothermal titration calorimetry (ITC),6 surface plasmon resonance (SPR),7 circular dichroism (CD),8 and protein aggregation-based differential static light scattering (DSLS).9,10 Despite their availability, each of these methods possesses its own advantages and drawbacks. For instance, although X-ray crystallography and NMR are powerful in providing structural information, not all targets form diffractable crystals, and the latter approach is generally restricted to small targets ( warfarin ≫ phenytoin (Figure 7). This affinity trend correlates well with those reported by NMR and a Biacore study as shown in Figure 7b.45,46 KD values measured by NMR titration for ibuprofen, warfarin, and phenytoin are 0.5 ± 1.0, 4.0 ± 2.8, and 131.6 ± 12.5 μM, respectively.46 Importantly, with our AuNPs assay, the dispersion status of AuNPs with HSA only provides a control to indicate the binding strength of a given ligand. As shown in Figure 7b (left region), the weak binding of phenytoin to HSA yields a similar dispersion status to that of HSA without a ligand. While a consistent trend was observed across the methods, the AuNPs colorimetric assay has advantages over the NMR and Biacore in terms of cost, simplicity, and amenability for high-throughput screening. Robustness of the AuNPs Assay Validated Using More NCI Compounds and Different Batches of AuNPs and FoxA1 Samples. We have found that the aggregation behavior of AuNPs in protein solutions (i.e., the flocculation test profile, e.g., Figure 1c) can vary slightly due to the batch-to-batch variation in AuNPs concentration and maybe protein purity. Figure S7 shows the comparison of two flocculation tests for two batches of AuNPs of 9.0 and 10.4 nM, using FoxA1 from a different purification. A slight shift of the AuNPs aggregation degree as a function of FoxA1 concentration is observed between the two tests. To validate the robustness of the AuNPs assay regardless of the possible sample variation, we have involved another six compounds from the NCI library, i.e., “group two” compounds (Lig9−Lig14). These compounds were first tested using Biacore SPR for their binding to FoxA1. It is confirmed that they all have limited affinity to FoxA1 (Figure 8c), relative to the two confirmed binders of Lig1 and 2368
dx.doi.org/10.1021/ac404241y | Anal. Chem. 2014, 86, 2361−2370
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Article
and equipment-free measurement of LMW ligand binding to proteins to facilitate drug discovery.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions ⊥
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS S.X. would like to acknowledge The Agency for Science, Technology and Research (A*STAR), Singapore, for the financial support under the Grant JCO 1131CFG001.
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Figure 8. (a and b) AuNPs assay using the second batch AuNPs (10.4 nM) for FoxA1 with “group two” compounds (Lig9−Lig14). The two binders (Lig1 and Lig2) and nonbinders (Lig4 and Lig5) of “group one” were also tested in this experiments as a reference. The cutoff r value was calculated using the average of FoxA1 alone and FoxA1-Lig4 and FoxA1-Lig5. (c) SPR Biacore T200 test of the binding of Lig 1, 2, 4, 5, and Lig9−Lig14 to FoxA1.
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CONCLUSION This is the first report in which proteins’ protection to AuNPs (measured as AuNPs’ dispersion/aggregation status) is used to determine LMW ligand-protein binding. The UV−vis spectra and solution color provide both qualitative screening of the LMW ligand binder and quantitative determination of ligand binding inducted protein stabilization. The results agree with those obtained with conventional and more expensive equipment-based methods both qualitatively and quantitatively. The generality of the assay has been demonstrated with two oncogenic transcription factors and an albumin protein and their respective LMW ligands. This assay requires no expensive equipment, fluorescent labeling, or laborious preparation steps. It can be an excellent complementary technique for large-scale 2369
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