Abstract
Differential scanning fluorimetry (DSF) is a technique that reports protein thermal stability via the selective recognition of unfolded states by fluorogenic dyes. However, DSF applications remain limited by protein incompatibilities with existing DSF dyes. Here we overcome this obstacle with the development of a protein-adaptive DSF platform (paDSF) that combines a dye library ‘Aurora’ with a streamlined procedure to identify protein–dye pairs on demand. paDSF was successfully applied to 94% (66 of 70) of proteins, tripling the previous compatibility and delivering assays for 66 functionally and biochemically diverse proteins, including 10 from severe acute respiratory syndrome coronavirus 2. We find that paDSF can be used to monitor biological processes that were previously inaccessible, demonstrated for the interdomain allostery of O-GlcNAc transferase. The chemical diversity and varied selectivities of Aurora dyes suggest that paDSF functionality may be readily extended. paDSF is a generalizable tool to interrogate protein stability, dynamics and ligand binding.
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Data availability
All data are available in the main text or Supplementary Information. A subset of the Aurora library is only available through a material transfer agreement with North Carolina State University. Raw paDSF data are available at ref. 35, and processed pDSF data at https://doi.org/10.5281/zenodo.10028702 (ref. 44). An annotated video of the primary liquid handling steps of a dye screen is available at ref. 23.
Code availability
All code and source data are available in Supplementary Information or upon request. DSFWorld is available at https://doi.org/10.5281/zenodo.8432909 (ref. 45).
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Acknowledgements
Specific material contributions are listed in Supplementary Information. The authors thank M. Jacobson, J. Fraser, K. Shokat, D. Wassarman, D. Schwarz, D. Elnatan, H. Shao, M. Moore and S. Williams for helpful discussions. Proteins were generously donated by A. Ambrose, A. Thwin, D. Wassarman, E. Thompson, A. Freeberg, G. Correy, H. Shao, H. Saunders, J. Swan, M. Janowska, M. Ramirz-Alvarado, M. Ravalin, M. Gupta, M. McCabe, M. Schoof, R. Freilich and V. Long from the labs of M. Arkin, A. Maria Cuervo, J. Fraser, J. Gestwicki, J. Gross, R. Klevit, G. Narlikar, K. Shokat, D. Southworth, R. Stroud, K. Verba, P. Walter and J. Ward. Several SARS-CoV-2 proteins were provided by the UCSF QBI Coronavirus Research Group (QCRG). We are grateful for additional support from M. Capps and Z. Karakesisoglu (Analytik Jena); C. Brown and D. Schubach (Exciton-Luxottica); D. Beniston (ChemBridge Corporation); K. Kitto, the Home Away students; and S. Gahbauer, S. Vigneron, J. Lee, D. Duncan and N. Saul (UCSF). Funding was provided by National Institute of Health U19 (J.E.G., AI171110); Sandler Program for Breakthrough Biomedical Research (J.E.G.); Tau Consortium (J.E.G.); National Science Foundation Graduate Research Fellowship (T.W., 1000259744); National Institute of Standards and Technology (N.R.V., 60NANB19D115); National Institutes of Health K99 (O.T.J., K99NS128717); National Institutes of Health F32 (E.C.C., F32AG076281); National Institutes of Health R01 (J.E.G., N.R.V., Z.M., 1R01GM141299‐01); National Institutes of Health R01 (C.L.P., GM107069, R01GM121507 and R35GM141859); GlycoNet, the Canadian Glycomics Network (D.J.V., CD-1); Canadian Cancer Society Research Institute (D.J.V., CCSRI-706825); and Canada Research Chairs program (D.J.V., Tier I CRC in Chemical Biology).
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Conceptualization: T.W., J.E.G., M.G.A. and D.J.V. Data curation: T.W. Formal analysis: T.W. and Z.J.G.-D. Funding acquisition: T.W. and J.E.G. Investigation: T.W., J.C.Y., A.S., M.G.A., A.S.W., Z.M., C.L.P., N.R.V. and D.F. Methodology: T.W., J.C.Y. and A.S. Visualization: T.W. Project administration: J.E.G. Supervision: J.E.G. Writing—original draft: T.W., E.C.C., O.T.J., Z.J.G.-D. and J.E.G. Writing—review and editing: T.W., M.G.A., D.J.V. and J.E.G.
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Extended data
Extended Data Fig. 1 Representative examples of paDSF screening results.
For full dye screen results for all proteins, including comparisons of raw paDSF curves collected for each protein with different dyes, see data availability statement. (a) Results of a dye screen against the SYPRO-incompatible protein nuclear-hormone receptor 23. Far left panel: representative unsuccessful DSF conducted with SYPRO Orange. Right panels: examples of successful paDSF data, collected with six different paDSF dyes. Plots show dyes at four different concentrations, demonstrating a dose-responsive relationship between RFU and dye concentration. (b) Representative examples of resolution of four archetypes of SYPRO Orange incompatibility by paDSF dyes. (c) Examples of successful paDSF data collected for four additional proteins. As in panel A, dyes are plots show dyes at four different concentrations, demonstrating a dose-responsive relationship between RFU and dye concentration.
Extended Data Fig. 2 Apparent melting temperatures for paDSF dyes and SYPRO Orange.
Plots include data for the 60 protein test set, as well as the tested 10 SARS-CoV2 proteins. (a) For the subset of tested proteins compatible with SYPRO Orange, comparison of Tmas reported by Aurora library dyes and SYPRO Orange. Tmas between SYPRO Orange and Aurora dyes for the same protein are strongly correlated, with a Pearson R value of 0.97, p = 2.2 e-16. Statistical significance was determined by one-way t-test. Data shown includes 2,211 unique pairings(b) Tmas reported by all hit dyes from the Aurora library for all tested proteins. Green points = Mean Tma between all hit Aurora dyes for a given protein. Error bars represent ± standard error of Tmas between different Aurora dyes. Experiments were performed in triplicate. For proteins compatible with SYPRO Orange, Tma reported by SYPRO Orange is displayed as orange ‘X’s. Grey points: Tma of minor unfolding transitions, defined as secondary or tertiary transitions with lower RFU in a given unfolding curve.
Extended Data Fig. 3 Screenshot of interactive online database for all dye screen data.
All dye screen data is presented in an interactive form at https://padsfdyes.shinyapps.io/Exp1243_heatmap_cache/. For full dye screen results for all proteins, including comparisons of raw paDSF curves collected for each protein with different dyes, see Extended Data Fig. 2.
Extended Data Fig. 4 Apparent thermal shifts with paDSF dyes for SARS-CoV2 proteins and resolution of screening artifacts.
(a) Thermal upshift in the presence of ADP ribose with nsp3 mac1, with 6 paDSF dyes and SYPRO orange. Error bars represent +/- standard deviation. Experiments were performed in triplicate. (b) Thermal upshift in the presence of GRL0617 with PLPro, with 3 paDSF dyes. Representative curves are shown. (c) Protein-independent activation of SYPRO Orange observed for compounds which produced uninterpretable raw DSF data from a 320-compound pyridine pilot screen using SYPRO Orange. Representative curves are shown. (d) Comparison of DSF data collected using SYPRO Orange and dye T004, demonstrating improved compound compatibility of T004 over SYPRO Orange. Individual duplicates are shown.
Extended Data Fig. 5 Raw and analyzed paDSF with OGT, catalytic domains and TPR domain.
(a) Structural model of full-length OGT, based on crystal structures of the TPR (PDB 1W3B) and catalytic (PDB 5C1D) domains. Cartoons of the (b) full-length OGT construct, which includes the full TPR and catalytic domains, (c) catalytic domain construct, which includes the full catalytic domain and TPR repeats 9–13.5, and (d) TPR domain construct, which includes TPR repeats 1-10. (e–h) paDSF results from the 10 hit dyes for OGT, with raw RFU data displayed alongside fitted models for (E) OGT alone, fitted to DSFworld Model 4 (two sigmoids, with initial RFU (F) catalytic domain alone, fitted to DSFworld Model 2 (one sigmoids, with initial RFU (G) TPR domain alone, fitted to DSFworld Model 2 (one sigmoids, with initial RFU (H) overlay of raw data and final fits for all three constructs. (i) Tmas for the three constructs, extracted using the fits displayed in panels A-D. Data are presented as the mean of three technical replicates +/- standard deviation. No significant (ns) difference was found between Tma OGT-1 and Tma cat, nor Tma OGT-2 and Tma TPR. Significance determined using two-tailed t-test, and a significance threshold of p < 0.01.
Extended Data Fig. 6 Thermal shifts with UDP-GlcNAc from TW408 paDSF.
paDSF results using dye TW408 to monitor titration of UDP-GlcNAc with OGT, catalytic domain, or TPR domain. (a) Schematic of interaction of UDP-GlcNAc with full length and isolated catalytic domain, but not TPR domain, of OGT. (b) Raw RFU for each construct with 0–50 µM UltraPure. UDP-GlcNAc. Top panels: full temperature range (30–80 °C), bottom panels: zoomed in on temperatures relevant to the unfolding transition. (c) Comparison of resulting ∆Tmas. Left panel: Tma OGT1 and Tma cat; right panel: Tma OGT2 and Tma TPR. Thermal upshift occurs in Tma OGT1 and Tma cat (Tma OGT1 Spearman estimate = 0.92, p = 1.13e-10; Tma cat Spearman estimate = 0.94, p = 1.48e-12); but not Tma OGT2 or Tma TPR (Tma OGT2 Spearman estimate = 0.095, p = 0.66; Tma TPR Spearman estimate = 0.005, p = 0.98). ns = not significant (p > 0.05); *** p < 0.0001. Statistical significance was determined by two-way t-test. Data are the mean of three technical replicates +/- standard deviation.
Extended Data Fig. 7 Thermal shifts with L4 from TW408 paDSF.
paDSF results using dye TW408 to monitor titration of L4 with OGT, catalytic domain, or TPR domain. (a) Schematic of L4 with the four tested conditions: full OGT, catalytic domain, TPR domain, and a 1:1 mix of catalytic and TPR domain. All constructs were tested at 1 µM final; 1:1 mix had 1 µM catalytic domain and 1 mM TPR domain. (b) Raw RFU for each construct in the presence of 0–10 µM L4. (c) Comparison of resulting Tmas. Left panel: Tma OGT1, Tma cat:TPR 1, and Tma cat; right panel: Tma OGT2, Tma cat:TPR 2, and Tma TPR. Significant thermal upshifts with L4 treatment observed with Tma OGT2, Tma cat:TPR 2, and Tma TPR (Tma OGT2 spearman estimate = 0,95, p = 2.3e-12; Tma cat:TPR 2 spearman estimate = 0.97, p = 4.7e-15; Tma TPR spearman estimate = 0.96, p = 3.6e-14). Significant thermal downshift with L4 observed in Tma OGT1 (data in Fig. 3d, included here for reference, Tma OGT1 spearman estimate = −0.92, p = 6.2e-11). No dose-responsive thermal shift with L4 observed in Tma cat or Tma cat:TPR 1 (Tma cat spearman estimate = −0.27, p = 0.2; Tma cat:TPR 1 spearman estimate = −0.34, p = 0.09). ns = not significant (p > 0.05); *** p < 0.0001. Statistical significance was determined by two-way t-test. Data are presented as the mean of three technical replicates +/- standard deviation.
Extended Data Fig. 8 OGT oligomerization state is not affected by L4, but L4 treatment increases the temperature sensitivity of OGT catalytic activity.
(a) Histograms of counts of MW from mass photometry, used to calculate molecular weights: OGT: 115 kDa, Catalytic domain; 82 kDa, TPR domain: 43, 86, 129 kDa (monomer, dimer, trimer). (b) OGT catalytic activity with a model substrate peptide (KKKYPGGSTPVSSANMM), with enzymatic activity allowed to proceed at 29, 32, 35, 38, 41, 44, 47, or 50 °C. Activity was measured as RLU using Promega UDP-Glo Assay, with higher RLU values corresponding to higher activity. Comparison of temperature-dependent loss of enzymatic activity (RLU) in the presence of increasing concentrations of L4. Solid lines and dots: OGT-containing samples; Dashed lines and Xs: protein-free control. (c) L4-dependent increase in temperature sensitivity of catalytic activity at 41 °C. In all panels, data are presented as the mean of three technical replicates +/- standard deviation.
Extended Data Fig. 9 Properties of the Aurora dye collection.
(a) Comparison of the chemical diversity of Aurora versus a representative diversity set of drug-like molecules that is used for high throughout drug screens. (b) cLogP of dyes and relationship to paDSF performance. (A) Comparison of cLogP, a calculated predictor of water solubility, to the paDSF performance of that dye. Filled circles: percent of tested proteins for which a dye was called a ‘hit’. Xs: percent of tested proteins for which a dye was called ‘sensitive’. No significant correlation was found between hit rate and cLogP (Pearson p value > 0.05, see panel for exact values). Statistical significance was determined with a two-way t-test. (c) Distribution of cLogP values for the full Aurora library (312 dyes), for reference. (d) Individual points represent a single dye, and dyes are arranged along the axis in order of increasing hit rate. Data are shown for the full Aurora library (312 dyes). Colored points indicate dyes which contain a given substructure, grey points indicate dyes lacking that substructure. A singe substructure is presented in each panel, and panels are shown only for sub-structures present in at least one dye. All points left of the grey vertical line, in the area labeled ‘Never hit’ were never a hit with any tested protein. Some dyes contain multiple substructures.
Extended Data Fig. 10 Additional analyses of the Aurora dyes.
(a) Distribution of dyes among the hit and sensitive dyes assigned for each protein. Individual histograms displaying the number of hit dyes for a given protein, binned by the global hit rates of the specific hit dyes. Total hit rates are divided into ten evenly spaced bins, containing hit rates of up to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of all tested proteins. Green bars: for ‘hit’ assignment. Grey bars: for ‘sensitive’ assignment. The presence of both selective and promiscuous dye hits in an individual dye screen appears as the presence of hits in both lower hit rate (for example < 20%) and higher hit rate (for example >40%) bins. Results for individual proteins are presented in individual panels, with the protein name indicated in the panel title. (b) Weak positive correlation between protein sequence similarity and dye fingerprint. Comparison of the relationship between primary sequence similarity and dye screen outcomes shows a weak positive Pearson’s correlation: 0.19, p-value 3.9e-20. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc analysis. Data shown includes 2,211 unique pairings between 67 proteins (72 proteins tested total - 5 proteins tested only as hetero-complexes; see Supplementary Methods). To provide context, the number of dyes screened in common between two proteins is indicated in point size and color. Smaller, darker points: fewer dyes screened in common. Larger, lighter points: more dyes screened in common. See figure key for exact mappings. Calculations of the Jaccard Index (y axis values) and pairwise sequence similarity (x axis values) are described in Supplementary Methods.
Supplementary information
Supplementary Information
Table of contents, Supplementary Notes 1–4 and legends for additional supplementary files.
Supplementary Table 1
A table of the key dye properties, such as hit percentage, fluorescence wavelength, catalog information and cLogP.
Supplementary Table 2
A table of proteins, with detailed information about the screening conditions (for example, buffer and concentration) for each.
Supplementary Data 1
Complete list of calculated properties of the proteins, such as hydrophobicity, secondary structure, molecular weight and sequence.
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Wu, T., Yu, J.C., Suresh, A. et al. Protein-adaptive differential scanning fluorimetry using conformationally responsive dyes. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02158-7
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DOI: https://doi.org/10.1038/s41587-024-02158-7
