Analysis of Protein Stability and Ligand Interactions by Thermal Shift Assay

Kathy Huynh1, Carrie L. Partch1

1 Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 28.9
DOI:  10.1002/0471140864.ps2809s79
Online Posting Date:  February, 2015
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Abstract

Purification of recombinant proteins for biochemical assays and structural studies is time‐consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real‐time PCR instruments. By screening a wide range of solution conditions and additives in a 96‐well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low‐cost screen to discover new protein‐ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small‐scale, high‐throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. © 2015 by John Wiley & Sons, Inc.

Keywords: differential scanning fluorimetry; TSA; ThermoFluor; thermal denaturation; buffer optimization; ligand screening

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Buffer Screen for Optimizing Protein Stability
  • Support Protocol 1: Determination of Melting Temperatures from Non‐Linear Fitting of Thermal Denaturation Data
  • Alternate Protocol 1: Additive Screen for Optimizing Protein Stability
  • Alternate Protocol 2: Ligand Screen for Discovering Protein‐Ligand Interactions
  • Reagents and solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Buffer Screen for Optimizing Protein Stability

  Materials
  • Commercial or in‐house buffer screen: 96‐well 2‐ml deep‐well block containing 5× buffer screen of choice (e.g., Hampton Research cat. no. HR2‐072 or see recipe and Table 28.9.1)
  • Purified protein (see recipe and Critical Parameters)
  • Dilution buffer (see recipe)
  • SYPRO Orange dye (e.g., Sigma‐Aldrich, cat. no. S5692)
  • 15‐ml polypropylene conical tubes (e.g., VWR, cat. no. 21008‐089)
  • Multichannel pipet (1‐50 μl) and reservoir trough
  • 96‐well plates specific for real‐time PCR instrument (e.g., Life Technologies, cat. no. 4346907)
  • Swinging bucket centrifuge with adapters for 96‐well plates (e.g., Eppendorf 5810 fitted with adapters A‐4‐81‐MTP)
  • Optically clear sealing film for 96‐well plates (e.g., VWR, cat. no. 33500‐696)
  • Adhesive aluminum sealing film for 96‐well plates (e.g., VWR, cat. no. 29445‐080)
  • Real‐time PCR instrument (e.g., Applied Biosystems ViiA7 with ViiA7 RUO software)
  • 96‐well deep‐well plates (e.g., VWR, cat. no. 37001‐518)
Table 8.9.1   MaterialsComposition of 96‐well Buffer Optimization Screen a

Well Buffer Salt Well Buffer Salt
A1 50 mM sodium citrate, pH 4.0 50 mM NaCl E1 50 mM sodium citrate, pH 4.0 250 mM NaCl
A2 50 mM sodium citrate, pH 4.5 50 mM NaCl E2 50 mM sodium citrate, pH 4.5 250 mM NaCl
A3 50 mM sodium citrate, pH 5.0 50 mM NaCl E3 50 mM sodium citrate, pH 5.0 250 mM NaCl
A4 50 mM sodium citrate, pH 5.5 50 mM NaCl E4 50 mM sodium citrate, pH 5.5 250 mM NaCl
A5 50 mM sodium citrate, pH 6.0 50 mM NaCl E5 50 mM sodium citrate, pH 6.0 250 mM NaCl
A6 50 mM sodium citrate, pH 6.5 50 mM NaCl E6 50 mM sodium citrate, pH 6.5 250 mM NaCl
A7 50 mM sodium citrate, pH 7.0 50 mM NaCl E7 50 mM sodium citrate, pH 7.0 250 mM NaCl
A8 50 mM MES, pH 5.5 50 mM NaCl E8 50 mM MES, pH 5.5 250 mM NaCl
A9 50 mM MES, pH 6.0 50 mM NaCl E9 50 mM MES, pH 6.0 250 mM NaCl
A10 50 mM MES, pH 6.5 50 mM NaCl E10 50 mM MES, pH 6.5 250 mM NaCl
A11 50 mM PIPES, pH 6.0 50 mM NaCl E11 50 mM PIPES, pH 6.0 250 mM NaCl
A12 50 mM PIPES, pH 6.5 50 mM NaCl E12 50 mM PIPES, pH 6.5 250 mM NaCl
B1 50 mM PIPES, pH 7.0 50 mM NaCl F1 50 mM PIPES, pH 7.0 250 mM NaCl
B2 50 mM PIPES, pH 7.5 50 mM NaCl F2 50 mM PIPES, pH 7.5 250 mM NaCl
B3 50 mM Bis‐Tris, pH 6.0 50 mM NaCl F3 50 mM Bis‐Tris, pH 6.0 250 mM NaCl
B4 50 mM Bis‐Tris, pH 6.5 50 mM NaCl F4 50 mM Bis‐Tris, pH 6.5 250 mM NaCl
B5 50 mM Bis‐Tris, pH 7.0 50 mM NaCl F5 50 mM Bis‐Tris, pH 7.0 250 mM NaCl
B6 50 mM MOPS, pH 6.5 50 mM NaCl F6 50 mM MOPS, pH 6.5 250 mM NaCl
B7 50 mM MOPS, pH 7.0 50 mM NaCl F7 50 mM MOPS, pH 7.0 250 mM NaCl
B8 50 mM MOPS, pH 7.5 50 mM NaCl F8 50 mM MOPS, pH 7.5 250 mM NaCl
B9 50 mM MOPS, pH 8.0 50 mM NaCl F9 50 mM MOPS, pH 8.0 250 mM NaCl
B10 50 mM L‐Arg/50 mM L‐Glu, pH 6.0 50 mM NaCl F10 50 mM L‐Arg/50 mM L‐Glu, pH 6.0 250 mM NaCl
B11 50 mM L‐Arg/50 mM L‐Glu, pH 6.5 50 mM NaCl F11 50 mM L‐Arg/50 mM L‐Glu, pH 6.5 250 mM NaCl
B12 50 mM L‐Arg/50 mM L‐Glu, pH 7.0 50 mM NaCl F12 50 mM L‐Arg/50 mM L‐Glu, pH 7.0 250 mM NaCl
C1 50 mM L‐Arg/50 mM L‐Glu, pH 7.5 50 mM NaCl G1 50 mM L‐Arg/50 mM L‐Glu, pH 7.5 250 mM NaCl
C2 50 mM sodium phosphate, pH 6.0 50 mM NaCl G2 50 mM sodium phosphate, pH 6.0 250 mM NaCl
C3 50 mM sodium phosphate, pH 6.5 50 mM NaCl G3 50 mM sodium phosphate, pH 6.5 250 mM NaCl
C4 50 mM sodium phosphate, pH 7.0 50 mM NaCl G4 50 mM sodium phosphate, pH 7.0 250 mM NaCl
C5 50 mM sodium phosphate, pH 7.5 50 mM NaCl G5 50 mM sodium phosphate, pH 7.5 250 mM NaCl
C6 50 mM sodium phosphate, pH 8.0 50 mM NaCl G6 50 mM sodium phosphate, pH 8.0 250 mM NaCl
C7 50 mM HEPES, pH 7.0 50 mM NaCl G7 50 mM HEPES, pH 7.0 250 mM NaCl
C8 50 mM HEPES, pH 7.5 50 mM NaCl G8 50 mM HEPES, pH 7.5 250 mM NaCl
C9 50 mM HEPES, pH 8.0 50 mM NaCl G9 50 mM HEPES, pH 8.0 250 mM NaCl
C10 50 mM Tris, pH 7.0 50 mM NaCl G10 50 mM Tris, pH 7.0 250 mM NaCl
C11 50 mM Tris, pH 7.5 50 mM NaCl G11 50 mM Tris, pH 7.5 250 mM NaCl
C12 50 mM Tris, pH 8.0 50 mM NaCl G12 50 mM Tris, pH 8.0 250 mM NaCl
D1 50 mM Tris, pH 8.5 50 mM NaCl H1 50 mM Tris, pH 8.5 250 mM NaCl
D2 50 mM Tris, pH 9.0 50 mM NaCl H2 50 mM Tris, pH 9.0 250 mM NaCl
D3 50 mM Bicine, pH 7.5 50 mM NaCl H3 50 mM Bicine, pH 7.5 250 mM NaCl
D4 50 mM Bicine, pH 8.0 50 mM NaCl H4 50 mM Bicine, pH 8.0 250 mM NaCl
D5 50 mM Bicine, pH 8.5 50 mM NaCl H5 50 mM Bicine, pH 8.5 250 mM NaCl
D6 50 mM Bicine, pH 9.0 50 mM NaCl H6 50 mM Bicine, pH 9.0 250 mM NaCl
D7 50 mM CHES, pH 8.5 50 mM NaCl H7 50 mM CHES, pH 8.5 250 mM NaCl
D8 50 mM CHES, pH 9.0 50 mM NaCl H8 50 mM CHES, pH 9.0 250 mM NaCl
D9 50 mM CHES, pH 9.5 50 mM NaCl H9 50 mM CHES, pH 9.5 250 mM NaCl
D10 50 mM CHES, pH 10.0 50 mM NaCl H10 50 mM CHES, pH 10.0 250 mM NaCl
D11 user‐determined buffer 50 mM NaCl H11 user‐determined buffer 250 mM NaCl
D12 user‐determined buffer 50 mM NaCl H12 user‐determined buffer 250 mM NaCl

 aConcentrations represent final concentration in thermal shift assay. Stocks should be prepared at 5× concentration.

Support Protocol 1: Determination of Melting Temperatures from Non‐Linear Fitting of Thermal Denaturation Data

  Materials
  • csv file containing raw fluorescence data from real‐time PCR instrument
  • Excel software (Microsoft)
  • Graphing software (e.g., GraphPad Prism 5.0, GraphPad Software)

Alternate Protocol 1: Additive Screen for Optimizing Protein Stability

  Additional Materials
  • 96‐well deep‐well block containing 5× additive screen of choice (see recipe and Table 28.9.2)
Table 8.9.2   Additional MaterialsComposition of 96‐well Additive Screen a

Well Additive Well Additive
A1 water E1 5 mM EDTA
A2 water E2 100 mM sodium fluoride
A3 water E3 100 mM potassium fluoride
A4 water E4 100 mM lithium chloride
A5 100 mM urea E5 100 mM potassium chloride
A6 250 mM urea E6 100 mM ammonium chloride
A7 500 mM urea E7 100 mM sodium iodide
A8 1 M urea E8 100 mM potassium iodide
A9 25 mM guanidine HCl E9 100 mM sodium bromide
A10 50 mM guanidine HCl E10 10 mM magnesium chloride
A11 100 mM guanidine HCl E11 10 mM calcium chloride
A12 250 mM guanidine HCl E12 5 mM manganese chloride
B1 500 mM guanidine HCl F1 5 mM nickel chloride
B2 1% (v/v) DMSO F2 5 mM iron (III) chloride
B3 2% (v/v) DMSO F3 5 mM zinc chloride
B4 2.5% (v/v) glycerol F4 5 mM cobalt chloride
B5 5% (v/v) glycerol F5 100 mM sodium formate
B6 10% (v/v) glycerol F6 100 mM sodium acetate
B7 15% (v/v) glycerol F7 100 mM sodium malonate
B8 20% (v/v) glycerol F8 100 mM sodium nitrate
B9 2.5% (v/v) D‐glucose F9 100 mM sodium thiocyanate
B10 5% (v/v) D‐glucose F10 100 mM sodium sulfate
B11 2.5% (v/v) sucrose F11 100 mM ammonium sulfate
B12 5% (v/v) sucrose F12 100 mM ammonium chloride
C1 2.5% (v/v) PEG400 G1 2 mM AMP + 5 mM MgCl 2
C2 5% (v/v) PEG400 G2 2 mM ADP + 5 mM MgCl 2
C3 2.5% (w/v) PEG1000 G3 2 mM ATP + 5 mM MgCl 2
C4 5% (w/v) PEG1000 G4 2 mM AMPPNP + 5 mM MgCl 2
C5 2.5% (w/v) PEG4000 G5 2 mM cAMP + 5 mM MgCl 2
C6 5% (w/v) PEG4000 G6 2 mM GDP + 5 mM MgCl 2
C7 2.5% (v/v) ethylene glycol G7 2 mM GTP + 5 mM MgCl 2
C8 5% (v/v) ethylene glycol G8 2 mM cGMP + 5 mM MgCl 2
C9 1 mM octyl glucoside G9 2 mM NAD + 5 mM MgCl 2
C10 2 mM CHAPS G10 2 mM NADH + 5 mM MgCl 2
C11 10 mM L‐proline G11 10 mM betaine
C12 50 mM L‐glycine G12 1 mM spermine
D1 25 mM L‐histidine H1 1 mM spermidine b
D2 50 mM L‐arginine H2 10 mM β‐mercaptoethanol b
D3 50 mM L‐glutamate H3 5 mM dithiothreitol b
D4 50 mM L‐Arg/50 mM L‐Glu H4 2 mM TCEP b
D5 25 mM L‐glutamine H5 user‐determined additive
D6 50 mM L‐lysine H6 user‐determined additive
D7 50 mM L‐cysteine H7 user‐determined additive
D8 50 mM taurine H8 user‐determined additive
D9 50 mM imidazole, pH 7.6 H9 user‐determined additive
D10 100 mM imidazole, pH 7.6 H10 user‐determined additive
D11 250 mM imidazole, pH 7.6 H11 user‐determined additive
D12 500 mM imidazole, pH 7.6 H12 user‐determined additive

 aConcentrations represent final concentration in thermal shift assay. Stocks should be prepared at 5× concentration.
 bAdd fresh each time.

Alternate Protocol 2: Ligand Screen for Discovering Protein‐Ligand Interactions

  Additional Materials
  • 96‐well plate containing 50× small‐molecule screen of choice (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
  Ablinger, E., Leitgeb, S., and Zimmer, A. 2013. Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant‐containing formulations. Int. J. Pharm. 441:255‐260.
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  Dupeux, F., Röwer, M., Seroul, G., Blot, D., and Márquez, J. 2011. A thermal stability assay can help to estimate the crystallization likelihood of biological samples. Acta Crystallogr. D Biol. Crystallogr. 67:915‐919.
  Ericsson, U., Hallberg, B., Detitta, G., Dekker, N., and Nordlund, P. 2006. Thermofluor‐based high‐throughput stability optimization of proteins for structural studies. Anal. Biochem. 357:289‐298.
  Fedorov, O., Niesen, F., and Knapp, S. 2012. Kinase inhibitor selectivity profiling using differential scanning fluorimetry. Methods Mol. Biol. 795:109‐118.
  Fukada, H. and Takahashi, K. 1998. Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride. Proteins 33:159‐166.
  Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 97:10.2A.1‐10.2A.44.
  Good, N., Winget, G., Winter, W., Connolly, T., Izawa, S., and Singh, R. 1966. Hydrogen ion buffers for biological research. Biochemistry 5:467‐477.
  Hawe, A., Sutter, M., and Jiskoot, W. 2008. Extrinsic fluorescent dyes as tools for protein characterization. Pharm. Res. 25:1487‐1499.
  Johnson, C. 2013. Differential scanning calorimetry as a tool for protein folding and stability. Arch. Biochem. Biophys. 531:100‐109.
  Kranz, J. and Schalk‐Hihi, C. 2011. Protein thermal shifts to identify low molecular weight fragments. Meth. Enzymol. 493:277‐298.
  Lo, M.‐C., Aulabaugh, A., Jin, G., Cowling, R., Bard, J., Malamas, M., and Ellestad, G. 2004. Evaluation of fluorescence‐based thermal shift assays for hit identification in drug discovery. Anal. Biochem. 332:153‐159.
  Matulis, D., Kranz, J., Salemme, F., and Todd, M. 2005. Thermodynamic stability of carbonic anhydrase: Measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44:5258‐5266.
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  Mezzasalma, T., Kranz, J., Chan, W., Struble, G., Schalk‐Hihi, C., Deckman, I., Springer, B., and Todd, M. 2007. Enhancing recombinant protein quality and yield by protein stability profiling. J. Biomol. Screen. 12:418‐428.
  Niesen, F., Berglund, H., and Vedadi, M. 2007. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2:2212‐2221.
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Key Reference
  Kranz and Schalk‐Hihi, 2011. See above.
  This review provides an overview of the thermal shift technique and a nice summary of the thermodynamic principles behind the estimation of Kd from thermal shift data in small‐molecule screens.
  Mashalidis, E., Śledź, P., Lang, S., and Abell, C. 2013. A three‐stage biophysical screening cascade for fragment‐based drug discovery. Nat. Protoc. 8:2309‐2324.
  This protocol outlines an experimental framework using the thermal shift assay as a first‐line screen for small‐molecule discovery in conjunction with ligand‐observed NMR and isothermal titration calorimetry. It also provides information on assembling and maintaining a library of small molecule fragments that are ideal for ligand discovery by thermal shift assay.
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Supplementary Material