Rescue of Misfolded Proteins and Stabilization by Small Molecules

  • Raymond C. Stevens
  • Javier Sancho
  • Aurora Martinez
Part of the Methods in Molecular Biology book series (MIMB, volume 648)


Increasing stability of functional proteins by binding small compounds and ions has long been used to extend shelf-life of protein formulations in the pharmacological and biotechnological industry. Likewise, the therapeutic application of small molecules for in vivo recovery and maintenance of structure and function of proteins is steadily increasing. Compounds that can rescue misfolded proteins by stimulating their correct folding and/or the stabilization of native-like conformations in vivo are referred to as pharmacological chaperones. Here we present thermal-shift and isothermal methods for the high-throughput screening of stabilizing pharmacological chaperones for soluble and membrane proteins. The effect of selected hit compounds on the kinetics of protein synthesis is further evaluated by an in vitro transcription–translation rapid translation system. These procedures can be integrated in an interdisciplinary and translational approach for the search of personalized pharmacological chaperones in genetic misfolding diseases.

Key words

Cytoplasmatic and membrane proteins High-throughput Experimental screening Protein stability Misfolding diseases Denaturation Microscale fluorescence Thermal-shift Melting temperature Compound libraries 



8-Anilino 1-naphthalene sulfonic acid


Concentration for half-maximal binding






Dimethyl sulfoxide


G protein-coupled receptor


Guanidinium hydrochloride




Emission wavelength


Excitation wavelength


Phenylalanine hydroxylase




Rapid translation system


Half-denaturation temperature





The authors would like to thank their respective groups, especially to Angel L. Pey, Nunilo Cremades, Adrián Velazquez-Campoy, Michael A. Hanson and Mark T. Griffith for discussions and skilful execution of experiments shown in Figs. 2 and 3.


  1. 1.
    Sanchez-Ruiz JM (2007) Ligand effects on protein thermodynamic stability. Biophys Chem 126:43–49PubMedCrossRefGoogle Scholar
  2. 2.
    Cremades N, Sancho J, Freire E (2006) The native-state ensemble of proteins provides clues for folding, misfolding and function. Trends Biochem Sci 31:494–496PubMedCrossRefGoogle Scholar
  3. 3.
    Maclean DS, Qian Q, Middaugh CR (2002) Stabilization of proteins by low molecular weight multi-ions. J Pharm Sci 91:2220–2229PubMedCrossRefGoogle Scholar
  4. 4.
    Meyer JD, Ho B, Manning MC (2002) Effects of conformation on the chemical stability of pharmaceutically relevant polypeptides. Pharm Biotechnol 13:85–107PubMedCrossRefGoogle Scholar
  5. 5.
    Ericsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P (2006) Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem 357:289–298PubMedCrossRefGoogle Scholar
  6. 6.
    Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221PubMedCrossRefGoogle Scholar
  7. 7.
    Alexandrov AI, Mileni M, Chien EY, Hanson MA, Stevens RC (2008) Microscale fluorescent thermal stability assay for membrane proteins. Structure 16:351–359PubMedCrossRefGoogle Scholar
  8. 8.
    Nayar R, Manning MC (2002) High throughput formulation: strategies for rapid development of stable protein products. Pharm Biotechnol 13:177–198PubMedCrossRefGoogle Scholar
  9. 9.
    Ulloa-Aguirre A, Janovick JA, Brothers SP, Conn PM (2004) Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease. Traffic 5:821–837PubMedCrossRefGoogle Scholar
  10. 10.
    Loo TW, Clarke DM (2007) Chemical and pharmacological chaperones as new therapeutic agents. Expert Rev Mol Med 9:1–18PubMedCrossRefGoogle Scholar
  11. 11.
    Pedemonte N, Lukacs GL, Du K, Caci E, Zegarra-Moran O, Galietta LJ, Verkman AS (2005) Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening. J Clin Invest 115:2564–2571PubMedCrossRefGoogle Scholar
  12. 12.
    Tropak MB, Blanchard JE, Withers SG, Brown ED, Mahuran D (2007) High-throughput screening for human lysosomal beta-N-Acetyl hexosaminidase inhibitors acting as pharmacological chaperones. Chem Biol 14:153–164PubMedCrossRefGoogle Scholar
  13. 13.
    Pey AL, Ying M, Cremades N, Velazquez-Campoy A, Scherer T, Thony B, Sancho J, Martinez A (2008) Identification of pharmacological chaperones as potential therapeutic agents to treat phenylketonuria. J Clin Invest 118:2858–2867PubMedCrossRefGoogle Scholar
  14. 14.
    McInnes C (2007) Virtual screening strategies in drug discovery. Curr Opin Chem Biol 11:494–502PubMedCrossRefGoogle Scholar
  15. 15.
    Tanrikulu Y, Schneider G (2008) Pseudoreceptor models in drug design: bridging ligand- and receptor-based virtual screening. Nat Rev Drug Discov 7:667–677PubMedCrossRefGoogle Scholar
  16. 16.
    Hanson MA, Cherezov V, Griffith MT, Roth CB, Jaakola VP, Chien EY, Velasquez J, Kuhn P, Stevens RC (2008) A specific cholesterol binding site is established by the 2.8 A structure of the human beta2-adrenergic receptor. Structure 16:897–905PubMedCrossRefGoogle Scholar
  17. 17.
    Desviat LR, Perez B, Ugarte M (2003) Investigation of folding and degradation of in vitro synthesized mutant proteins in the cytosol. Methods Mol Biol 232:257–263PubMedGoogle Scholar
  18. 18.
    Martinez A, Calvo AC, Teigen K, Pey AL (2008) Chapter 3 Rescuing proteins of low kinetic stability by chaperones and natural ligands: phenylketonuria, a case study. Prog Nucleic Acid Res Mol Biol 83:89–134Google Scholar
  19. 19.
    Hanson MA, Stevens RC (2009) Discovery of new GPCR biology: one receptor structure at a time. Structure 17:8–14PubMedCrossRefGoogle Scholar
  20. 20.
    Martínez A, Knappskog PM, Olafsdottir S, Døskeland AP, Eiken HG, Svebak RM, Bozzini M, Apold J, Flatmark T (1995) Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases Isolation and characterization of the wild-type enzyme. Biochem J 306:589–597PubMedGoogle Scholar
  21. 21.
    Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Yao XJ, Weis WI, Stevens RC, Kobilka BK (2007) GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318:1266–1273PubMedCrossRefGoogle Scholar
  22. 22.
    Irun MP, Maldonado S, Sancho J (2001) Stabilization of apoflavodoxin by replacing hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or Gln. Protein Eng 14:173–181PubMedCrossRefGoogle Scholar
  23. 23.
    Senisterra GA, Soo Hong B, Park HW, Vedadi M (2008) Application of high-throughput isothermal denaturation to assess protein stability and screen for ligands. J Biomol Screen 13:337–342PubMedCrossRefGoogle Scholar
  24. 24.
    Thony B, Calvo AC, Scherer T, Svebak RM, Haavik J, Blau N, Martinez A (2008) Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase. J Neurochem 106:672–681PubMedCrossRefGoogle Scholar
  25. 25.
    Thony B, Ding Z, Martinez A (2004) Tetrahydrobiopterin protects phenylalanine hydroxylase activity in vivo: implications for tetrahydrobiopterin-responsive hyperphenylalaninemia. FEBS Lett 577:507–511PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Raymond C. Stevens
    • 1
  • Javier Sancho
    • 2
  • Aurora Martinez
    • 3
  1. 1.Department of Molecular BiologyThe Scripps Research InstituteLa JollaUSA
  2. 2.Departamento de Bioquimica y Biologia Molecular y Celular, Facultad de CienciasUniversidad de ZaragozaZaragozaSpain
  3. 3.Department of BiomedicineUniversity of BergenBergenNorway

Personalised recommendations