Over the past 15 years, a series of energetics-based techniques have been developed for the thermodynamic analysis of protein folding and stability. These techniques include tability of npurified roteins from ates of amide H/D change (SUPREX), pulse proteolysis, tability of roteins from ates of idation (SPROX), slow histidine H/D exchange, lysine amidination, and quantitative cysteine reactivity (QCR). The above techniques, which are the subject of this review, all utilize chemical or enzymatic modification reactions to probe the chemical denaturant– or temperature-induced equilibrium unfolding properties of proteins and protein-ligand complexes. They employ various mass spectrometry-, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)-, and optical spectroscopy–based readouts that are particularly advantageous for high-throughput and in some cases multiplexed analyses. This has created the opportunity to use protein folding and stability measurements in new applications such as in high-throughput screening projects to identify novel protein ligands and in mode-of-action studies to identify protein targets of a particular ligand.


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Literature Cited

  1. Wyman J, Gill SJ. 1.  1990. Binding and Linkage Mill Valley, CA: Univ. Sci. Books330
  2. Greene RF, Pace CN. 2.  1974. Urea and guanidine-hydrochloride denaturation ribonuclease, lysozyme, α-chymotrypsin, and β-lactoglobulin. J. Biol. Chem. 249:5388–93 [Google Scholar]
  3. Pace CN.3.  1986. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131:266–80 [Google Scholar]
  4. Bai YW, Milne JS, Mayne L, Englander SW. 4.  1994. Protein stability parameters measured by hydrogen-exchange. Proteins 20:4–14 [Google Scholar]
  5. Jelesarov I, Bosshard HR. 5.  1999. Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition. J. Mol. Recognit. 12:3–18 [Google Scholar]
  6. Leavitt S, Freire E. 6.  2001. Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr. Opin. Struct. Biol. 11:560–66 [Google Scholar]
  7. Tang L, Roulhac PL, Fitzgerald MC. 7.  2007. H/D exchange and mass spectrometry-based method for biophysical analysis of multidomain proteins at the domain level. Anal. Chem. 79:8728–39 [Google Scholar]
  8. DeArmond PD, Xu Y, Strickland EC, Daniels KG, Fitzgerald MC. 8.  2011. Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach. J. Proteome Res. 10:4948–58 [Google Scholar]
  9. Strickland EC, Geer MA, Tran DT, Adhikari J, West GM. 9.  et al. 2013. Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation. Nat. Protoc. 8:148–61 [Google Scholar]
  10. West GM, Tucker CL, Xu T, Park SK, Han XM. 10.  et al. 2010. Quantitative proteomics approach for identifying protein-drug interactions in complex mixtures using protein stability measurements. Proc. Natl. Acad. Sci. USA 107:9078–82 [Google Scholar]
  11. Schellman JA.11.  1975. Macromolecular binding. Biopolymers 14:999–1018 [Google Scholar]
  12. Waldron TT, Murphy KP. 12.  2003. Stabilization of proteins by ligand binding: application to drug screening and determination of unfolding energetics. Biochemistry 42:5058–64 [Google Scholar]
  13. Ghaemmaghami S, Fitzgerald MC, Oas TG. 13.  2000. A quantitative, high-throughput screen for protein stability. Proc. Natl. Acad. Sci. USA 97:8296–301 [Google Scholar]
  14. Frego L, Gautschi E, Martin L, Davidson W. 14.  2006. The determination of high-affinity protein/inhibitor binding constants by electrospray ionization hydrogen/deuterium exchange mass spectrometry. Rapid Commun. Mass Spectrom. 20:2478–82 [Google Scholar]
  15. Liyanage R, Devarapalli N, Puckett LM, Phan NH, Gidden J. 15.  et al. 2009. Comparison of two ESI-MS based H/D exchange methods for extracting protein folding energies. Int. J. Mass Spectrom. 287:96–104 [Google Scholar]
  16. Powell KD, Fitzgerald MC. 16.  2001. Measurements of protein stability by H/D exchange and matrix-assisted laser desorption/ionization mass spectrometry using picomoles of material. Anal. Chem. 73:3300–4 [Google Scholar]
  17. Powell KD, Wang MZ, Silinski P, Ma LY, Wales TE. 17.  et al. 2003. The accuracy and precision of a new H/D exchange- and mass spectrometry-based technique for measuring the thermodynamic stability of proteins. Anal. Chim. Acta 496:225–32 [Google Scholar]
  18. Powell KD, Fitzgerald MC. 18.  2003. Accuracy and precision of a new H/D exchange- and mass spectrometry-based technique for measuring the thermodynamic properties of protein-peptide complexes. Biochemistry 42:4962–70 [Google Scholar]
  19. Dai SY, Gardner MW, Fitzgerald MC. 19.  2005. Protocol for the thermodynamic analysis of some proteins using an H/D exchange- and mass spectrometry-based technique. Anal. Chem. 77:693–97 [Google Scholar]
  20. Powell KD, Wales TE, Fitzgerald MC. 20.  2002. Thermodynamic stability measurements on multimeric proteins using a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based method. Protein Sci. 11:841–51 [Google Scholar]
  21. Esswein ST, Florance HV, Baillie L, Lippens J, Barran PE. 21.  2010. A comparison of mass spectrometry based hydrogen deuterium exchange methods for probing the cyclophilin A cyclosporin complex. J. Chromat. A 1217:6709–17 [Google Scholar]
  22. Wang MZ, Shetty JT, Howard BA, Campa MJ, Patz EF, Fitzgerald MC. 22.  2004. Thermodynamic analysis of cyclosporin A binding to cyclophilin A in a lung tumor tissue lysate. Anal. Chem. 76:4343–48 [Google Scholar]
  23. Ma L, Fitzgerald MC. 23.  2003. A new H/D exchange- and mass spectrometry-based method for thermodynamic analysis of protein-DNA interactions. Chem. Biol. 10:1205–13 [Google Scholar]
  24. Tang L, Hopper ED, Tong Y, Sadowsky JD, Peterson KJ. 24.  et al. 2007. H/D exchange- and mass spectrometry-based strategy for the thermodynamic analysis of protein-ligand binding. Anal. Chem. 79:5869–77 [Google Scholar]
  25. Roulhac PL, Powell KD, Dhungana S, Weaver KD, Mietzner TA. 25.  et al. 2004. SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange) analysis of the thermodynamics of synergistic anion binding by ferric-binding protein (FbpA), a bacterial transferrin. Biochemistry 43:15767–74 [Google Scholar]
  26. Tong Y, Wuebbens MM, Rajagopalan KV, Fitzgerald MC. 26.  2005. Thermodynamic analysis of subunit interactions in Escherichia coli molybdopterin synthase. Biochemistry 44:2595–601 [Google Scholar]
  27. Williams JC, Roulhac PL, Roy AG, Vallee RB, Fitzgerald MC, Hendrickson WA. 27.  2007. Structural and thermodynamic characterization of a cytoplasmic dynein light chain-intermediate chain complex. Proc. Natl. Acad. Sci. USA 104:10028–33 [Google Scholar]
  28. Xu Y, Schmitt S, Tang LJ, Jakob U, Fitzgerald MC. 28.  2010. Thermodynamic analysis of a molecular chaperone binding to unfolded protein substrates. Biochemistry 49:1346–53 [Google Scholar]
  29. Hopper ED, Pittman AMC, Fitzgerald MC, Tucker CL. 29.  2008. In vivo and in vitro examination of stability of primary hyperoxaluria-associated human alanine: glyoxylate aminotransferase. J. Biol. Chem. 283:30493–502 [Google Scholar]
  30. Frankel BA, Tong Y, Bentley ML, Fitzgerald MC, McCafferty DG. 30.  2007. Mutational analysis of active site residues in the Staphylococcus aureus transpeptidase SrtA. Biochemistry 46:7269–78 [Google Scholar]
  31. Reid CW, Brewer D, Clarke AJ. 31.  2004. Substrate binding affinity of Pseudomonas aeruginosa membrane-bound lytic transglycosylase B by hydrogen-deuterium exchange MALDI MS. Biochemistry 43:11275–82 [Google Scholar]
  32. Reid CW, Blackburn NT, Clarke AJ. 32.  2006. Role of arginine residues in the active site of the membrane-bound lytic transglycosylase B from Pseudomonas aeruginosa. Biochemistry 45:2129–38 [Google Scholar]
  33. Powell KD, Fitzgerald MC. 33.  2004. High-throughput screening assay for the tunable selection of protein ligands. J. Comb. Chem. 6:262–69 [Google Scholar]
  34. Hopper ED, Roulhac PL, Campa MJ, Patz EF Jr, Fitzgerald MC. 34.  2008. Throughput and efficiency of a mass spectrometry-based screening assay for protein-ligand binding detection. J. Am. Soc. Mass Spectrom. 19:1303–11 [Google Scholar]
  35. Dearmond PD, West GM, Anbalagan V, Campa MJ, Patz EF Jr, Fitzgerald MC. 35.  2010. Discovery of novel cyclophilin A ligands using an H/D exchange- and mass spectrometry-based strategy. J. Biomol. Screen. 15:1051–62 [Google Scholar]
  36. Roulhac PL, Weaver KD, Adhikari P, Anderson DS, DeArmond PD. 36.  et al. 2008. Ex vivo analysis of synergistic anion binding to FbpA in Gram-negative bacteria. Biochemistry 47:4298–305 [Google Scholar]
  37. Ghaemmaghami S, Oas TG. 37.  2001. Quantitative protein stability measurement in vivo. Nat. Struct. Biol. 8:879–82 [Google Scholar]
  38. Reichmann D, Xu Y, Cremers CM, Ilbert M, Mittelman R. 38.  et al. 2012. Order out of disorder: working cycle of an intrinsically unfolded chaperone. Cell 148:947–57 [Google Scholar]
  39. Tang LJ, Sundaram S, Zhang JM, Carlson P, Matathia A. 39.  et al. 2013. Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry. MAbs 5:114–25 [Google Scholar]
  40. Hopper ED, Pittman AM, Tucker CL, Campa MJ, Patz EF Jr, Fitzgerald MC. 40.  2009. Hydrogen/deuterium exchange- and protease digestion-based screening assay for protein-ligand binding detection. Anal. Chem. 81:6860–67 [Google Scholar]
  41. Park C, Marqusee S. 41.  2005. Pulse proteolysis: a simple method for quantitative determination of protein stability and ligand binding. Nat. Methods 2:207–12 [Google Scholar]
  42. Na YR, Park C. 42.  2009. Investigating protein unfolding kinetics by pulse proteolysis. Protein Sci. 18:268–76 [Google Scholar]
  43. Chiu J, Tillett D, March PE. 43.  2006. Mutation of Phe102 to Ser in the carboxyl terminal helix of Escherichia coli thioredoxin affects the stability and processivity of T7 DNA polymerase. Proteins 64:477–85 [Google Scholar]
  44. Hnizda A, Spiwok V, Jurga V, Kozich V, Kodicek M, Kraus JP. 44.  2010. Cross-talk between the catalytic core and the regulatory domain in cystathionine β-synthase: study by differential covalent labeling and computational modeling. Biochemistry 49:10526–34 [Google Scholar]
  45. Hnizda A, Majtan T, Liu L, Pey AL, Carpenter JF. 45.  et al. 2012. Conformational properties of nine purified cystathionine β-synthase mutants. Biochemistry 51:4755–63 [Google Scholar]
  46. Hnizda A, Jurga V, Rakova K, Kozich V. 46.  2012. Cystathionine beta-synthase mutants exhibit changes in protein unfolding: conformational analysis of misfolded variants in crude cell extracts. J. Inherit. Metab. Dis. 35:469–77 [Google Scholar]
  47. Okada J, Koga Y, Takano K, Kanaya S. 47.  2012. Slow unfolding pathway of hyperthermophilic Tk-RNase H2 examined by pulse proteolysis using the stable protease Tk-subtilisin. Biochemistry 51:9178–91 [Google Scholar]
  48. Roychaudhuri R, Yang MF, Condron MM, Teplow DB. 48.  2012. Structural dynamics of the amyloid β-protein monomer folding nucleus. Biochemistry 51:3957–59 [Google Scholar]
  49. Hanes MS, Ratcliff K, Marqusee S, Handel TM. 49.  2010. Protein-protein binding affinities by pulse proteolysis: application to TEM-1/BLIP protein complexes. Protein Sci. 19:1996–2000 [Google Scholar]
  50. Velkov T.50.  2009. Thermodynamics of lipophilic drug binding to intestinal fatty acid binding protein and permeation across membranes. Mol. Pharm. 6:557–70 [Google Scholar]
  51. Schlebach JP, Kim MS, Joh NH, Bowie JU, Park C. 51.  2011. Probing membrane protein unfolding with pulse proteolysis. J. Mol. Biol. 406:545–51 [Google Scholar]
  52. London E, Khorana HG. 52.  1982. Denaturation and renaturation of bacteriorhodopsin in detergents and lipid-detergent mixtures. J. Biol. Chem. 257:7003–11 [Google Scholar]
  53. Chen GQ, Gouaux E. 53.  1999. Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: evaluation of reversible unfolding conditions. Biochemistry 38:15380–87 [Google Scholar]
  54. Kim MS, Song J, Park C. 54.  2009. Determining protein stability in cell lysates by pulse proteolysis and Western blotting. Protein Sci. 18:1051–59 [Google Scholar]
  55. Liu PF, Kihara D, Park C. 55.  2011. Energetics-based discovery of protein-ligand interactions on a proteomic scale. J. Mol. Biol. 408:147–62 [Google Scholar]
  56. Keseler IM, Collado-Vides J, Gama-Castro S, Ingraham J, Paley S. 56.  et al. 2005. EcoCyc: a comprehensive database resource for Escherichia coli. Nucleic Acids Res. 33:D334–37 [Google Scholar]
  57. Chang Y, Schlebach JP, VerHeul RA, Park C. 57.  2012. Simplified proteomics approach to discover protein-ligand interactions. Protein Sci. 21:1280–87 [Google Scholar]
  58. West GM, Tang L, Fitzgerald MC. 58.  2008. Thermodynamic analysis of protein stability and ligand binding using a chemical modification- and mass spectrometry-based strategy. Anal Chem 80:4175–85 [Google Scholar]
  59. West GM, Thompson JW, Soderblom EJ, Dubois LG, Dearmond PD. 59.  et al. 2010. Mass spectrometry-based thermal shift assay for protein-ligand binding analysis. Anal. Chem. 82:5573–81 [Google Scholar]
  60. Strickland EC, Geer MA, Hong J, Fitzgerald MC. 60.  2014. False-positive rate determination of protein target discovery using a covalent modification- and mass spectrometry-based proteomics platform. J. Am. Soc. Mass Spectrom. 25:132–40 [Google Scholar]
  61. DeArmond PD, West GM, Huang HT, Fitzgerald MC. 61.  2011. Stable isotope labeling strategy for protein-ligand binding analysis in multi-component protein mixtures. J. Am. Soc. Mass Spectrom. 22:418–30 [Google Scholar]
  62. 62.  Deleted in proof
  63. Tran DT, Banerjee S, Alayash AI, Crumbliss AL, Fitzgerald MC. 63.  2012. Slow histidine H/D exchange protocol for thermodynamic analysis of protein folding and stability using mass spectrometry. Anal. Chem. 84:1653–60 [Google Scholar]
  64. Miyagi M, Nakazawa T. 64.  2008. Determination of pKa values of individual histidine residues in proteins using mass spectrometry. Anal. Chem. 80:6481–87 [Google Scholar]
  65. Baud F, Karlin S. 65.  1999. Measures of residue density in protein structures. Proc. Natl. Acad. Sci. USA 96:12494–99 [Google Scholar]
  66. Isom DG, Vardy E, Oas TG, Hellinga HW. 66.  2010. Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity. Proc. Natl. Acad. Sci. USA 107:4908–13 [Google Scholar]
  67. Xu Y, Falk IN, Hallen MA, Fitzgerald MC. 67.  2011. Mass spectrometry- and lysine amidination-based protocol for thermodynamic analysis of protein folding and ligand binding interactions. Anal. Chem. 83:3555–62 [Google Scholar]
  68. Isom DG, Marguet PR, Oas TG, Hellinga HW. 68.  2011. A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity. Proteins 79:1034–47 [Google Scholar]

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