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Using circular dichroism spectra to estimate protein secondary structure

Abstract

Circular dichroism (CD) is an excellent tool for rapid determination of the secondary structure and folding properties of proteins that have been obtained using recombinant techniques or purified from tissues. The most widely used applications of protein CD are to determine whether an expressed, purified protein is folded, or if a mutation affects its conformation or stability. In addition, it can be used to study protein interactions. This protocol details the basic steps of obtaining and interpreting CD data, and methods for analyzing spectra to estimate the secondary structural composition of proteins. CD has the advantage that measurements may be made on multiple samples containing ≤20 μg of proteins in physiological buffers in a few hours. However, it does not give the residue-specific information that can be obtained by x-ray crystallography or NMR.

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Figure 1: CD spectra of polypeptides and proteins with representative secondary structures.
Figure 2: Circular dichroism spectra of lysozyme in 10 mM sodium phosphate pH 7.0.

References

  1. Velluz, L., Legrand, M. & Grosjean, M. Optical Circular Dichroism: Principles, Measurements, and Applications (Verlag Chemie Academic Press Inc., New York and London, 1965).

    Google Scholar 

  2. Beychok, S. Circular dichroism of biological macromolecules. Science 154, 1288–1299 (1966).

    CAS  Article  Google Scholar 

  3. Sreerama, N. & Woody, R.W. Computation and analysis of protein circular dichroism spectra. Methods Enzymol. 383, 318–351 (2004).

    CAS  Article  Google Scholar 

  4. Holzwarth, G. & Doty, P. The ultraviolet circular dichroism of polypeptides. J. Am. Chem. Soc. 87, 218–228 (1965).

    CAS  Article  Google Scholar 

  5. Greenfield, N. & Fasman, G.D. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 8, 4108–4116 (1969).

    CAS  Article  Google Scholar 

  6. Venyaminov, S., Baikalov, I.A., Shen, Z.M., Wu, C.S. & Yang, J.T. Circular dichroic analysis of denatured proteins: inclusion of denatured proteins in the reference set. Anal. Biochem. 214, 17–24 (1993).

    CAS  Article  Google Scholar 

  7. Bovey, F.A. & Hood, F.P. Circular dichroism spectrum of poly-L-proline. Biopolymers 5, 325–326 (1967).

    CAS  Article  Google Scholar 

  8. Tiffany, M.L. & Krimm, S. Effect of temperature on the circular dichroism spectra of polypeptides in the extended state. Biopolymers 11, 2309–2316 (1972).

    CAS  Article  Google Scholar 

  9. Woody, R.W. Circular dichroism and conformation of unordered polypeptides. Adv. Biophys. Chem. 2, 31–79 (1992).

    Google Scholar 

  10. Rucker, A.L. & Creamer, T.P. Polyproline II helical structure in protein unfolded states: lysine peptides revisited. Protein Sci. 11, 980–985 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Nafie, L.A. Infrared and Raman vibrational optical activity: theoretical and experimental aspects. Annu. Rev. Phys. Chem. 48, 357–386 (1997).

    CAS  Article  Google Scholar 

  12. Jackson, M. & Mantsch, H.H. The use and misuse of FTIR spectroscopy in the determination of protein structure. Crit. Rev. Biochem. Mol. Biol. 30, 95–120 (1995).

    CAS  Article  Google Scholar 

  13. Cooper, E.A. & Knutson, K. Fourier transform infrared spectroscopy investigations of protein structure. Pharm. Biotechnol. 7, 101–143 (1995).

    CAS  Article  Google Scholar 

  14. Pelton, J.T. & McLean, L.R. Spectroscopic methods for analysis of protein secondary structure. Anal. Biochem. 277, 167–176 (2000).

    CAS  Article  Google Scholar 

  15. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951).

    CAS  PubMed  Google Scholar 

  16. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254 (1976).

    CAS  Article  Google Scholar 

  17. Hennessey, J.P. Jr. & Johnson, W.C. Jr. Information content in the circular dichroism of proteins. Biochemistry 20, 1085–1094 (1981).

    CAS  Article  Google Scholar 

  18. Lang, C.A. Simple microdetermination of Kjeldahl nitrogen in biological material. Anal. Chem. 30, 1692–1694 (1958).

    CAS  Article  Google Scholar 

  19. Jaenicke, L. A rapid micromethod for the determination of nitrogen and phosphate in biological material. Anal. Biochem. 61, 623–627 (1974).

    CAS  Article  Google Scholar 

  20. Brahms, S. & Brahms, J. Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. J. Mol. Biol. 138, 149–178 (1980).

    CAS  Article  Google Scholar 

  21. Reed, J. & Reed, T.A. A set of constructed type spectra for the practical estimation of peptide secondary structure from circular dichroism. Anal. Biochem. 254, 36–40 (1997).

    CAS  Article  Google Scholar 

  22. Saxena, V.P. & Wetlaufer, D.B. A new basis for interpreting the circular dichroic spectra of proteins. Proc. Natl Acad. Sci. USA 68, 969–972 (1971).

    CAS  Article  Google Scholar 

  23. Chen, Y.H., Yang, J.T. & Chau, K.H. Determination of the helix and β form of proteins in aqueous solution by circular dichroism. Biochemistry 13, 3350–3359 (1974).

    CAS  Article  Google Scholar 

  24. Chang, C.T., Wu, C.S. & Yang, J.T. Circular dichroic analysis of protein conformation: inclusion of the β-turns. Anal. Biochem. 91, 13–31 (1978).

    CAS  Article  Google Scholar 

  25. Yang, J.T., Wu, C.S. & Martinez, H.M. Calculation of protein conformation from circular dichroism. Methods Enzymol. 130, 208–269 (1986).

    CAS  Article  Google Scholar 

  26. Greenfield, N.J. Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Anal. Biochem. 235, 1–10 (1996).

    CAS  Article  Google Scholar 

  27. Provencher, S.W. & Glöckner, J. Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20, 33–37 (1981).

    CAS  Article  Google Scholar 

  28. Manavalan, P. & Johnson, W.C. Jr. Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Anal. Biochem. 167, 76–85 (1987).

    CAS  Article  Google Scholar 

  29. Toumadje, A., Alcorn, S.W. & Johnson, W.C. Jr. Extending CD spectra of proteins to 168 nm improves the analysis for secondary structures. Anal. Biochem. 200, 321–331 (1992).

    CAS  Article  Google Scholar 

  30. Toumadje, A. & Johnson, W.C. Jr. Effects of relative band intensity on prediction of protein secondary structure from CD. Anal. Biochem. 211, 258–260 (1993).

    CAS  Article  Google Scholar 

  31. Johnson, W.C. Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins 35, 307–312 (1999).

    CAS  Article  Google Scholar 

  32. Sreerama, N. & Woody, R.W. A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal. Biochem. 209, 32–44 (1993).

    CAS  Article  Google Scholar 

  33. Sreerama, N. & Woody, R.W. Protein secondary structure from circular dichroism spectroscopy. Combining variable selection principle and cluster analysis with neural network, ridge regression and self-consistent methods. J. Mol. Biol. 242, 497–507 (1994).

    CAS  PubMed  Google Scholar 

  34. Sreerama, N., Venyaminov, S.Y. & Woody, R.W. Estimation of the number of α-helical and β-strand segments in proteins using circular dichroism spectroscopy. Protein Sci. 8, 370–380 (1999).

    CAS  Article  Google Scholar 

  35. Sreerama, N., Venyaminov, S.Y. & Woody, R.W. Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal. Biochem. 287, 243–251 (2000).

    CAS  Article  Google Scholar 

  36. Sreerama, N. & Woody, R.W. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem. 287, 252–260 (2000).

    CAS  Article  Google Scholar 

  37. Böhm, G., Muhr, R. & Jaenicke, R. Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng. 5, 191–195 (1992).

    Article  Google Scholar 

  38. Andrade, M.A., Chacón, P., Merelo, J.J. & Morán, F. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng. 6, 383–390 (1993).

    CAS  Article  Google Scholar 

  39. Unneberg, P., Merelo, J.J., Chacón, P. & Morán, F. SOMCD: method for evaluating protein secondary structure from UV circular dichroism spectra. Proteins 42, 460–470 (2001).

    CAS  Article  Google Scholar 

  40. Perczel, A., Park, K. & Fasman, G.D. Analysis of the circular dichroism spectrum of proteins using the convex constraint algorithm: a practical guide. Anal. Biochem. 203, 83–93 (1992).

    CAS  Article  Google Scholar 

  41. Chen, Y.H. & Yang, J.T. A new approach to the calculation of secondary structures of globular proteins by optical rotatory dispersion and circular dichroism. Biochem. Biophys. Res. Commun. 44, 1285–1291 (1971).

    CAS  Article  Google Scholar 

  42. Chen, Y.H., Yang, J.T. & Martinez, H.M. Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry 11, 4120–4131 (1972).

    CAS  Article  Google Scholar 

  43. Sreerama, N. & Woody, R.W. Poly(pro)II helices in globular proteins: identification and circular dichroic analysis. Biochemistry 33, 10022–10025 (1994).

    CAS  Article  Google Scholar 

  44. Perczel, A., Hollósi, M., Tusnády, G. & Fasman, G.D. Convex constraint analysis: a natural deconvolution of circular dichroism curves of proteins. Protein Eng. 4, 669–679 (1991).

    CAS  Article  Google Scholar 

  45. Greenfield, N.J. & Hitchcock-DeGregori, S.E. Conformational intermediates in the folding of a coiled-coil model peptide of the N-terminus of tropomyosin and α-tropomyosin. Protein Sci. 2, 1263–1273 (1993).

    CAS  Article  Google Scholar 

  46. Safar, J., Roller, P.P., Gajdusek, D.C. & Gibbs, C.J. Jr. Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity. Protein Sci. 2, 2206–2216 (1993).

    CAS  Article  Google Scholar 

  47. Greenfield, N.J. Using circular dichroism, collected as a function of temperature, to determine the thermodynamics of protein unfolding and binding interactions. Nat. Protoc. doi: 10.1038/nprot.2006.204 (2006).

  48. Adler, A.J., Greenfield, N.J. & Fasman, G.D. Circular dichroism and optical rotatory dispersion of proteins and polypeptides. Methods Enzymol. 27, 675–735 (1973).

    CAS  Article  Google Scholar 

  49. Rosenkranz, H. Circular dichroism of helical and nonhelical proteins. A review of the limits of the methods for calculating secondary structure. Z. Klin. Chem. Klin. Biochem. 12, 222 (1974).

    CAS  PubMed  Google Scholar 

  50. Johnson, W.C. Jr. Protein secondary structure and circular dichroism: a practical guide. Proteins 7, 205–214 (1990).

    CAS  Article  Google Scholar 

  51. Woody, R.W. Circular dichroism. Methods Enzymol. 246, 34–71 (1995).

    CAS  Article  Google Scholar 

  52. Kelly, S.M., Jess, T.J. & Price, N.C. How to study proteins by circular dichroism. Biochim. Biophys. Acta 1751, 119–139 (2005).

    CAS  Article  Google Scholar 

  53. Miles, A.J. & Wallace, B.A. Synchrotron radiation circular dichroism spectroscopy of proteins and applications in structural and functional genomics. Chem. Soc. Rev. 35, 39–51 (2006).

    CAS  Article  Google Scholar 

  54. Venyaminov, S.Y. & Yang,, J.T. in Circular Dichroism and the Conformational Analysis of Biomolecules (ed. Fasman, G.D.) (Plenum Press, New York and London, 1996).

    Google Scholar 

  55. Greenfield, N.J. Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism. Nat. Protoc. doi: 10.1038/nprot.2006.229 (2006).

  56. Greenfield, N.J. The use of circular dichroism to study the kinetics of protein folding and unfolding. Nat. Protoc. doi: 10.1038/nprot.2006.244 (2006).

  57. Miles, A.J., Whitmore, L. & Wallace, B.A. Spectral magnitude effects on the analyses of secondary structure from circular dichroism spectroscopic data. Protein Sci. 14, 368–374 (2005).

    CAS  Article  Google Scholar 

  58. Edelhoch, H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948–1954 (1967).

    CAS  Article  Google Scholar 

  59. Mihalyi, E. Numerical values of the absorbances of the aromatic amino acids in acid, neutral, and alkaline solutions. J. Chem. Eng. Data 13, 179–182 (1968).

    CAS  Article  Google Scholar 

  60. Goa, J. A micro biuret method for protein determination. Scand. J. Clin. Lab. Invest. 5, 218–222 (1953).

    CAS  Article  Google Scholar 

  61. Konno, T. Conformational diversity of acid-denatured cytochrome c studied by a matrix analysis of far-UV CD spectra. Protein Sci. 7, 975–982 (1998).

    CAS  Article  Google Scholar 

  62. Melberg, S.G. & Johnson, W.C. Jr. Changes in secondary structure follow the dissociation of human insulin hexamers: a circular dichroism study. Proteins 8, 280–286 (1990).

    CAS  Article  Google Scholar 

  63. Savitsky, A. & Golay, M.J.E. Smoothing and dIfferentiation of data by simplified least squares procedures. Anal. Chem. 36, 1627–1639 (1964).

    Article  Google Scholar 

  64. Bentz, H., Bächinger, H.P., Glanville, R. & Kühn, K. Physical evidence for the assembly of A and B chains of human placental collagen in a single triple helix. Eur. J. Biochem. 92, 563–567 (1978).

    CAS  Article  Google Scholar 

  65. Steiner, R.F. Structural transitions of lysozyme. Biochim. Biophys. Acta 79, 51–63 (1964).

    CAS  PubMed  Google Scholar 

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Acknowledgements

I thank M.A. Andrade, W.C. Johnson, Jr., N. Sreerama, R.W. Woody, S. Venyaminov, and especially the late Gerald D. Fasman for supplying versions of their software for analyzing CD data for use, analysis and distribution. The research was supported by a National Institutes of Health grant, GM-36326 to N.J.G. and to Sarah E. Hitchcock-DeGregori and by the Circular Dichroism Facility at Robert Wood Johnson Medical School (UMDNJ).

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Greenfield, N. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1, 2876–2890 (2006). https://doi.org/10.1038/nprot.2006.202

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