Determining the CD Spectrum of a Protein

Roger Pain1

1 Jozef Stefan Institute, Ljubljana
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.6
DOI:  10.1002/0471140864.ps0706s38
Online Posting Date:  January, 2005
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This unit describes the theory behind circular dichroism (CD) and deals with considerations regarding instrumentation and reagents for CD spectrometry. A protocol is provided that outlines the steps in recording a CD spectrum and two support protocols explain the interpretation of near‐UV and far‐UV CD spectra.

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Recording a CD Spectrum
  • Support Protocol 1: Interpretation of Near‐UV CD Spectra
  • Support Protocol 2: Interpretation of Far‐UV CD Spectra
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Recording a CD Spectrum

  • Buffer solution
  • Clarified protein solution for analysis
  • Nitrogen supply
  • CD spectrometer (calibrated) and cells
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Literature Cited

   Bayley, P.M. 1980. Circular dichroism and optical rotation. In An Introduction to Spectroscopy for Biochemists (S.B. Brown, ed.) pp. 148‐235. Academic Press, London.
   Chaffotte, A.F., Guillou, Y., and Goldberg, M.E. 1992. Kinetic resolution of peptide bond and side chain far‐UV circular dichroism during the folding of hen egg white lysozyme. Biochemistry 31:9694‐9702.
   Chen, G.C. and Yang, J.T. 1977. Two‐point calibration of circular dichrometer with D‐10‐camphorsulfonic acid. Anal. Lett. 10:1195‐1207.
   Craig, S., Hollecker, M., Creighton, T.E., and Pain, R.H. 1985. Single amino acid mutations block a late step in the folding of β‐lactamase from Staphylococcus aureus. J. Mol. Biol. 185:681‐687.
   Craig, S., Pain, R.H., Schmeissner, U., Virden, R., and Wingfield, P.T. 1989. Determination of the contributions of individual aromatic residues to the CD spectrum of IL‐1β using site directed mutagenesis. Int. J. Peptide Protein Res. 33:256‐262.
   Farrell, H.M. Jr., Wickham, E.D., Unruh, J.J., Qi, P.X., and Hoagland, P.D. 2001. Secondary structural studies of bovine caseins: Temperature dependence of β‐casein structure as analyzed by circular dichroism and FTIR spectroscopy and correlation with micellization. Food Hydrocolloids 15:341‐354.
   Gray, D.M., Gray, C.W., Mou, T.C., and Wen, J.D. 2002. CD of single‐stranded, double‐stranded, and G‐quartet nucleic acids in complexes with a single‐stranded DNA‐binding protein. Enantiomer 7:49‐58.
   Greenfield, N.J. 1996. Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Anal. Biochem. 235:1‐10.
   Herzberg, O., Kapadia, G., Blanco, B., Smith, T.S., and Coulson, A. 1991. Structural basis for the inactivation of the P54 mutant of β‐lactamase from Staphylococcus aureus PC1. Biochemistry 30:9503‐9509.
   Holzwarth, G. and Doty, P. 1965. The ultraviolet circular dichroism of proteins. J. Am. Chem. Soc. 87:218‐228.
   Huang, X., Nakanishi, K., and Berova, N. 2000. Porphyrins and metalloporphyrins: Versatile circular dichroic reporter groups for structural studies. Chirality 12:237‐255.
   Johnson, W.C. Jr. 1987. The circular dichroism of carbohydrates. Adv. Carbohydr. Chem. Biochem. 45:73‐124.
   Johnson, W.C. 1990. Protein secondary structure and circular dichroism: A practical guide. Proteins: Struct., Funct., Genet. 7:205‐214.
   Johnson, W.C. 1999. Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins: Struct., Funct., Genet. 35:307‐312.
   Kamal, J.K. and Behere, D.V. 2002. Thermal and conformational stability of seed coat soybean peroxidase. Biochemistry 41:9034‐9042.
   Kidric, M., Fabian, H., Brzin, J., Popovic, T., and Pain, R.H. 2002. Folding, stability, and secondary structure of a new dimeric cysteine proteinase inhibitor. Biochem. Biophys. Res. Commun. 297:962‐967.
   Koepf, E.K., Petrassi, H.M., Sudol, M., and Kelly, J.W. 1999. WW: An isolated three‐stranded antiparallel β‐sheet domain that unfolds and refolds reversibly; evidence for a structured hydrophobic cluster in urea and GdnHCl and a disordered thermal unfolded state. Protein Sci. 8:841‐853.
   Kuwajima, K. and Arai, M. 2000. The molten globule state: The physical picture and biological significance. In Mechanisms of Protein Folding (R.H. Pain, ed.) pp. 138‐174. Oxford University Press, Oxford.
   Manning, M.C. 1989. Underlying assumptions in the estimation of secondary structure content in proteins by circular dichroism spectroscopy—a critical review. J. Pharmacol. Biomed. Anal. 7:1103‐1119.
   Ogawa, M., Kanamaru, J., Miyashita, H., Tamiya, T., and Tsuchiya, T. 1995. α‐Helical structure of fish actomyosin: Changes during setting. J. Food Sci. 60:297‐299.
   Pain, R.H., Lah, T., and Turk, V. 1985. Conformation and processing of cathepsin D. Biosci. Rep. 5:957‐967.
   Provencher, S.W. and Glöckner, J. 1981. Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20:33‐37.
   Schmid, F.X. 1989. Spectral methods of characterizing protein conformation and conformational changes. In Protein Structure (T.E. Creighton, ed.) pp. 251‐285. IRL Press, Oxford.
   Sreerama, N., Manning, M.C., Powers, M.E., Zhang, J.X., Goldenberg, D.P., and Woody, R.W. 1999a. Tyrosine, phenylalanine, and disulfide contributions to the circular dichroism of proteins: Circular dichroism spectra of wild‐type and mutant bovine pancreatic trypsin inhibitor. Biochemistry 38:10814‐10822.
   Sreerama, N., Venyaminov, S.Y., and Woody, R.W. 1999b. Estimation of the number of α‐helical and β‐strand segments in proteins using circular dichroism spectroscopy. Protein Sci. 8:370‐380.
   Strickland, E.H. 1974. Aromatic contributions to circular dichroism spectra of proteins. C.R.C. Crit. Rev. Biochem. 2:113‐175.
   Swords, N.A. and Wallace, B.A. 1993. Circular dichroism analyses of membrane proteins: Examination of environmental effects on bacteriorhodopsin spectra. Biochem. J. 289:215‐219.
   Wingfield, P., Graber, P., Moonen, P., Craig, S., and Pain, R.H. 1988. The conformation and stability of recombinant‐derived granulocyte‐macrophage colony stimulating factors. Eur. J. Biochem. 173:65‐72.
   Yang, J.T., Wu, C.‐S., and Martinez, H.M. 1986. Calculation of protein conformation from circular dichroism. Methods Enzymol. 130:208‐269.
Key References
   Bayley, 1980. See above
  A good introduction to the principles and practice of circular dichroism.
   Johnson, 1990. See above.
  A good account of the practice of far‐UV CD spectroscopy and a description and assessment of the determination of protein secondary structure content.
   Sears, D.W. and Beychok, S. 1973. Circular dichroism. In Physical Principles and Techniques of Protein Chemistry, Part C (S.J. Leach, ed.) pp. 445‐593. Academic Press, New York and London.
  A comprehensive review of the theory of CD, together with a useful discussion of near‐UV spectra in particular for selected proteins.
   Strickland, 1974. See above.
  An older but still excellent account of the practice, spectroscopic basis, and interpretation of the near‐UV CD spectroscopy of proteins and model compounds.
   Yang et al., 1986. See above.
  A good account of the practice of far‐UV CD spectroscopy and a description and assessment of the determination of protein secondary structure content.
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