Circular Dichroism in Protein Folding Studies

David T. Clarke1

1 Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, Oxfordshire, United Kingdom
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 28.3
DOI:  10.1002/0471140864.ps2803s70
Online Posting Date:  November, 2012
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Protein folding is a biological process of both fundamental significance and practical importance, and protein misfolding is implicated in a number of serious diseases of both humans and animals. The study of protein folding requires a technique that is able to monitor changes in protein structure in solution, with millisecond time resolution. Ultraviolet circular dichroism (CD) is such a technique, providing information on both secondary and tertiary protein structure. This unit describes the procedures for performing CD experiments for the study of protein folding, and identifies commonly encountered problems and their solutions. Curr. Protoc. Protein Sci. 70:28.3.1‐28.3.17. © 2012 by John Wiley & Sons, Inc.

Keywords: protein folding; circular dichroism; stopped‐flow; protein structure

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Table of Contents

  • Introduction
  • Basic Protocol 1: Characterization of Samples Using Steady‐State CD Spectroscopy
  • Support Protocol 1: Accurate Determination of Cell Pathlength
  • Basic Protocol 2: Assessment of the Conditions to be Used for the Stopped‐Flow Experiment
  • Support Protocol 2: Analysis of Steady‐State CD Spectra for Secondary Structure Content
  • Basic Protocol 3: Running a Stopped‐Flow CD Experiment
  • Support Protocol 3: Testing Performance of Stopped‐Flow Apparatus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Characterization of Samples Using Steady‐State CD Spectroscopy

  • CD spectrometer. These are available from a number of manufacturers (e.g., Jasco Corporation, Applied Photophysics, or Olis Inc.). The details of operation vary, so the experimenter should consult the operation manual in addition to the generic protocol given below. It is assumed that the spectrometer is well maintained and calibrated. Guidelines for identification of common instrument problems are given in the troubleshooting section below.
  • CD sample cells. Short‐pathlength cells are required for far‐UV CD to minimize absorption of light at the shorter wavelengths by water, buffer components, and denaturants. Cells suitable for CD are available in two types: bottle cells, which are sealed units into which the sample is introduced through a filling hole (e.g., Hellma Analytics 121 series), and demountable cells, which consist of two plates between which a drop of the sample is sandwiched (e.g., Hellma Analytics 124 series). In general, demountable cells are preferred for very short pathlengths (<0.5 mm), as the bottle cells are difficult to fill and require more sample. Calibration of cell pathlength is important with short‐pathlength cells, and is described in protocol 2.
  • Sample buffer for folded state. The choice of buffer depends on the requirements of the protein under investigation. Where possible, the concentrations of buffer salts should be kept to a minimum to avoid absorbance problems, particularly for far‐UV CD. Low‐concentration phosphate buffers (10 to 20 mM) are suitable. Addition of salts such as sodium chloride will increase absorbance and therefore reduce wavelength range and signal‐to‐noise of the short‐wavelength data.
  • Purified protein in sample buffer. Preferably at a concentration between 0.5 and 10 mg/ml. Protein concentration should be known as accurately as possible for secondary structure analysis.
  • Denaturant solution at a range of concentrations. For example, from 0.5 to 6.0 M. Generally, urea is preferred because it does not alter ionic strength. If the protein does not unfold in saturated urea solution, guanidine hydrochloride can be used. Because high concentrations are required, the highest grade reagent obtainable should be used.
  • Protein in denaturant solutions.

Support Protocol 1: Accurate Determination of Cell Pathlength

  • Cells to be used for CD measurement
  • Spectrophotometer or CD spectrometer set up to measure absorbance

Basic Protocol 2: Assessment of the Conditions to be Used for the Stopped‐Flow Experiment

  • CD data (from protocol 1)
  • Software for simple manipulation of CD spectra. Standard spreadsheet software can be used for this purpose. A specialized software package for processing CD data, cdtool, is also available, and can be downloaded from (Lees et al., )

Support Protocol 2: Analysis of Steady‐State CD Spectra for Secondary Structure Content

  • Computer loaded with CD data for analysis, with Internet access

Basic Protocol 3: Running a Stopped‐Flow CD Experiment

  • Stopped‐flow CD apparatus (e.g., Applied Photophysics SX20, Bio‐Logic SFM‐2000)
  • Sample dilution buffer (see Materials section of protocol 1)
  • Protein dissolved in denaturant—denaturant concentration will have been determined from the steady‐state measurements described in protocol 1; select a protein concentration appropriate to the pathlength of the stopped‐flow apparatus, using the guidelines in protocol 1, step 3
  • Protein dissolved in dilution buffer, at the same concentration as the protein in denaturant

Support Protocol 3: Testing Performance of Stopped‐Flow Apparatus

  • 0.5 mM 2,6‐dichloroindophenol (DCIP; see recipe; this stock solution is diluted down to a range of concentrations as described in the steps below and in Reagents and Solutions)
  • 100 mM ascorbic acid/0.1 M NaCl, pH 5.0 (see recipe)
  • Stopped‐flow apparatus set up to measure absorbance
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Literature Cited

   Akiyama, S., Takahashi, S., Ishimori, K., and Morishima, I. 2000. Stepwise formation of alpha‐helices during cytochrome c folding. Nat. Struct. Biol. 7:514‐520.
   Andrade, M.A., Chacon, P., Merelo, J.J., and Moran, F. 1993. Evaluation of secondary structure of proteins from uv circular‐dichroism spectra using an unsupervised learning neural‐network. Protein Eng. 6:383‐390.
   Arai, M., Iwakura, M., Matthews, C.R., and Bilsel, O. 2011. Microsecond subdomain folding in dihydrofolate reductase. J. Mol. Biol. 410:329‐342.
   Bai, Y. (ed.) 2006. Protein Folding Protocols. Humana Press, New York.
   Clarke, D.T. and Jones, G. 2004. CD12: A new high‐flux beamline for ultraviolet and vacuum‐ultraviolet circular dichroism on the SRS, Daresbury. J. Synchrotron Radiat. 11:142‐149.
   Correa, D.H.A. and Ramos, C.H.I. 2009. The use of circular dichroism spectroscopy to study protein folding, form and function. African J. Biochem. Res. 3:164‐173.
   Dobson, C.M. and Fersht, A.R. (eds.) 1996. Protein Folding. Cambridge University Press, Cambridge, U.K.
   Dumont, C., Emilsson, T., and Gruebele, M. 2009. Reaching the protein folding speed limit with large, sub‐microsecond pressure jumps. Nat. Methods 6:U515‐U570.
   Dyer, R.B., Gai, F., and Woodruff, W.H. 1998. Infrared studies of fast events in protein folding. Acc. Chem. Res. 31:709‐716.
   Fasman, G.D. (ed.) 2010. Circular Dichroism and the Conformational Analysis of Biomolecules. Springer, New York.
   Gruenewald, B. and Knoche, W. 1978. Pressure jump method with detection of optical‐rotation and circular‐dichroism. Rev. Sci. Instrum. 49:797‐801.
   Huang, C.Y., Balakrishnan, G., and Spiro, T.G. 2005. Early events in apomyoglobin unfolding probed by laser T‐jump/UV resonance Raman spectroscopy. Biochemistry 44:15734‐15742.
   Jenkins, D.C., Pearson, D.S., Harvey, A., Sylvester, I.D., Geeves, M.A., and Pinheiro, T.J.T. 2009. Rapid folding of the prion protein captured by pressure‐jump. Eur. Biophys. J. Biophys. Lett. 38:625‐635.
   Jones, G.R. and Clarke, D.T. 2004. Applications of extended ultra‐violet circular dichroism spectroscopy in biology and medicine. Faraday Discuss. 126:223‐236.
   Kelly, S.M. and Price, N.C. 1997. The application of circular dichroism to studies of protein folding and unfolding. Biochim. Biophys. Acta 1338:161‐185.
   Khuc, M.T., Mendonca, L., Sharma, S., Solinas, X., Volk, M., and Hache, F. 2011. Measurement of circular dichroism dynamics in a nanosecond temperature‐jump experiment. Rev. Sci. Instrum. 82:054302.
   Lees, J.G., Smith, B.R., Wien, F., Miles, A.J., and Wallace, B.A. 2004. CDtool an integrated software package for circular dichroism spectroscopic data processing, analysis, and archiving. Anal. Biochem. 332:285‐289.
   Manavalan, P. and Johnson, W.C. 1985. Protein secondary structure from circular‐dichroism spectra. J. Biosci. 8:141‐149.
   Manavalan, P. and Johnson, W.C. 1987. Variable selection method improves the prediction of protein secondary structure from circular‐dichroism spectra. Anal. Biochem. 167:76‐85.
   Manolopoulos, S., Clarke, D., Derbyshire, G., Jones, G., Read, P., and Torbet, M. 2004. A new multichannel detector for proteomics studies and circular dichroism. Nucl. Instrum. Meth. Phys. Res. A 531:302‐306.
   Maxwell, K.L., Wildes, D., Zarrine‐Afsar, A., de los Rios, M.A., Brown, A.G., Friel, C.T., Hedberg, L., Horng, J.C., Bona, D., Miller, E.J., Vallee‐Belisle, A., Main, E.R.G., Bemporad, F., Qiu, L.L., Teilum, K., Vu, N.D., Edwards, A.M., Ruczinski, I., Poulsen, F.M., Kragelund, B.B., Michnick, S.W., Chiti, F., Bai, Y.W., Hagen, S.J., Serrano, L., Oliveberg, M., Raleigh, D.P., Wittung‐Stafshede, P., Radford, S.E., Jackson, S.E., Sosnick, T.R., Marqusee, S., Davidson, A.R., and Plaxco, K.W. 2005. Protein folding: Defining a “standard” set of experimental conditions and a preliminary kinetic data set of two‐state proteins. Protein Sci. 14:602‐616.
   Miles, A.J., Janes, R.W., Brown, A., Clarke, D.T., Sutherland, J.C., Tao, Y., Wallace, B.A., and Hoffmann, S.V. 2008. Light flux density threshold at which protein denaturation is induced by synchrotron radiation circular dichroism beamlines. J. Synchrotron Radiat. 15:420‐422.
   Munoz, V. (ed.) 2008. Protein Folding, Misfolding and Aggregation: Classical Themes and Novel Approaches. Royal Society of Chemistry, London, U.K.
   Nienhaus, G.U. 2009. Single‐molecule fluorescence studies of protein folding. In Methods in Molecular Biology, vol. 490 (J.W. Shriver, ed.) pp. 311‐337. Humana Press, Totowa, N.J.
   Norden, B., Rodger, A., and Dafforn, T. 2010. Linear Dichroism and Circular Dichroism: A Textbook on Polarized‐Light Spectroscopy. Royal Society of Chemistry, London, U.K.
   Noronha, M., Gerbelova, H., Faria, T.Q., Lund, D.N., Smith, D.A., Santos, H., and Macanita, A.L. 2010. Thermal unfolding kinetics of ubiquitin in the microsecond‐to‐second time range probed by Tyr‐59 fluorescence. J. Phys. Chem. B 114:9912‐9919.
   Ovadi, J. and Orosz, F. (eds.) 2009. Protein folding and misfolding: Neurodegenerative diseases Springer, New York.
   Pollack, L., Tate, M.W., Finnefrock, A.C., Kalidas, C., Trotter, S., Darnton, N.C., Lurio, L., Austin, R.H., Batt, C.A., Gruner, S.M., and Mochrie, S.G.J. 2001. Time resolved collapse of a folding protein observed with small angle x‐ray scattering. Phys. Rev. Lett. 86:4962‐4965.
   Provencher, S.W. and Glockner, J. 1981. Estimation of globular protein secondary structure from circular‐dichroism. Biochemistry 20:33‐37.
   Rami, B.R. and Udgaonkar, J.B. 2001. pH‐jump‐induced folding and unfolding studies of barstar: Evidence for multiple folding and unfolding pathways. Biochemistry 40:15267‐15279.
   Royer, C.A. 2006. Probing protein folding and conformational transitions with fluorescence. Chem. Rev. 106:1769‐1784.
   Sato, S., Luisi, D.L., and Raleigh, D.P. 2000. pH jump studies of the folding of the multidomain ribosomal protein L9: The structural organization of the N‐terminal domain does not affect the anomalously slow folding of the C‐terminal domain. Biochemistry 39:4955‐4962.
   Sreerama, N., Venyaminov, S.Y., and Woody, R.W. 1999. Estimation of the number of alpha‐helical and beta‐strand segments in proteins using circular dichroism spectroscopy. Protein Sci. 8:370‐380.
   Tonomura, B., Nakatani, H., Ohnishi, M., Yamaguchiito, J., and Hiromi, K. 1978. Test reactions for a stopped‐flow apparatus reduction of 2,6‐dichlorophenolindophenol and potassium ferricyanide by l‐ascorbic‐acid. Anal. Biochem. 84:370‐383.
   Vanstokkum, I.H.M., Spoelder, H.J.W., Bloemendal, M., Vangrondelle, R., and Groen, F.C.A. 1990. Estimation of protein secondary structure and error analysis from circular‐dichroism spectra. Anal. Biochem. 191:110‐118.
   Wallace, B.A. and Janes, R.W. (eds.) 2009. Modern Techniques for Circular Dichroism and Synchrotron Radiation Circular Dichroism Spectroscopy. IOS Press, Amsterdam.
   Whitmore, L. and Wallace, B.A. 2004. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 32:W668‐W673.
   Whitmore, L. and Wallace, B.A. 2008. Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases. Biopolymers 89:392‐400.
   Woody, R.W. 1996. Theory of circular dichroism of proteins. In Circular Dichroism and the Conformational Analysis of Biomolecules (G.D. Fasman, ed.). Plenum Press, New York.
Internet Resources
  Download site for cdtools specialized software for CD data processing.
  Dichroweb on‐line analysis of far‐UV CD spectra.
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