Biophysical Reviews

, Volume 9, Issue 5, pp 517–527 | Cite as

Going deep into protein secondary structure with synchrotron radiation circular dichroism spectroscopy

  • Patricia S. Kumagai
  • Ana P. U. Araujo
  • Jose L. S. Lopes


Circular dichroism (CD) spectroscopy is a fast, powerful, well-established, and widely used analytical technique in the biophysical and structural biology community to study protein secondary structure and to track changes in protein conformation in different environments. The use of the intense light of a synchrotron beam as the light source for collecting CD measurements has emerged as an enhanced method, known as synchrotron radiation circular dichroism (SRCD) spectroscopy, that has several advantages over the conventional CD method, including a significant spectral range extension for data collection, deeper access to the lower limit (cut-off) of conventional CD spectroscopy, an improved signal-to-noise ratio to increase accuracy in the measurements, and the possibility to collect measurements in highly absorbing solutions. In this review, we discuss different applications of the SRCD technique by researchers from Latin America. In this context, we specifically look at the use of this method for examining the secondary structure and conformational behavior of proteins belonging to the four main classes of the hierarchical protein domain classification CATH (Class, Architecture, Topology, Homology) database, focusing on the advantages and improvements associated with SRCD spectroscopy in terms of characterizing proteins composed of different structural elements.


Circular dichroism spectroscopy Conformational changes Protein conformation Protein secondary structure Synchrotron radiation circular dichroism spectroscopy 



JLSL and PSK are grateful for beamtime access at the AU-CD beamline at the ASTRID2 (Aarhus, Denmark) and at the UV-CD12 at t he ANKA (Karlsruhe, Germany) synchrotrons. All authors thank Prof. BA Wallace for supporting the implementation of SRCD in Brazil and for helpful discussions.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Bremer A, Wolff M, Thalhammer A, Hincha DK (2017) Folding of intrinsically disordered plant LEA proteins is driven by glycerol-induced crowding and the presence of membranes. FEBS J 284(6):919–936. doi:10.1111/febs.14023 CrossRefPubMedGoogle Scholar
  2. Bürck J, Wadhwani P, Fanghänel S, Ulrich AS (2016) Oriented circular dichroism: A method to characterize membrane-active peptides in oriented lipid bilayers. Acc Chem Res 49(2):184–192. doi:10.1021/acs.accounts.5b00346 CrossRefPubMedGoogle Scholar
  3. Cowieson NP, Miles AJ, Robin G, Forwood JK, Kobe B, Martin JL, Wallace BA (2008) Evaluating protein:protein complex formation using synchrotron radiation circular dichroism spectroscopy. Proteins 70(4):1142–1146CrossRefPubMedGoogle Scholar
  4. Drechsler A, Miles AJ, Norton RS, Wallace BA, Separovic F (2009) Effect of lipid on the conformation of the N-terminal region of equinatoxin II: A synchrotron radiation circular dichroism spectroscopic study. Eur Biophys J 39(1):121–127. doi:10.1007/s00249-009-0445-x CrossRefPubMedGoogle Scholar
  5. Feng Y, Yu W, Li X, Zhou Y, Hu J, Liu X (2013) Structural insight into Golgi membrane stacking by GRASP65 and GRASP55 proteins. J Biol Chem 288(39):28418–28427. doi:10.1074/jbc.M113.478024 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Garcia AF, Garcia W, Nonato MC, Araújo AP (2008) Structural stability and reversible unfolding of recombinant porcine S100A12. Biophys Chem 134(3):246–253. doi:10.1016/j.bpc.2008.02.013 CrossRefPubMedGoogle Scholar
  7. Garcia AF, Lopes JLS, Costa-Filho AJ, Wallace BA, Araujo APU (2013) Membrane interactions of S100A12 (Calgranulin C). PLoS One 8(12):e82555. doi:10.1371/journal.pone.0082555 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1(6):2876–2890. doi:10.1038/nprot.2006.202 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Hussain R, Jávorfi T, Rudd TR, Siligardi G (2016) High-throughput SRCD using multi-well plates and its applications. Sci Rep 6:38028. doi:10.1038/srep38028 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Jasanoff A, Fersht AR (1997) Mechanism of helix induction by trifluoroethanol: A framework for extrapolating the helix-forming properties of peptides from trifluoroethanol/water mixtures back to water. Biochemist 36:8413. doi:10.1021/bi9707133 CrossRefGoogle Scholar
  11. Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751(2):119–139CrossRefPubMedGoogle Scholar
  12. Kinseth MA, Anjard C, Fuller D, Guizzunti G, Loomis WF, Malhotra V (2007) The Golgi-associated protein GRASP is required for unconventional protein secretion during development. Cell 130:524–534. doi:10.1016/j.cell.2007.06.029 CrossRefPubMedGoogle Scholar
  13. Kumagai PS, DeMarco R, Lopes JLS (2017) Advantages of synchrotron radiation circular dichroism spectroscopy to study intrinsically disordered proteins. Eur Biophys J. doi 10.1007/s00249-017-1202-1
  14. Lees JG, Miles AJ, Wien F, Wallace BA (2006) A reference database for circular dichroism spectroscopy covering fold and secondary structure space. Bioinformatics 22(16):1955–1962CrossRefPubMedGoogle Scholar
  15. Lima MA, Hughes AJ, Veraldi N, Rudd TR, Hussain R, Brio AS, Chavante SF, Tersariol II, Siligardi G, Nader HB, Yates EA (2013) Antithrombin stabilisation by sulfated carbohydrates correlates with anticoagulant activity. Med Chem Commun 4:870–873. doi:10.1039/C3MD00048F CrossRefGoogle Scholar
  16. Lopes JLS, Miles AJ, Whitmore L, Wallace BA (2014a) Distinct circular dichroism spectroscopic signatures of polyproline II and unordered secondary structures: Applications in secondary structure analyses. Protein Sci 23(12):1765–1772. doi:10.1002/pro.2558 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Lopes JLS, Nobre TM, Cilli EM, Beltramini LM, Araujo APU, Wallace BA (2014b) Deconstructing the DGAT1 enzyme: Binding sites and substrate interactions. Biochim Biophys Acta 1838(12):3145–3152. doi:10.1016/j.bbamem.2014.08.017 CrossRefPubMedGoogle Scholar
  18. Lopes JLS, Orcia D, Araujo APU, DeMarco R, Wallace BA (2013) Folding factors and partners for the intrinsically disordered protein micro-exon gene 14 (MEG-14). Biophys J 104(11):2512–2520. doi:10.1016/j.bpj.2013.03.063 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lopes JLS, Yoneda JS, Martins JM, DeMarco R, Jameson DM, Castro AM, Bossolan NR, Wallace BA, Araujo APU (2016) Environmental factors modulating the stability and enzymatic activity of the Petrotoga mobilis esterase (PmEst). PLoS One 11(6):e0158146. doi:10.1371/journal.pone.0158146 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Matsuo K, Gekko K (2013) Circular-dichroism and synchrotron-radiation circular-dichroism spectroscopy as tools to monitor protein structure in a lipid environment. Methods Mol Biol 974:151–176. doi:10.1007/978-1-62703-275-9_8 CrossRefPubMedGoogle Scholar
  21. Mendes LFS, Garcia AF, Kumagai PS, Morais FR, Melo FA, Kmetzsch L, Vainstein MH, Rodrigues ML, Costa-Filho AJ (2016) New structural insights into Golgi reassembly and stacking protein (GRASP) in solution. Sci Rep 6:29976. doi:10.1038/srep29976 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Micsonai A, Wien F, Kernya L, Lee YH, Goto Y, Réfrégiers M, Kardos J (2015) Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci USA 112(24):E3095–E3103. doi:10.1073/pnas.1500851112 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Miles AJ, Drechsler A, Kristan K, Anderluh G, Norton RS, Wallace BA, Separovic F (2008) The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: A synchrotron radiation circular dichroism spectroscopic study. Biochim Biophys Acta 1778(10):2091–2096. doi:10.1016/j.bbamem.2008.04.001 CrossRefPubMedGoogle Scholar
  24. Miles AJ, Wallace BA (2016) Circular dichroism spectroscopy of membrane proteins. Chem Soc Rev 45(18):4859–4872. doi:10.1039/c5cs00084j CrossRefPubMedGoogle Scholar
  25. Miller WC, Miles AJ, Wallace BA (2016) Structure of the C-terminal domain of the prokaryotic sodium channel orthologue NsvBa. Eur Biophys J 45(8):807–814CrossRefPubMedGoogle Scholar
  26. Orcia D, Zeraik AE, Lopes JLS, Macedo JNA, Santos CR, Oliveira KC, Anderson L, Wallace BA, Verjovski-Almeida S, Araujo APU, DeMarco R (2017) Interaction of an esophageal MEG protein from schistosomes with a human S100 protein involved in inflammatory response. Biochim Biophys Acta 1861(1 Pt A):3490–3497. doi:10.1016/j.bbagen.2016.09.015 CrossRefPubMedGoogle Scholar
  27. Orengo CA, Michie AD, Jones S, Jones DT, Swindells MB, Thornton JM (1997) CATH-a hierarchic classification of protein domain structures. Structure 5(8):1093–1108CrossRefPubMedGoogle Scholar
  28. Poklar Ulrih N (2017) Analytical techniques for the study of polyphenol-protein interactions. Crit Rev Food Sci Nutr 57(10):2144–2161. doi:10.1080/10408398.2015.1052040 CrossRefPubMedGoogle Scholar
  29. Powl AP, O’Reilly AO, Miles AJ, Wallace BA (2010) Synchrotron radiation circular dichroism spectroscopy-defined structure of the C-terminal domain of NaChBac and its role in channel assembly. Proc Natl Acad Sci USA 107(32):14064–14069. doi:10.1073/pnas.1001793107 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Ranjbar B, Gill P (2009) Circular dichroism techniques: Biomolecular and nanostructural analyses- a review. Chem Biol Drug Des 74(2):101–120. doi:10.1111/j.1747-0285.2009.00847.x CrossRefPubMedGoogle Scholar
  31. Recveur-Brechot V, Bourhis JM, Uversky VN, Canard B, Longhi S (2006) Assessing protein disorder and induced folding. Proteins 62:24–45CrossRefGoogle Scholar
  32. Ruskamo S, Chukhlieb M, Vahokoski J, Bhargav SP, Liang F, Kursula I, Kursula P (2012) Juxtanodin is an intrinsically disordered F-actin-binding protein. Sci Rep 2:899. doi:10.1038/srep00899 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Sreerama N, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: Comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287(2):252–260CrossRefPubMedGoogle Scholar
  34. Sutherland JC, Emrick A, France LL, Monteleone DC, Trunk J (1992) Circular dichroism user facility at the National Synchrotron Light Source: Estimation of protein secondary structure. BioTechniques 13(4):588–590PubMedGoogle Scholar
  35. Truschel ST, Sengupta D, Foote A, Heroux A, Macbeth MR, Linstedt AD (2011) Structure of the membrane-tethering GRASP domain reveals a unique PDZ ligand interaction that mediates Golgi biogenesis. J Biol Chem 286(23):20125–20129. doi:10.1074/jbc.C111.245324 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Uversky VN (2009) Intrinsically disordered proteins and their environment: Effects of strong denaturants, temperate pH, counter ions, membranes, binding partners, osmolytes, and macromolecular crowding. Protein J 28:305–325. doi:10.1007/s10939-009-9201-4 CrossRefPubMedGoogle Scholar
  37. van der Lee R, Buljan M, Lang B, Weatheritt RJ et al (2014) Classification of intrinsically disordered regions and proteins. Chem Rev 114(13):6589–6631. doi:10.1021/cr400525m CrossRefPubMedPubMedCentralGoogle Scholar
  38. Wada A (1976) The alpha-helix as an electric macro-dipole. Adv Biophys:1–63Google Scholar
  39. Wallace BA (2000) Conformational changes by synchrotron radiation circular dichroism spectroscopy. Nat Struct Biol 7(9):708–709. doi:10.1038/78915 CrossRefPubMedGoogle Scholar
  40. Wallace BA, Wien F, Miles AJ, Lees JG, Hoffmann SV, Evans P, Wistow GJ, Slingsby C (2004) Biomedical applications of synchrotron radiation circular dichroism spectroscopy: Identification of mutant proteins associated with disease and development of a reference database for fold motifs. Faraday Discuss 126:237–243CrossRefPubMedGoogle Scholar
  41. Wallace BA (2009) Protein characterisation by synchrotron radiation circular dichroism spectroscopy. Q Rev Biophys 42(4):317–370. doi:10.1017/S003358351000003X CrossRefPubMedGoogle Scholar
  42. Wallace BA, Janes RW (eds) (2009) Modern techniques for circular dichroism and synchrotron radiation circular dichroism spectroscopy. Advances in biomedical spectroscopy, vol 1. IOS Press, AmsterdamGoogle Scholar
  43. Whitmore L, Wallace BA (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 32(Web Server issue):W668–73.Google Scholar
  44. Yoneda JS, Miles AJ, Araujo APU, Wallace BA (2017) Differential dehydration effects on globular proteins and intrinsically disordered proteins during film formation. Protein Sci 26(4):718726. doi:10.1002/pro.3118 CrossRefGoogle Scholar

Copyright information

© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Patricia S. Kumagai
    • 1
  • Ana P. U. Araujo
    • 1
  • Jose L. S. Lopes
    • 2
  1. 1.Instituto de Física de São CarlosUniversidade de São PauloSão PauloBrazil
  2. 2.Departamento Física Aplicada, Instituto de Física,Universidade de São PauloSão PauloBrazil

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