Advertisement

Enzyme–Ligand Interaction Monitored by Synchrotron Radiation Circular Dichroism

  • Rohanah HussainEmail author
  • Charlotte S. Hughes
  • Giuliano SiligardiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2089)

Abstract

CD spectroscopy is the essential tool to quickly ascertain in the far-UV region the global conformational changes, the secondary structure content, and protein folding and in the near-UV region the local tertiary structure changes probed by the local environment of the aromatic side chains, prosthetic groups (hemes, flavones, carotenoids), the dihedral angle of disulfide bonds, and the ligand chromophore moieties, the latter occurring as a result of protein–ligand binding interaction. Qualitative and quantitative investigations into ligand-binding interactions in both the far- and near-UV regions using CD spectroscopy provide unique and direct information whether induced conformational changes upon ligand binding occur and of what nature that are unattainable with other techniques such as fluorescence, ITC, SPR, and AUC.

This chapter provides an overview of how to perform circular dichroism (CD) experiments, detailing methods, hints and tips for successful CD measurements. Descriptions of different experimental designs are discussed using CD to investigate ligand-binding interactions. This includes standard qualitative CD measurements conducted in both single-measurement mode and high-throughput 96-well plate mode, CD titrations, and UV protein denaturation assays with and without ligand.

The highly collimated micro-beam available at B23 beamline for synchrotron radiation circular dichroism (SRCD) at Diamond Light Source (DLS) offers many advantages to benchtop instruments. The synchrotron light source is ten times brighter than a standard xenon arc light source of benchtop instruments. The small diameter of the synchrotron beam can be up to 160 times smaller than that of benchtop light beams; this has enabled the use of small aperture cuvette cells and flat capillary tubes reducing substantially the amount of volume sample to be investigated. Methods, hints and tips, and golden rules to measure good quality, artifact-free SRCD and CD data will be described in this chapter in particular for the study of protein–ligand interactions and protein photostability.

Key words

Circular dichroism Ligand binding Titration Binding constant UV denaturation Protein stability Data processing 

Notes

Acknowledgement

We like to thank Diamond Light Source for access to B23 beamline (CM12182, CM14484, CM16778, CM19680).

References

  1. 1.
    Javorfi T, Hussain R, Myatt D, Siligardi G (2010) Measuring circular dichroism in a capillary cell using the B23 synchrotron radiation CD beamline at Diamond Light Source. Chirality 22(1E):E149–E153PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Hussain R, Javorfi T, Siligardi G (2012) Circular dichroism beamline B23 at the Diamond Light Source. J Synchrotron Radiat 19(1):132–135PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Hussain R, Jávorfi T, Rudd TR, Siligardi G (2016) High-throughput SRCD using multi-well plates and its applications. Sci Rep 6(1):38028PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Hussain R, Jávorfi T, Siligardi G (2012) Spectroscopic analysis: synchrotron radiation circular dichroism. In: Carreira EM, Yamamoto H (eds) Comprehensive chirality, vol 8. Elsevier, Amsterdam, pp 438–448CrossRefGoogle Scholar
  5. 5.
    Hussain R, Longo E, Siligardi G, Hussain R, Longo E, Siligardi G (2018) UV-denaturation assay to assess protein photostability and ligand-binding interactions using the high photon flux of diamond B23 beamline for SRCD. Molecules 23(8):1906PubMedCentralCrossRefGoogle Scholar
  6. 6.
    Hughes CS, Longo E, Phillips-Jones MK, Hussain R (2017) Characterisation of the selective binding of antibiotics vancomycin and teicoplanin by the VanS receptor regulating type A vancomycin resistance in the enterococci. Biochim Biophys Acta Gen Subj 1861(8):1951–1959PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Phillips-Jones MK, Patching SG, Edara S, Nakayama J, Hussain R, Siligardi G (2013) Interactions of the intact FsrC membrane histidine kinase with the tricyclic peptide inhibitor siamycin I revealed through synchrotron radiation circular dichroism. Phys Chem Chem Phys 15(2):444–447PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Siligardi G, Hussain R, Patching SG, Phillips-Jones MK (2014) Ligand- and drug-binding studies of membrane proteins revealed through circular dichroism spectroscopy. Biochim Biophys Acta 1838(1 Pt A):34–42PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Hussain R, Harding SE, Hughes CS, Ma P, Patching SG, Edara S et al (2016) Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions. Biochem Soc Trans 44(3):810–823PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Bettaney KE, Sukumar P, Hussain R, Siligardi G, Henderson PJF, Patching SG (2013) A systematic approach to the amplified expression, functional characterization and purification of inositol transporters from Bacillus subtilis. Mol Membr Biol 30(1):3–14PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Runti G, Lopez Ruiz Mdel C, Stoilova T, Hussain R, Jennions M, Choudhury HG et al (2013) Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7(1-35). J Bacteriol 195(23):5343–5351PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Patching S, Edara S, Ma P (2012) Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism. Biochem Biophys Acta 1818(7):1595–1602PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Hassan KA, Jackson SM, Penesyan A, Patching SG, Tetu SG, Eijkelkamp BA et al (2013) Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins. Proc Natl Acad Sci U S A 110(50):20254–20259PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Kalverda AP, Gowdy J, Thompson GS, Homans SW, Henderson PJF, Patching SG (2014) TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor. Mol Membr Biol 31(4):131–140PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Hughes CS, Longo E, Phillips-Jones MK, Hussain R (2017) Quality control and biophysical characterisation data of VanSA. Data Br 1(14):41–47CrossRefGoogle Scholar
  16. 16.
    Hussain R, Benning K, Javorfi T, Longo E, Rudd TR, Pulford B et al (2015) CDApps: integrated software for experimental planning and data processing at beamline B23, Diamond Light Source. J Synchrotron Radiat 22(2):465–468PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751(2):119–139PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Hennessey JP, Johnson WC (1981) Information content in the circular dichroism of proteins. Biochemistry 20(5):1085–1094PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Provencher SW, Glöckner J (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20(1):33–37PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    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):252–260PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    van Stokkum IHM, Spoelder HJW, Bloemendal M, van Grondelle R, Groen FCA (1990) Estimation of protein secondary structure and error analysis from circular dichroism spectra. Anal Biochem 191(1):110–118PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1(6):2876–2890PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Micsonai A, Wien F, Kernya L, Lee Y-H, Goto Y, Réfrégiers M, Kardos J (2015) Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci U S A 112(24):E3095–E3103PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Lobley A, Whitmore L, Wallace BA (2002) DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra. Bioinformatics 18(1):211–212PubMedCrossRefGoogle Scholar
  25. 25.
    Siligardi G, Hussain R (1998) Biomolecules interactions and competitions by non-immobilised ligand interaction assay by circular dichroism. Enantiomer 3(2):77–87PubMedPubMedCentralGoogle Scholar
  26. 26.
    Kumar S (2006) Organic chemistry spectroscopy of organic compounds. Guru Nanak Dev University, AmritsarGoogle Scholar
  27. 27.
    Iyer KS, Klee WA (1973) Direct spectrophotometric measurement of the rate of reduction of disulfide bonds. The reactivity of the disulfide bonds of bovine-lactalbumin. J Biol Chem 248(2):707–710PubMedPubMedCentralGoogle Scholar
  28. 28.
    Seo A, Jackson JL, Schuster JV, Vardar-Ulu D (2013) Using UV-absorbance of intrinsic dithiothreitol (DTT) during RP-HPLC as a measure of experimental redox potential in vitro. Anal Bioanal Chem 405(19):6379–6384PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Han JC, Han GY (1994) A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol. Anal Biochem 220(1):5–10PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Wingfield PT (2001) Use of protein folding reagents. Curr Protoc Protein Sci. Appendix 3:Appendix 3AGoogle Scholar
  31. 31.
    Knubovets T, Osterhout JJ, Klibanov AM (1999) Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies. Biotechnol Bioeng 63(2):242–248PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Lide DR (2010) In: Haynes WM (ed) CRC handbook of chemistry and physics, 91th edn. CRC Press (Taylor and Francis Group), Boca Raton, FLGoogle Scholar
  33. 33.
    Zinna F, Resta C, Gorecki M, Pescitelli G, Di Bari L, Javorfi T, Hussain R, Siligardi G (2017) Circular dichroism imaging: mapping the local supramolecular order in thin films of chiral functional polymers. Macromolecules 50(5):2054–2060CrossRefGoogle Scholar
  34. 34.
    Johnson WC (1985) Circular dichroism and its empirical application to biopolymers. Methods Biochem Anal 31:61–163PubMedPubMedCentralGoogle Scholar
  35. 35.
    Lindon JC, Tranter GE, Koppenaal D (2016) Encyclopedia of spectroscopy and spectrometry. 3rd ed. 287 pGoogle Scholar
  36. 36.
    Damon AJH, Kresheck GC (1982) Influence of surfactants on the conformation of β-lactoglobulin B using circular dichroism. Biopolymers 21(5):895–908PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Plangger H, Scheibenzuber M, Blümelhuber G, Meyer-Pittroff R (2003) Influence of high pressure on the secondary structure of poly-l-lysine. In: Winter R (ed) Advances in high pressure bioscience and biotechnology II. Springer, Berlin, HeidelbergGoogle Scholar
  38. 38.
    Greenfield NJ (2007) Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc 1(6):2527–2535CrossRefGoogle Scholar
  39. 39.
    Gutiérrez-Mejía FA, van IJzendoorn LJ, Prins MWJ (2015) Surfactants modify the torsion properties of proteins: a single molecule study. New Biotechnol 32(5):441–449CrossRefGoogle Scholar
  40. 40.
    Sun C, Yang J, Wu X, Huang X, Wang F, Liu S (2005) Unfolding and refolding of bovine serum albumin induced by cetylpyridinium bromide. Biophys J 88(5):3518–3524PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Hayashi R, Kakehi Y, Kato M, Tanimizu N, Ozawa S, Matsumoto M (2002) Circular dichroism under high pressure. Prog Biotechnol 19:583–590Google Scholar
  42. 42.
    Brown EM, Groves ML (1985) Effect of temperature on the circular dichroism spectra of-β2-microglobulins. FEBS Lett 184(1):36–39PubMedCrossRefPubMedCentralGoogle Scholar
  43. 43.
    Hussain R, Jávorfi T, Siligardi G (2012) Circular dichroism beamline B23 at the Diamond Light Source. J Synchrotron Radiat 19(1):132–135PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Clarke DT, Jones G (2004) CD12: a new high-flux beamline for ultraviolet and vacuum-ultraviolet circular dichroism on the SRS, Daresbury. J Synchrotron Radiat 11(2):142–149PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Tanaka M, Yagi-Watanabe K, Kaneko F, Nakagawa K (2009) First observation of natural circular dichroism spectra in the extreme ultraviolet region using a polarizing undulator-based optical system and its polarization characteristics. J Synchrotron Radiat 16(4):455–462PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Prodromou C, Siligardi G, O’Brien R, Woolfson DN, Regan L, Panaretou B, Ladbury JE, Piper PW, Pearl LH (1999) Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones. EMBO J 18(3):754–762PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Singleton DG, Hussain R, Siligardi G, Kumar P, Hrdlicka PJ, Berova N et al (2016) Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling. Org Biomol Chem 14(1):149–157PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2020

Authors and Affiliations

  1. 1.Diamond Light Source Ltd.ChiltonUK

Personalised recommendations