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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Abstract

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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Materials

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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Figures

  •   FigureFigure 7.6.1 Relation between absorbance and circular dichroism for a polypeptide chain in the helical conformation. (A) The absorbance spectrum of poly‐γ‐methyl‐L‐glutamate in the α‐helical form, with an assignment of its three constituent transitions. (B) The far‐UV CD spectrum of the same compound, deconvoluted on the assumption that the shapes of the absorbance and dichroic bands are directly related (broken lines). The solid line is the observed spectrum and the filled circles the sum of the three bands. In general, the sign of a CD band may be either positive or negative, and its intensity does not necessarily follow that of the absorbance band. (From Holzwarth and Doty, .)
  •   FigureFigure 7.6.2 The two near‐UV absorbance transitions for tryptophan. The near‐UV absorption spectrum for N‐stearyl tryptophan n‐hexyl ester dissolved in methylcyclohexane at 24°C (solid line) has been resolved into the 1La and 1Lb bands. (Drawn from Strickland, .)
  •   FigureFigure 7.6.3 The relation of ellipticity to the differential absorption of circularly polarized radiation. The oscillating radiation sine wave, OI, is proceeding out of the plane of the paper towards the viewer. (A) Plane‐polarized radiation is made up of left‐ and right‐handed circularly polarized components, OL and OR, respectively. Absorption by a chromophore in a nonchiral environment results in an equal reduction in intensity of each component, whose resultant is a vector oscillating only in the vertical plane—i.e., plane‐polarized radiation. (B) Interaction of the radiation with a chiral chromophore leads to unequal absorption, so that combination of the emerging vectors, OL′ and OR′, leads to a resultant that describes an elliptical path as it progresses out of the plane of the paper. The ratio of the major and minor axes of the ellipse is expressed by tan θ, thus defining ellipticity. The major axis of the ellipse makes an angle (ϕ) with the original plane, which defines the optical rotation. This figure thus demonstrates the close relation between optical rotation and circular dichroism.
  •   FigureFigure 7.6.4 The contributions of different aromatic residues in a folded protein to circular dichroism. Interleukin 1β contains one tryptophan and four tyrosine residues. Their individual contributions have been resolved using site‐directed mutagenesis. (A) The near‐UV CD spectra of the tyrosine residues show that, although Tyr 68 makes a significant contribution to the negative ellipticity, indicating its buried, asymmetric environment, the contributions resulting from residues 24 and 90 are little different from that of free tyrosine. The derived spectrum of Tyr 121 is interesting in that it clearly contains a contribution arising from tryptophan (Fig. ), indicating its close interaction with Trp 120. (B) The contribution from the tryptophan is seen to dominate, reflecting its nonpolar environment. The intensity of the protein spectrum is less intense than it might otherwise be, because of the opposing signs of the individual spectra. Spectra were measured using a 1‐nm bandwidth (Craig et al., ).
  •   FigureFigure 7.6.5 The near‐UV CD spectra of granulocyte/macrophage‐colony stimulating factor (GM‐CSF). (A) Comparison of the spectrum of the native (N) murine GM‐CSF (with one tryptophan residue, which is exposed, and four tyrosine residues, 2.5 of which are exposed) with those for the protein unfolded in 3.25 M guanidine⋅HCI (U) and refolded from this solution (R) by dialysis against 3 M urea. The intense negative spectrum of the folded protein, with a maximum at 278 nm, is characteristic of tyrosine, with contributions from phenylalanine. The intensity is probably a result of the interaction of Tyr 81 and Tyr 82. The absence of a band at 292 nm shows that the Trp 14 makes little contribution, as expected from its exposed location. Refolding restores the detailed tertiary structure of the native protein. (B) Reduction (RED) of native human GM‐CSF (with two tryphtophan residues, one of which is exposed, and two tyrosine residues, which are buried) reduces the ellipticity of the native protein (N) to that of the fully unfolded protein (U). The secondary structure is however, unaffected (see inset for far‐UV CD spectra). Notice that, in contrast to the murine protein, human GMCSF exhibits a less intense, but positive, near‐UV CD band at 290 nm with little fine structure, implying a contribution from a 1La transition of Trp 123 and indicating the buried nature of this residue. Spectra were measured using 0.01‐mm path‐length cells and 2‐nm bandwidth in the far‐UV, and 10‐mm path‐length cells with 1‐mm bandwidth in the near‐UV (Wingfield et al., ).
  •   FigureFigure 7.6.6 Far‐UV CD spectra. (A) Seed coat soybean peroxidase under native and denaturing conditions (data from Kamal and Behere, ). Protein concentration 6 µM and path length 1 mm. The troughs at 208 and 222 nm are characteristic of a high content of α‐helix. Unlike the aromatic and heme residues (Fig. B), this secondary structure is not completely disrupted at 90°C, but requires a strong chemical denaturant. The cut‐off in measurements in 8 M GdmCl is due to the strong absorbance of the latter in the far UV, making it impossible to make measurements at lower wavelengths. (B) Bovine β‐casein as a function of temperature (data from Farrell et al., ). Protein concentrations in the range of 0.18 mg/ml; path length 0.50 mm; bandwidth 1.5 nm. Note the minimum at 199 nm whose intensity and wavelength shift at higher temperatures, suggesting low cooperativity of the structure in solution. Note also the appearance at higher temperatures of an apparent contribution from aromatic asymmetry centered on ∼226 nm. (C) Clitocypin—a protease inhibitor from mushroom (data from Kidric et al., ). Protein concentration 0.1 mg/ml in 50 mM phosphate, pH 6.6; path length 1 mm; bandwidth 2 nm; scan step nm; dwell time at each step 20 sec; four repeats. Note the minimum around 200 nm and peak at 189 nm due to the high content of β structure. The intense peak at 231 nm, which disappears on thermal denaturation, is due to an aromatic contribution, probably due to a tryptophan‐tryptophan interaction in the native protein. The symmetry of this peak (inset) suggests a single chromophore transition. (D) Immunoglobulin G. Protein concentration 0.03 mg/ml; path length 1 mm; bandwidth 1 nm; scan step nm; dwell time at each step sec (i.e., scan speed 12 nm/min). The minimum at 216 nm and peak at 194 nm are characteristic of the classical β‐structure CD spectrum, in contrast to those of casein and clitocypin.
  •   FigureFigure 7.6.7 CD spectrum of D‐(+)‐10‐camphorsulfonic acid (CSA) in water. The vertical bars represent variations of ±1.5% and ± 5%. The broken line represents the extrapolation of a gaussian band. Commercial CSA was twice recrystallized. (From Chen and Yang, .)
  •   FigureFigure 7.6.8 Obtaining the corrected near‐UV CD spectrum for hen egg white lysozyme. The protein and baseline spectra were collected using a 10‐mm cylindrical cell and 0.5 mg/ml protein in 0.067 M phosphate buffer, pH 6.0. Instrument settings were 1‐nm bandwidth, 0.2‐nm step size, scan speed 2 nm/min, time constant 8 sec (scan speed × time constant = 0.27 nm). Protein solution and buffer were scanned once each. The spectra were smoothed, a sample of the fit being shown in the inset. Reproducibility of the instrument and of the state of the cell are demonstrated by the coincidence of the ellipticity above 300 nm. The corrected spectrum was obtained by subtraction, using the instrument software.
  •   FigureFigure 7.6.9 The distribution of intensity of radiation passing through the sample cell. Radiation emerging from a monochromator set at a wavelength corresponding to the peak maximum will contain components at other wavelengths, with intensities described by the curve. The bandwidth (b) is the width of the distribution curve at half the peak height. Its magnitude will depend on the exit slit width. The setting of the slit width is usually programmed to change during scanning, so as to give a constant bandwidth and hence constant spectral resolution.
  •   FigureFigure 7.6.10 The near‐UV CD spectra of cathepsin D. The solid line represents the porcine enzyme and the dashed line represents the bovine enzyme, each cleaved into two chains which remain associated; the dotted line represents the bovine intact enzyme. The spectra were recorded using 10‐mm and 20‐mm path‐length cells, with enzyme concentrations of 0.8 to 0.4 g/liter. The far‐UV CD and fluorescence spectra of the cleaved and intact enzymes are not significantly different (Pain et al., ).
  •   FigureFigure 7.6.11 The near‐UV absorbance transition dipole moment of the tyrosine sidechain. The 1Lb transition is seen to be perpendicular to the axis of rotation of the phenolic ring. Interactions of the transition with the environment, expressed as an enhanced CD band, will be sensitive to rotation of the sidechain around this axis.
  •   FigureFigure 7.6.12 Effect of mutations detected by CD. The far‐UV CD spectra (A) show that the secondary structure of β‐lactamase PC1 (solid line) from Staphylococcus aureus is essentially unaffected by point mutations P2 (Thr 140→Ile; dashed line) and P54 (Asp 146→Asn; dotted line). The crystallographic structure of P54 (Herzberg et al., ) confirms that, apart from a loop region, the main body of the molecule that contains the thirteen tyrosine residues is very closely similar to that in the wild‐type enzyme. The intensity of the tyrosine ellipticity (B) is, however, markedly decreased in each of the mutants, the lower thermodynamic stabilities of which support the interpretation of increased dynamics (Craig et al., ).
  •   FigureFigure 7.6.13 A convincing artifact. In an attempt to study the conformational consequences of adding an acceptor, D‐glutamine, to a DD‐peptidase, far‐UV CD spectra were recorded for the enzyme in the presence of increasing concentrations of glutamine: a = 0 mM, b = 3 mM, c = 7 mM, d = 10 mM, e = 20 mM, f = 30 mM, g = 50 mM, and h = 95 mM. The enzyme concentration was 0.1 mg/ml, equivalent to ∼10−3 M peptide bond, in 10 mM sodium phosphate pH 7.2. A 2‐mm cell path length was used.

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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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