Circular Dichroism to Study Protein Interactions

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Abstract
Table of Contents
Figures
Literature Cited

Abstract

Circular dichroism (CD) is a powerful technique for studying the structures of proteins in solution, as well as structural changes that may occur when proteins bind to ligands. Changes in CD signals accompanying complex formation can be used to determine the strength of the interaction and to provide information about the nature and extent of the conformational changes involved. This unit outlines the type of information available from CD studies and describes how such experiments should be carried out to ensure that reliable information is obtained.

Keywords: circular dichroism; protein structure; conformational change; ligand binding

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Strategic Planning
Basic Protocol 1: CD Titration to Monitor Binding
Basic Protocol 2: Analysis of CD Data to Determine Binding Constants
Support Protocol 1: Analysis of Far‐UV CD Data to Determine Secondary Structure Content
Support Protocol 2: Assessment of Stability of Protein During Titration
Support Protocol 3: Choice of Buffer and Solvent System
Support Protocol 4: Determination of Protein Concentration
Support Protocol 5: Calibration of CD Instrument
Support Protocol 6: Choice of Instrument Parameters
Commentary
Literature Cited
Figures
Tables

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Materials

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Figures

Figure 20.10.1 CD spectra of the molybdate‐sensing protein ModE from E. coli in the absence (solid line) and presence (dashed line) of 1 mM molybdate. Panels A and B represent the near‐UV and far‐UV CD spectra, respectively. The data are adapted from Anderson et al. () and Boxer et al. ().

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Figure 20.10.2 Far‐UV CD spectra of the adenylyl cyclase toxin of Bordetella pertussis (CyaA) in the absence (solid line) and presence (dashed line) of 1 mM calcium chloride. The data are adapted from Cheung et al. (unpub. observ.).

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Figure 20.10.3 Analysis of binding data. Panel A illustrates nonlinear fitting to Equation . The resultant values are Δθ_max = 15.97 mdeg (standard error 0.03 mdeg), and K _d = 34.9 µM (standard error 0.2 µM). Panel B shows a Scatchard plot according to Equation . In this plot, the slope = −1/ K _d ; hence, K _d can be calculated to be 35 µM. The intercept on the y ‐axis = Δθ_max ; hence, Δθ_max = 16 mdeg. Whereas the nonlinear fitting is useful for indicating the errors in the estimates of the binding parameters, the Scatchard plot can indicate whether there are systematic deviations from the hyperbolic binding model. In this example, the ligand concentration is assumed to be much larger than that of the protein, so that [L]_free can be set equal to [L_t ] in the plots.

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Figure 20.10.4 Analysis of specimen binding data using the Hill plot according to Equation . The slope of the plot is the Hill coefficient; this can be calculated to be 2.5, indicating a significant degree of cooperativity in ligand binding. From the point of intersection of line with the x ‐axis, the value of K can be calculated to be 5500 µM (Equation ).

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Literature Cited

	Anderson, L.A., Palmer, T., Price, N.C., Bornemann, S., Boxer, D.H., and Pau, R.N. 1997. Characterisation of the molybdenum‐responsive ModE regulatory protein and its binding to the promoter region of the modABCD (molybdenum transport) operon of Escherichia coli. Eur. J. Biochem. 246:119‐126.
	Baldwin, G.S., Kelly, S.M., Price, N.C., Wilson, G.W., Connolly, B.A., and Hornby, D.P. 1994. Ligand‐induced conformational states of a deoxy‐cytidine specific DNA methyltransferase. J. Mol. Biol. 235:545‐553.
	Boxer, D.H., Zhang, H., Gourley, D.G., Hunter, W.N., Kelly, S.M., and Price, N.C. 2004. Sensing of remote oxyanion binding at the DNA binding domain of the molybdate‐dependent transcription regulator, ModE. Org. Biomol. Chem. 2:2829‐2837.
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Internet Resources
	http://www.gla.ac.uk/ibls/pcf/circular_dichroism.htm
	The home page of the Scottish Circular Dichroism Facility contains an explanation of the theory and practice of CD and has links to other important CD sites.
	http://www.cryst.bbk.ac.uk/cdweb/html/home.html
	The DICHROWEB server for online analysis of far‐UV CD spectra (Lobley et al., ; Whitmore and Wallace, ).
	http://us.expasy.org/tools/protparam.html
	The ProtParam tool within the ExPASy Website calculates the extinction coefficient for a native protein according to the equation proposed by Pace et al. () (Equation ). Also see UNIT .