DICHROISMCIRCULAR

Circular DichroismCircular dichroism (CD) spectroscopy is a form of light absorption spectroscopy that measures the difference in absorbance of right- and left-circularly polarized light (rather than the commonly used absorbance of isotropic light) by a substance.

Jasco J-810 Circular Dichroism System

• Definitions of asymmetry• What is optical Activity• What is polarized light

Important Basic concepts

Circular Dichroism (CD) phenomena that result when asymmetrical molecules interact with plane polarized light.

Asymmetry

Optical activity

Light

Polarized Light

Types of polarized light

Circular Polarized Light

Instrumentation

Prism Polarizer

• The function of purging the CD instrument with nitrogen is to remove oxygen from the lamp housing, monochromator, and the sample chamber.

• The presence of oxygen is detrimental for two reasons. When deep ultraviolet light strikes oxygen, ozone is produced. Ozone causes degradation of optics and can cause respiratory problems. The second reason for removing oxygen is that oxygen absorbs deep UV light, thus reducing the light available for the measurement.

Nitrogen purging

• Additives, buffers and stabilizing compounds: Any

compound which absorbs in the region of interest (250 –190 nm) should be avoided (see below).• Protein solution: The protein solution should

contain only those chemicals necessary to maintain protein stability, and at the lowest concentrations possible. Any additional protein or peptide will contribute to the CD signal.• Data collection: Initial experiments are useful to

establish the best conditions for the "real" experiment. Cells of 0.5 mm path length offer a good starting point.

Sample preparation and measurement

Typical Initial Conditions:

• Protein Concentration: 0.5 mg/ml• Cell Path Length: 0.5 mm• Stabilizers (Metal ions, etc.): minimum• Buffer Concentration : 5 mM or as low

as possible while maintaining protein stability

Cutoff Wavelengths For Common Solvents and Buffers

CD Data Analysis

CD Data Analysis

IL-IR

IL-IR

CD Data Analysis

• The molar ellipticity [] is related to the difference in extinction coefficients

[] = 3298 Δε.• Here [] has the standard units of degrees

cm2 dmol -1 • The molar ellipticity has the units degrees

deciliters mol-1 decimeter-1.

Result Table

CD spectra of calf thymus DNA titrated with BPSQ; at (a) r 0 ¼ 0; (b) 0.08; (c) 0.16; (d) 0.24; (e) 0.40; and (g) 0.72.

Resultant Spectra

A. Estimation of protein and nucleic acid conformation

B. Determination of conformational changes due to the interactions of asymmetric moleculese.g.1. Protein-protein interactions2. Protein-DNA interactions3. Protein-Ligand interactions4. DNA-Ligand interactions

C. Determination of the thermodynamics of folding and unfolding of proteins and nucleicacids

Applications of CD spectroscopy

D. Determination of binding constants by:1. direct titrations2. serial dilutions of complexes3. changes in stability to thermal or chemical denaturation

E. Kinetics of folding and unfolding of macromolecules

Circular Dichroism of Proteins

• It has been shown that CD spectra between 260 and approximately 180 nm can be analyzed for the different secondary structural types: alpha helix, parallel and anti-parallel beta sheets, turns, and other.

• A number of excellent review articles are available describing the technique and its application (Woody, 1985 and Johnson, 1990).

• Modern secondary structure determination by CD are reported to achieve accuracies of 0.97 for helices, 0.75 for beta sheet, 0.50 for turns, and 0.89 for other structure types (Manavalan & Johnson, 1987).

CD Signal of Proteins• For proteins we will be mainly concerned

with absorption in the ultraviolet region of the spectrum from the peptide bonds (symmetric chromophores) and amino acid sidechains in proteins.

• Protein chromophores can be divided into three classes: the peptide bond, the amino acid sidechains, and any prosthetic groups.

• The lowest energy transition in the peptide chromophore is an n → p* transition observed at 210 - 220 nm with very weak intensity (emax~100).

---- *p p → * ~`1p 90 nm emax~7000

----n n → * 208-210, 191-193 p nm emax~100

----p

Resultant Spectra

Chromophore Analysis

CD Spectra of Protein secondary Structures

Far UV CD Spectra of Proteins[

] x1

0-3

deg

rees

cm2

dm

ol

-1

CD Spectra Fit

• In a first approximation, a CD spectrum of a protein or polypeptide can be treated as a sum of three components: a-helical, b-sheet, and random coil contributions to the spectrum.

• At each wavelength, the ellipticity (θ) of the spectrum will contain a linear combination of these components:

(1)

• θT is the total measured ellipticity, θh the contribution from helix, θs for sheet, θc for coil, and the corresponding χ the fraction of this contribution.

CD Spectra Fit• As we have three unknowns in this equation,

a measurement at 3 points (different wavelengths) would suffice to solve the problem for χ, the fraction of each contribution to the total measured signal.

• We usually have many more data points available from our measurement (e.g., a whole CD spectrum, sampled at 1 nm intervals from 190 to 250 nm). In this case, we can try to minimize the total deviation between all data points and calculated model values. This is done by a minimization of the sum of residuals squared (s.r.s.), which looks as follows :

Resultant Spectra

Resultant Spectra

DICHROWEBhttp://www.cryst.bbk.ac.uk/cdweb/html/home.html

Using CD to Monitor 3º Structure of Proteins

• CD bands in the near UV region (260 – 350 nm) are observed in a folded protein where aromatic side chains are immobilized in an asymmetric environment.

• The CD of aromatic residues is very small in the absence of ordered structure (e.g. short peptides).

• The signs, magnitudes, and wavelengths of aromatic CD bands cannot be calculated; they depend on the immediate structural and electronic environment of the immobilized chromophores.

• The near-UV CD spectrum has very high sensitivity for the native state of a protein. It can be used as a finger-print of the correctly folded conformation.

 Peptide binding to nucleic acids

CD spectra before and after mixing SCM and Mj HSP16.5. 

Protein-protein interactions

Summary of CD

• Circular dichroism spectroscopy is used to gain information about the secondary structure and folded state of proteins and polypeptides in solution.

Benefits:

• Uses very little sample (200 ul of 0.5 mg/ml solution in standard cells)

• Non-destructive • Relative changes due to influence of

environment on sample (pH, denaturants, temperature, etc.) can be monitored accurately.

Drawbacks:

• Interference with solvent absorption in the UV region

• Only very dilute, non-absorbing buffers allow measurements below 200 nm

• Absolute measurements subject to a number of experimental errors

• Average accuracy of fits about +/- 10%

• CD spectropolarimeter is relatively expensive