BioPHYSICAL chemistry

Determination of secondary structure using circular dichroism

Chiral molecules can be distinguished based upon their inter-

action with polarized light. Light can be linearly polarized, in

which case the electromagnetic fields oscillate back and forth

along a line. Alternatively, light can be polarized circularly

when the field rotates as the light propagates, so that when

the light is viewed down the path of travel, the electro-

magnetic fields are observed to rotate in a circle in either a

right-handed or left-handed direction. For chiral molecules,

right- and left-handed circularly polarized light travels differ-

ently in an effect termed circular birefringence. As a result,

chiral molecules show circular dichroism, namely a difference

in their absorbance for left- and right-handed polarized light. In a

circular dichroism spectrum, the wavelength dependence is

measured for the difference in the absorbance for the right-

and left-handed polarized light. For proteins, the effect of this

optical activity is evident in a circular dichroism spectrum

that is largely determined by the secondary structure of the

protein (Figure 13.12). For example, αhelices show a much

stronger peak near 190 nm than βstrands or random coils.

By fitting a spectrum of a protein, it is possible to estimate the

relative contributions of secondary structure. In addition to being

used for structure prediction, circular dichroism is a useful measure of

the extent to which a previously unfolded protein has been folded, and

thus can be used in thermal or chemical denaturation studies.

Research direction: modeling protein structures and folding

Identification of the interactions that stabilize protein structures has pro-

vided the framework for the development of computational models of

protein structure. Such models are becoming increasingly more sophisti-

cated and are now routinely run for protein-structure determination

(Chapter 15). To provide an accurate representation of the protein, these

models include terms that reflect bond stretching, bending, and rotation.

Although bond lengths and angles are formally determined by inter-

actions of electrons and nuclei as described by quantum mechanics, these

interactions can be treated by simple physical models. For example, the

bond-stretching potential, V(r), is determined by calculating the distance

for each covalent bond, r, and comparing that distance to an ideal 9 alue,

rstandard(eqn 13.13). A similar expression can be written for the bending

involving each angle θthat can be defined in terms of two neighboring

284 PART 2 QUANTUM MECHANICS AND SPECTROSCOPY

0

190 210 230 250

Random coil

α Helix

Circular dichroism signalβ Sheet

Wavelength (nm)

Figure 13.12
Circular dichroism
spectra of αhelices,
βsheets, and
random coils.