Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Absorption and CD Spectroscopy 51
Fig. 3. Far-UV CD spectra of apo-calmodulin recorded at five-degree intervals over
the temperature range 15 (�) to 75°C (�).
Some of these features are illustrated in Fig. 3, which shows the far-UV CD
spectrum of apo-calmodulin as a function of temperature in the range 15–75°C.
At low temperature the spectrum shows bands characteristic of the α-helix; heating
causes progressive unfolding of the protein, the α-helical bands are lost and
the bands characteristic of random or disordered structure appear.
5. Several approaches have been employed in attempts to determine the secondary
structure content of proteins from their far-UV CD spectra (for reviews, see refs.
1,2,4,5,15). Early methods attempted to analyze CD spectra as linear combinations
of reference (or basis) spectra for individual secondary structure elements
that were derived from the spectra of model polypeptides or proteins. More modern
methods analyze experimental CD curves more directly as linear combinations
of the spectra of proteins whose structure has been determined by X-ray
diffraction. All methods, even the oldest, give a reasonable estimate of α-helix
content. CONTIN, VARSLC, and SELCON are all generally reliable; these, and
other available methods have been discussed in several articles (1,15–18; see
Note 12). The best starting point for anyone interested in these methods is the
excellent review article by Greenfield (17). The validity of the various underlying
assumptions in the calculation of secondary structure content from CD have been