Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

50 Martin and Bayley
Fig. 2. Near-UV CD spectra of apo-calmodulin (�), Ca 4-calmodulin (�), and the
complex of Ca 4-calmodulin with an 18-residue peptide from the calmodulin binding
domain of skeletal myosin light-chain kinase (�).
and 293 nm. Studies with nontryptophan-containing peptides show that the spectrum
of Ca 4-calmodulin remains effectively unchanged in the complex.
4. Far-UV CD spectra depend upon the secondary structure content of the protein
and are generally easier to interpret. Characteristic features of the spectra of different
protein classes may be summarized as follows (15):
• All-α proteins show an intense negative band with two peaks (208 and 222 nm)
and a strong positive band (191–193 nm). The intensities of these bands reflect
α-helical content. ∆ε mrw values for a totally helical protein would be of the
order of –11 M –1 cm –1 (208/222 nm) and +21 M –1 cm –1 (191–193 nm).
• Regular all-β proteins usually have a single negative band (210–225 nm,
∆ε mrw –1 to –2.5 M –1 cm –1 ) and a stronger positive band (190–200 nm, ∆ε mrw
2–6 M –1 cm –1 ). Intensities are significantly lower than for all-α proteins.
• Unordered peptides and denatured proteins have a strong negative band
(195–200 nm, ∆ε mrw –4 to –8 M –1 cm –1 ) and a much weaker band (either
negative or positive) between 215 and 230 nm (∆ε mrw +0.5 to –2.5 M –1 cm –1 ).
• α+β and α/β proteins generally have spectra dominated by the α-helical component
and, therefore, often show bands at 222, 208, and 190–195 nm. In
some cases, there may be a single broad minimum between 210 and 220 nm
because of overlapping α-helical and β-sheet contributions. Intensities depend
on the α-helical content.