Figure 9

Secondary structure and melting temperature of the LLER protein. A. The far-UV spectra (195 - 245 nm) of the LLER protein were recorded with an Aviv circular dichroism (CD) spectrometer at the indicated temperatures. The percentage of alpha-helical content at the various temperatures was calculated and is shown in the key. B. The melting curve of the BCCL2 protein was generated by plotting the alpha-helical content as a function of temperature. Note the biphasic shape of the curve. C. Approximately 2 μg of the BCCL2 protein was incubated at the indicated temperatures for 5 minutes prior to the addition of 1 mM glutaraldehyde. Incubation at the same temperature was continued for another 8 minutes, after which the reaction was quenched by addition of excess 0.1 M Tris-HCl, pH 7.5 buffer. A control reaction without the addition of glutaraldehyde was also performed at each temperature. The reaction mixtures were boiled in Laemmli buffer and analyzed on a 4-12% SDS-polyacrylamide gel. M, molecular weight markers.