Secondary structure and dynamics of glucagon in solution.

A correlation between the secondary structure of glucagon determined by circular dichroism and its dynamic behaviour as obtained from picosecond fluorescence anisotropy is demonstrated. The CD data show that the percentage of alpha-helix decreases with increasing temperature, but the rotational relaxation time of the glucagon increases with temperature. These observations suggest that the protein's shape changes with temperature in such a way that its volume is larger at 38 degrees C than at 5.5 degrees C. The fluorescence anisotropy of glucagon decays biexponentially at each temperature studied and at 26 degrees C the rotation lifetimes are 1670 and 307 ps at pH 10.2 and 2147 and 517 ps at pH 2.2. It is proposed that the shorter decays are due to the restricted motion of the single tryptophan residue while rotation of the whole protein is responsible for the longer decays. The calculated rotational diffusion coefficient, Dw, of the tryptophan residue is much smaller, (ie. has a larger apparent volume) than that of a free tryptophan in solution. The hydrophobic interactions between residues Phe-22 to Leu-26 are probably responsible for the larger apparent volume in the protein compared to solution and will stabilize this part of the protein. The rotational diffusion of aggregated glucagon is also discussed.