Inclusion body formation of urogastrone and some fusion derivatives and relation to intracellular stability

to the P-axis and exposed to solvent in the dimer, while 248 is on a helix at the Q-axis and will become exposed to solvent as the Q-axis dimer forms monomers. All measurements are of solutions at equilibrium attained after at least 2 h incubation in the guanidinium HCl. The figure reports the intensity of the fluorescence of both tryptophans decreases at 0.3 M-guanidinium HCI and in the same range the enzyme activity, measured in cuvettes containing guanidinium HCI, NADH and pyruvate is lost. The fluorescence lifetimes and the fluorescence anisotropies were also measured in one experiment with the single photon counting instrument described by Birch et al. (1987). One emission photomultiplier measured total fluorescence at the polarization magic angle of 55 deg., while the other measured fluorescence in 30 s intervals when a motor-driven prism was either vertically or horizontally orientated to the vertically polarized excitation beam. The correlation time (7,) of the long lifetime (7.5 ns) tryptophan-203 in buffer (39 ns) is characteristic of the known dimer state, while in 1 Mguanidinium HCI (22 ns) it is typical of a globular monomer. We conclude the globular dimer is active and the monomer so prepared is enzymically inactive. Tryptophan-203 fluorescence only senses one further change in environment which is half-complete at 1.8 Mguanidinium HCI. At higher concentrations its anisotropy correlation time plateaus at 4 ns with, to judge by the zero limiting anisotropy (rx . ) , no local restriction to motion. Tryptophan-248 on the other hand, senses two further phase changes at 1.2 M and 2.8 M-guanidinium HC1, and only at the second phase (2.8 M) does the limiting, long time anisotropy decrease to zero. Between these concentrations the residue is carried by a structure which allows rapid (3 ns), but limited amplitude (r , = 0.054), motion. Whatever the nature of these structures, these measurements identify four reversible and stable intermediates during the folding of the polypeptide chain of this enzyme. In buffer, tryptophan-248 is on a helix, while 203 is carried on an extended chain. The explanation that the helix retains its folded structure at higher guanidinium HCl concentrations (2.8 M) than the chain (1.8 M) can be tested with other specific tryptophan reporters engineered at neighbouring sites on the helix 3G.