The molecular chaperonin cpn60 displays local flexibility that is reduced after binding with an unfolded protein.

Steady-state fluorescence polarization was used to examine the chaperonin cpn60 that was covalently labeled with pyrene. Two compounds, 1-pyrenesulfonyl chloride or N-(1-pyrene)maleimide, were used to incorporate up to 8 mol of pyrene per mol of cpn60 14-mer. The fluorescence lifetime of the cpn60-pyrenesulfonyl chloride conjugate exhibited a double exponential decay: 5.36 ns, with a fractional contribution to the intensity of 7%, and 48.77 ns, with a fractional contribution to the intensity of 93%. These yield a second-order average lifetime of 45.58 ns at 20 degrees C. Analysis of the fluorescence polarization data for the pyrene probe by the Perrin-Weber treatment revealed the existence of two components that account for the depolarization. The fast component accounted for 24% of the depolarization at 20 degrees C. The rotational relaxation time for the cpn60 14-mer derived from the low viscosity part of the Perrin-Weber plot which accentuates the slow motion gave rho h = 1113 +/- 55 ns. When this value of rho h is compared with the rho h calculated based on the Stokes radius of cpn60 from ultracentrifugation, rho Stokes, it leads to rho h/rho Stokes = 0.4 which is considerably smaller than the value expected (rho h/rho Stokes = 1) or actually found in the cpn60-rhodanese complex (rho h/rho Stokes = 0.93). These considerations and the observed presence of the fast motion suggest that cpn60 is not a rigid protein. Analysis of the polarization data as a function of temperature, which is weighted more toward the fast motion, showed that the rotational relaxation time assessed by temperature variation is greatly increased (from 552.5 to 2591 ns) for the complex of cpn60 with partially folded rhodanese (34-kDa monomeric protein). No change in rho h was observed upon formation of the cpn60.ATP complex (rho h = 556.9 ns). These data indicate that there is local motion in the cpn60 14-mer molecule that can be frozen by formation of a binary complex with partially folded proteins. This conclusion is in keeping with results showing that the structure of cpn60 is generally stabilized when it forms complexes with passenger proteins (Mendoza, J. A., and Horowitz, P. M. (1994) J. Biol. Chem. 269, 25963-25965).