FLEXIBILITY INVOLVING THE INTERMOLECULAR DITYROSYL CROSS-LINKS OF ENZYMATICALLY POLYMERIZED CALMODULIN

The role of dityrosine as a fluorescent crossbridge between adjacent c almodulin molecules within the high molecular mass polymers that are g enerated by Arthromyces peroxidase-catalyzed cross-linking [Malencik, D. A., and Anderson, S. R. (1996) Biochemistry 35, 4375] has been exam ined in frequency domain fluorescence anisotropy studies. Measurements on a polymer fraction possessing a range of molecular masses > 96000 in NaDodSO(4) polyacrylamide gel electrophoresis demonstrate predomina ting fast local rotations involving the dityrosyl moieties. Normal dis tribution analyses of the results show peak: rotational correlation ti mes of 0.6 ns (zero Ca2+) and 1.2 ns (+Ca2+), values that are smaller than the principal correlation times determined for the global rotatio n of the free calmodulin monomer in either the presence or absence of Ca2+. The intermolecularly cross-linked segments of the polymers retai n a degree of the mobility that is characteristic of the tyrosine-cont aining sequences of native calmodulin. The half-widths of the normal d istribution curves range from 13 ns (zero Ca2+) to similar to 90 ns (5 mM Ca2+), thus encompassing varying rates of segmental motion within the polymers. When Ca2+ is present, possible contributions from the gl obal rotations of polymer molecules are detected near the operating li mits of the method. Experiments with the intramolecularly cross-linked calmodulin monomer give global rotational correlation times of 7.9 ns (zero Ca2+) and 11.4 n (+Ca2+), which compare to values of 7.2 ns and 9.9 ns found previously in time domain measurements [Small, E. W., an d Anderson, S. R. (1988) Biochemistry 27, 419]. Rotations of apparent phi(2) = 0.2 to 0.3 ns also are detected, accounting for 31% (-Ca2+) t o 23% (+Ca2+) of the anisotropy.