Mk. Helms et al., FLEXIBILITY INVOLVING THE INTERMOLECULAR DITYROSYL CROSS-LINKS OF ENZYMATICALLY POLYMERIZED CALMODULIN, Biochemistry, 37(23), 1998, pp. 8378-8384
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.