Ma. Markus et al., LOCAL MOBILITY WITHIN VILLIN 14T PROBED VIA HETERONUCLEAR RELAXATION MEASUREMENTS AND A REDUCED SPECTRAL DENSITY MAPPING, Biochemistry, 35(6), 1996, pp. 1722-1732
Villin 14T, a representative domain from the actin severing and bundli
ng protein villin, binds calcium ions and actin monomers. To begin to
understand the contributions of mobility to the villin-calcium and vil
lin-actin interactions, relaxation rates for magnetization involving t
he amide nitrogens and protons have been measured for N-15-labeled vil
lin 14T in solution. Although we have measured the complete set of rat
es required for a full spectral density map, difficulties in the accur
ate measurement of relaxation rates for antiphase coherence and two-sp
in order led us to consider a reduced mapping formalism. From the redu
ced spectral density map, a model-free analysis, or directly from the
measured N-x,N-y relaxation rates, local variations in mobility along
the backbone of villin 14T have been revealed. Fast motions are eviden
t not only at the amino and carboxyl termini but also in the turn betw
een strands beta 4 and beta 5 of the central beta-sheet and in the tur
n between helix alpha 3 and strand beta 7. Slower motions are suggeste
d for the turn between strands beta 2 and beta 3. Motions on the micro
second to millisecond time scale have been probed directly by examinin
g the dependence of the proton transverse relaxation rate on the spin-
locking field strength. Leu(11) shows a strong dependence on field str
ength, implying conformational exchange with a time constant of 125 +/
- 69 mu s. The backbone at the actin-binding interface appears to be r
ather rigid.