LOCAL MOBILITY WITHIN VILLIN 14T PROBED VIA HETERONUCLEAR RELAXATION MEASUREMENTS AND A REDUCED SPECTRAL DENSITY MAPPING

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.