Local Mobility within Villin 14T Probed via Heteronuclear Relaxation Measurements and a Reduced Spectral Density Mapping

• Published in: Biochemistry (Easton) 1996-02, Vol.35 (6), p.1722-1732
• Main Authors: Markus, Michelle A, Dayie, Kwaku T, Matsudaira, Paul, Wagner, Gerhard
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Amino Acid Sequence; Binding Sites; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Electron Spin Resonance Spectroscopy; Magnetic Resonance Spectroscopy; Microfilament Proteins - chemistry; Microfilament Proteins - genetics; Microfilament Proteins - metabolism; Models, Molecular; Molecular Sequence Data; Molecular Structure; Protein Binding; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi951933o

Villin 14T, a representative domain from the actin severing and bundling protein villin, binds calcium ions and actin monomers. To begin to understand the contributions of mobility to the villin−calcium and villin−actin interactions, relaxation rates for magnetization involving the amide nitrogens and protons have been measured for 15N-labeled villin 14T in solution. Although we have measured the complete set of rates required for a full spectral density map, difficulties in the accurate measurement of relaxation rates for antiphase coherence and two-spin order led us to consider a reduced mapping formalism. From the reduced spectral density map, a model-free analysis, or directly from the measured N x,y relaxation rates, local variations in mobility along the backbone of villin 14T have been revealed. Fast motions are evident not only at the amino and carboxyl termini but also in the turn between strands β4 and β5 of the central β-sheet and in the turn between helix α3 and strand β7. Slower motions are suggested for the turn between strands β2 and β3. Motions on the microsecond to millisecond time scale have been probed directly by examining the dependence of the proton transverse relaxation rate on the spin-locking field strength. Leu11 shows a strong dependence on field strength, implying conformational exchange with a time constant of 125 ± 69 μs. The backbone at the actin-binding interface appears to be rather rigid.