Mobility of NH bonds in DNA-binding protein HU of shape Bacillus stearothermophilus from reduced spectral density mapping analysis at multiple NMR fields

Abstract

The dynamics of the backbone NH bonds of protein HU from Bacillus stearothermophilus (HUBst) have been characterized using measurements of cross-relaxation, longitudinal and transverse relaxation rates$({\text{R}}_{\text{N}} ({\text{H}}_{\text{z}} \leftrightarrow {\text{N}}_{\text{z}} ),{\text{R}}_{\text{N}} {\text{(N}}_{\text{z}} ){\text{ and R}}_{\text{N}} ({\text{N}}_{{\text{x,y}}} ))$ at 11.7, 14.1 and 17.6 T. Linear regression of the values$2{\text{R}}_{\text{N}} ({\text{N}}_{{\text{x,y}}} ) - {\text{R}}_{\text{N}} ({\text{N}}_{\text{Z}} )$ with the squared Larmor frequency ω ²_N has revealed global exchange processes, which contributed on the order of 0.5–5.0 s^-1to the transverse relaxation rate. Subsequently, the experimental values${\text{R}}_{\text{N}} ({\text{N}}_{{\text{x,y}}} )$ were corrected for these exchange contributions. A reduced spectral density mapping procedure has been employed with the experimental relaxation rates and seven values of the spectral density function J(ω) have been extracted. These spectral densities have been fitted within the framework of the model-free approach. The densities agree well with an axially symmetric rotational diffusion tensor with a diffusion anisotropy D_∥/D_⊥ of 1.15, indicating that the flexible arms of HUBst do not significantly contribute to the rotational diffusion. The overall correlation time is 8.9 ± 0.6 ns/rad. The fast internal motions of most of the NH bonds in the core display order parameters ranging between 0.74 and 0.83 and internal correlation times between 1 and 20 ps. For the residues in the DNA-binding β-arms, an extended version of the model function has been used. The slow internal motions show correlation times of 1–2 ns. The concomitant order parameters (0.3–0.6) are lower than those observed on the fast time scale, indicating that the flexibility of the β-arms is mainly determined by the slower internal motions. A substantial decrease of the generalized order parameters in the β-arms starting at residues Arg⁵⁵ and Ser⁷⁴, opposite on both strands of the β-ribbon arms, has been explained as a ‘hinge’ motion. A comparison of the order parameters for free and DNA-bound protein has demonstrated that the slow hinge motions largely disappear when HU binds DNA.