Thermal coefficients of the methyl groups within ubiquitin

Abstract

Physiological processes such as protein folding and molecular recognition are intricately linked to their dynamic signature, which is reflected in their thermal coefficient. In addition, the local conformational entropy is directly related to the degrees of freedom, which each residue possesses within its conformational space. Therefore, the temperature dependence of the local conformational entropy may provide insight into understanding how local dynamics may affect the stability of proteins. Here, we analyze the temperature dependence of internal methyl group dynamics derived from the cross-correlated relaxation between dipolar couplings of two CH bonds within ubiquitin. Spanning a temperature range from 275 to 308 K, internal methyl group dynamics tend to increase with increasing temperature, which translates to a general increase in local conformational entropy. With this data measured over multiple temperatures, the thermal coefficient of the methyl group order parameter, the characteristic thermal coefficient, and the local heat capacity were obtained. By analyzing the distribution of methyl group thermal coefficients within ubiquitin, we found that the N-terminal region has relatively high thermostability. These results indicate that methyl groups contribute quite appreciably to the total heat capacity of ubiquitin through the regulation of local conformational entropy.

The unusual internal motion of the villin headpiece subdomain

“…In contrast to previous studies of methyl dynamics across different temperatures, a particularly sharp temperature dependence of is observed, with an average value of −5.9 ± 1.5 × 10 −3 K −1 across methyl residues. This value is significantly larger than that in previous studies on ubiquitin and a calmodulin–peptide complex, where the observed d /d T values were −2.3 and −2.5 × 10 −3 K −1 , respectively .…”

“…The average temperature derivative of the values obtained in the solid state data is −2.2 ± 1.2 × 10 −3 K −1 and compares with −5.9 ± 1.5 × 10 −3 K −1 obtained in solution. Interestingly, the slope of the solid state data correlates very well with previous studies of temperature‐dependent methyl dynamics in the solution state . Significant differences between the solution and solid‐state lambda values for HP36 are seen (Fig.…”

Structural Integrity of the A147T Polymorph of Mammalian TSPO

“…In order to find out if the A147T substitution influences the motional properties of the side chains of mTSPO and thus modulates the entropic component of the interaction with PK11195, we probed the dynamics of the methyl groups of mTSPO in complex with (R)-PK11195. Cross-correlated relaxation rates (σ obs ) between dipolar couplings of two CH bonds were measured for well separated cross peaks in 2D 1 H-13 C HSQC spectra [21]. 19 methyl groups distributed across the entire structure could be analyzed ( Figure 3A and Supporting Information Figure S3).…”

“…19 methyl groups distributed across the entire structure could be analyzed ( Figure 3A and Supporting Information Figure S3). As more rigid methyl groups have larger σ obs rates [21], the methyl groups of A50, I52 and L56 are most rigid. Moreover, comparison of σ obs rates between wild-type protein and the A147 variant showed that the motional properties of the methyl groups were retained despite the A147 substitution ( Figure 3A).…”

“…CCR rates in methyl groups were measured on 13 C/ 15 N-labelled protein in complex with (R)-PK11195 (samples in D 2 O) using constant-time 1 H-13 C HSQC experiments. Collected intensities were analyzed as described previously [21].…”