Helix mobility and recognition function of the rat thyroid transcription factor 1 homeodomain - Hints from ¹⁵N-NMR relaxation studies

The backbone dynamics of the ¹⁵N-labeled homeodomain of the rat thyroid transcription factor 1 has been studied by 2D NMR spectroscopy. Longitudinal (R₁) and transverse (R₂) ¹⁵N relaxation rate constants and steady-state {¹H}-¹⁵N NOEs were measured at 11.7 T. These data were analyzed by both the model-free formalism and the reduced spectral density mapping (RSDM) approaches. The global rotational correlation time, τ_m, of the thyroid transcription factor 1 homeodomain in aqueous solution at 286 K was found to be 10.51 ± 0.05 ns by model-free formalism and 9.85 ± 1.79 ns by RSDM calculation. The homogeneity of the values of the overall correlation time calculated from the individual (R₂/R₁) ratios suggested a good degree of isotropy of the global molecular motion, consistent with the similar global τ_m results obtained with the two different methods. Tyr25 was found to undergo slow conformational exchange by both methods, whereas this contribution was identified also for Lys21, Gln22, Ile38 and His52 only by RSDM. With both methods, the C-terminal fragment of helix III was found to be more flexible than the preceding N-terminal portion, with slightly different limits between rigid and mobile moieties. Additionally, Arg53 appeared to be characterized by an intermediate motional freedom between the very flexible N-terminal and C-terminal residues and the structured core of the molecule, suggesting the occurrence of a hinge point. Finally, slow-time-scale motions observed at the end of helix I, at the end of helix II and within helix III appear to be consistent with typical fraying transitions at helical C-termini.