The homeodomain fold is a relatively simple DNA/RNA-binding protein structure consisting of 60 aminoacids which form three a-helices and bind to the nucleic acid with a helix-turn-helix motif. The domain was found in many transcription factors involved in several aspects of embryonic development, regulating cell fate and development plan of the body .
Our main goal consisted in searching for water-protein contacts observable by NMR. A doubly labeled mutant K52E of the drosophila Engrailed homeodomain was prepared for an NMR analysis. Standard 3D NMR spectra (HNCACB and CBCACONH) were measured for the backbone assignment. Protein solvation was monitored at various temperatures by recording homonuclear 2D NOESY spectra with high resolution in the indirect dimension. Several NOESY cross peaks were observed at the water frequency, indicating water molecules tightly bound to protein residues . The movement of these water molecules is significantly slowed down, giving rise to an NOE signal. In order to distinguish exchangeable water molecules from bulk water and from tightly bound water molecules, a CLEANEX-PM experiment was conducted .
NOESY experiments showed that residues S13, R18, R32, R56 are tightly bound to water molecules. Several residues in exchange between bulk and bound water (R6, R21, R27, R33 and R34) have been revealed by the CLEANEX-PM experiment.
This study was supported by the Czech Science Foundation, grant no. GA14-14654S. This work was realized in CEITEC - Central European Institute of Technology with research infrastructure supported by the project CZ.1.05/1.1.00/02.0068 financed from European Regional Development Fund.