Backbone dynamics of a short PU.1 ETS domain

The resonance assignments, secondary structure and backbone dynamics of the ETS domain of the transcription factor PU.1 have been determined for the f ree protein in solution by NMR spectroscopy. The secondary structure for th e free ETS domain is similar to that observed in the crystal structure of t he PU.1 protein complexed with DNA, except that helix alpha 2 and recogniti on helix alpha 3 are shorter for the free protein in solution. Backbone dyn amics of the protein have been examined using amide hydrogen-deuterium exch ange and N-15 laboratory-frame spin relaxation measurements. A significant probability of local unfolding of helix alpha 2, which precedes the loop-he lix-loop DNA recognition domain, is inferred from the very fast hydrogen-de uterium exchange for amide protons in this helix. The 15N relaxation measur ements indicate that the protein is partially oligomerized at a concentrati on of 2.5 mM, but monomeric at a concentration of 0.3 mM. The 15N relaxatio n data for the low concentration sample were interpreted, using the model-f ree formalism, to provide insight into protein dynamics on picosecond-nanos econd and microsecond-millisecond time scales. High flexibility of the prot ein backbone is observed for the residues in the loop between alpha 2 and a 3. This loop is variable in length and in structure within the class of win ged helix proteins and is partially responsible for binding to DNA. The dyn amic properties observed for alpha 2, alpha 3 and the intervening loop may indicate a correlation between protein plasticity in particular structural elements and recognition of specific DNA sequences. (C) 1999 Academic Press .