Local structural preferences and dynamics restrictions in the urea-denatured state of SUMO-1: NMR characterization

Abstract

We have investigated by multidimensional NMR the structural and dynamic characteristics of the urea-denatured state of activated SUMO-1, a 97-residue protein belonging to the growing family of ubiquitin-like proteins involved in post-translational modifications. Complete backbone amide and ¹⁵N resonance assignments were obtained in the denatured state by using HNN and HN(C)N experiments. These enabled other proton assignments from TOCSY-HSQC spectra. Secondary H^α chemical shifts and ¹H-¹H NOE indicate that the protein chain in the denatured state has structural preferences in the broad β-domain for many residues. Several of these are seen to populate the (Φ ,ψ) space belonging to polyproline II structure. Although there is no evidence for any persistent structures, many contiguous stretches of three or more residues exhibit structural propensities suggesting possibilities of short-range transient structure formation. The hetero-nuclear ¹H-¹⁵N NOEs are extremely weak for most residues, except for a few at the C-terminal, and the ¹⁵N relaxation rates show sequence-wise variation. Some of the regions of slow motions coincide with those of structural preferences and these are interspersed by highly flexible residues. The implications of these observations for the early folding events starting from the urea-denatured state of activated SUMO-1 have been discussed.