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 Halpha chemical shifts and 1H-1H NOE indicate that the protein chain in the denatured state has structural preferences in the broad beta-domain for many residues. Several of these are seen to populate the (phi,psi) 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.

FIGURE 1

(A) Sequential walk through the F₁–F₃ planes of HNN spectrum of SUMO-1 in 8 M urea at pH 5.6 and 27°C. Sequential connectivities are shown for Gly-19 to Leu-24 stretch. The F₂ (¹⁵N) values are shown at the top for each strip. The black and red contours indicate positive and negative peaks. The distinct Gly plane serves as the start point in the sequential assignment. (B) Summary of sequential assignment obtained from HNN walk along the primary sequence of SUMO-1. Gly shown in green worked as start/check point; Pro shown in red worked as break point.

FIGURE 2

(¹H-¹⁵N) HSQC spectrum of SUMO-1 in 8 M urea at pH 5.6 and 27°C. Residue-specific assignment for each peak is marked on the spectrum.

FIGURE 3

(A) Sequence-corrected secondary chemical shift for H^α (Δδ(H^α)) in SUMO-1 at 8 M urea at pH 5.6 and 27°C. The sequence-corrected random coil values used are those determined by Schwarzinger et al. (70). The native secondary structural elements are shown on the top of the figure and the segments showing structural propensities are indicated by solid bars. (B) Selected region from a high resolution (¹H-¹⁵N) HSQC spectrum of SUMO-1 in 8 M urea at pH 5.6 and 27°C. The splitting in the peaks was used to measure ³J (H^N-H^α) coupling constants. (C) Secondary coupling constants (see text) are plotted against sequence for SUMO-1. (D) Far-UV CD spectra of SUMO-1 at pH 5.6 and 27°C, in the absence of urea (shaded circles), and in 8 M urea (open circles).

FIGURE 4

Secondary temperature coefficients. Deviation of amide proton temperature coefficients from the random coil temperature coefficient (magnitude mode) at pH 5.6 plotted against the sequence. The native secondary structure elements are indicated at the top.

FIGURE 5

Summary of sequence-specific ¹H-¹H connectivities obtained from the NOESY-¹⁵N-¹H HSQC spectrum.

FIGURE 6

Spectra showing ¹⁵N-¹H heteronuclear NOE of SUMO-1 in 8 M urea at pH 5.6 and 27°C. (A) Reference spectrum without irradiation of protons; (B) spectrum with irradiation of protons for 3 s; and (C) spectrum of the folded protein (pH 7.4, 27°C) with proton irradiation as in B. In B, a cross section through the peak for Q94 (Q94 is enclosed in a box) is shown to demonstrate the good sensitivity in the spectrum. The red and black contours indicate negative and positive peaks.

FIGURE 7

¹⁵N relaxation rates plotted against sequence for SUMO-1 in 8 M urea at pH 5.6 and 27°C. (A) Longitudinal (R₁) and (B) transverse (R₂) relaxation rates. The structural propensities seen in Fig. 3 A are also indicated in B with solid bars for ready reference. Solid circles in A and B identify residues with high flexibility.