Long-lived states to monitor protein unfolding by proton NMR

Abstract

The relaxation of long-lived states (LLS) corresponds to the slow return to statistical thermal equilibrium between symmetric and antisymmetric proton spin states. This process is remarkably sensitive to the presence of external spins and can be used to obtain information about partial unfolding of proteins. We detected the appearance of a destabilized conformer of ubiquitin when urea is added to the protein in its native state. This conformer shows increased mobility in the C-terminus, which significantly extends the lifetimes of proton LLS magnetisation in Ser-65. These changes could not be detected by conventional measurements of T(1) and T(2) relaxation times of protons, and would hardly be sensed by carbon-13 or nitrogen-15 relaxation measurements. Conformers with similar dynamic and structural features, as revealed by LLS relaxation times, could be observed, in the absence of urea, in two ubiquitin mutants, L67S and L69S.

Figure 1

¹⁵N -¹H HSQC spectra of WT ubiquitin (measured at ¹⁵N natural abundance) at pH 7 before (green) and after (red) addition of 8 M urea. The experiments were carried out at 18.8 T (800 MHz for protons) and T = 298 K. Shown insets outline signals of residues undergoing significant shifts.

Figure 2

Residues in the β-strand structure of ubiquitin and selected H-bond partners. The residues that show weighted (see text) chemical shift differences larger than the average value for all the amino acids in the ¹⁵N-¹H HSQC spectra of Fig. 1 are marked by rectangles.

Figure 3

Intensities of signals of Gly-75, Gly-76 (left) and Ser-65 (right) as a function of the concentration of urea, using a constant sustaining interval τ_LLS = 1 s. Error bars reflect noise in the spectra. The Ser-65 signal intensity increases, while the Gly-75, Gly-76 signal intensities decrease.

Figure 4

Region of a ¹H - ¹H TOCSY spectrum of the L69S mutant of ubiquitin. The chemical shifts of Ser-69 and Ser-65 are shown. The experiments were carried out at 14.1 T (600 MHz for protons) with a mixing interval τ_m = 80 ms. The inset shows a 1D spectrum of L69S ubiquitin obtained after transfer into observable magnetisation from an LLS comprising the three protons H^α, H^β1, and H^β2 of Ser-65. The LLS was sustained during τ_LLS = 1 s by a train of Sinc pulses.