Deuterium isotope shifts for backbone ¹H, ¹⁵N and ¹³C nuclei in intrinsically disordered protein α-synuclein

Abstract

Intrinsically disordered proteins (IDPs) are abundant in nature and characterization of their potential structural propensities remains a widely pursued but challenging task. Analysis of NMR secondary chemical shifts plays an important role in such studies, but the output of such analyses depends on the accuracy of reference random coil chemical shifts. Although uniform perdeuteration of IDPs can dramatically increase spectral resolution, a feature particularly important for the poorly dispersed IDP spectra, the impact of deuterium isotope shifts on random coil values has not yet been fully characterized. Very precise (2)H isotope shift measurements for (13)C(α), (13)C(β), (13)C', (15)N, and (1)H(N) have been obtained by using a mixed sample of protonated and uniformly perdeuterated α-synuclein, a protein with chemical shifts exceptionally close to random coil values. Decomposition of these isotope shifts into one-bond, two-bond and three-bond effects as well as intra- and sequential residue contributions shows that such an analysis, which ignores conformational dependence, is meaningful but does not fully describe the total isotope shift to within the precision of the measurements. Random coil (2)H isotope shifts provide an important starting point for analysis of such shifts in structural terms in folded proteins, where they are known to depend strongly on local geometry.

Figure 1

Spectra acquired on a mixture of protonated and perdeuterated aS. (a) Expanded region of the 900 MHz TROSY-HSQC spectrum. The upfield components in the ¹⁵N dimension of each pair of resonances lack the partially resolved ³J_HH splitting seen on the downfield components and correspond to the perdeuterated form of the protein. With the exception of H(D) of T22 and T59, all ²H isotope shifts observed in this spectrum are negative. (b) Expanded region of a projection on the ¹³C/¹⁵N plane of the 600 MHz constant-time NUS TROSY-HNCACB spectrum. Dashed lines connect peaks originating from perdeuterated and protonated aS. The P128 C^α peaks are observed via residue S129. P128 and S129 peaks are connected by horizontal dotted lines. Upfield shifted components of each pair correspond to the perdeuterated form of the protein.

Figure 2

Overlays of two spectra acquired on ¹H/¹⁵N-Lys, perdeuterated aS (red) and on a mixture of protonated and perdeuterated aS (black). (a) Expanded region of the 900 MHz TROSY-HSQC spectrum, where the spectrum of the mixed sample presents a different region of the same spectrum shown in Figure 1a. Small downfield shoulders on the red correlations observed for Val residues originate from incomplete perdeuteration of their sidechains. Resonances for Lys residues in the ¹H/¹⁵N-Lys, perdeuterated aS (red) spectrum are more downfield than those of the fully protonated component of the mixed sample spectrum (black), indicating that perdeuteration of the residue preceding Lys causes a downfield ²H isotope shift for both ¹⁵N and ¹H^N. In the ¹H/¹⁵N-Lys, perdeuterated aS spectrum the Lys peaks are displaced from their positions in perdeuterated aS by a nearly uniform amount. However, the displacement of Lys peaks from their positions in fully protonated aS is very different for K12 compared to K32 and K102. This results from the fact that K12 is preceded by Ala, while K32 and K102 are preceded by Gly, and the neighboring-residue contribution of Gly on ¹⁵N is about double that of Ala. (b) Expanded region of a projection of the constant-time 600 MHz NUS TROSY-HNCO spectrum on the ¹³C/¹⁵N plane. Resonances are labeled by the observed amide pair following the ¹³C'. The small, mostly downfield change in chemical shift relative to the broad component in the mixed sample (black) corresponds to the intraresidue ²H isotope shift effect on the ¹³C' of the residue preceding Lys.

Figure 3

Correlation plots of the total deuterium isotope shifts for (A) ¹H^N, (B) ¹⁵N, and (C) ¹³C’. Experimental values (vertical axis) are plotted against values predicted by using eqs 2–4 with the coefficients of Tables 2–4. Pairwise root mean sqare differences between measured (exp) and predicted (pred) H(D), N(D), and C'(D) isotope shifts are 2, 14, and 5 ppb, respectively.