Advances in studying protein disorder with solid-state NMR

Abstract

Solution NMR is a key tool to study intrinsically disordered proteins (IDPs), whose importance for biological function is widely accepted. However, disordered proteins are not limited to solution and are also found in non-soluble systems such as fibrils and membrane proteins. In this Trends article, I will discuss how solid-state NMR can be used to study disorder in non-soluble proteins. Techniques based on dipolar couplings can study static protein disorder which either occurs naturally as e.g. in spider silk or can be induced by freeze trapping IDPs or unfolded proteins. In this case, structural ensembles are directly reflected by a static distribution of dihedral angels that can be determined by the distribution of chemical shifts or other methods. Techniques based on J-couplings can detect dynamic protein disorder under MAS. In this case, only average chemical shifts are measured but disorder can be characterized with a variety of data including secondary chemical shifts, relaxation rates, paramagnetic relaxation enhancements, or residual dipolar couplings. I describe both technical aspects and examples of solid-state NMR on protein disorder and end the article with a discussion of challenges and opportunities of this emerging field.

Keywords: Frozen solution; Intrinsically disordered proteins; Protein dynamics; Protein folding; Scalar coupling based methods; Solid-state NMR.

Figure 1:

DOQSY spectra can determine the distribution of dihedral angles in silk. a) Experimental 2D DOQSY spectra of 1-¹³C alanine-labeled N. edulis silk. b) best fitting theoretical DOQSY spectrum. c) Resulting probability density P (ϕ, ψ) describing dihedral angle distribution. (Adapted with permission from Ref. [34]. Copyright (2002) National Academy of Sciences.)

Figure 2:

Principle of studying IDPs in frozen solution. Solution NMR can only investigate the dynamic average of the rapid conformational changes inherent to IDPs (Solution). In contrast, frozen solution solid-state NMR can directly observe the conformational ensemble of an IDPs (Frozen Solution). Here, not only an average chemical shift is observed but each conformation gives rise to its own set of chemical shifts. Artwork by Erin N. Johnson.

Figure 3:

Examples of frozen solution to study protein folding and protein disorder. a) Apparatus for rapid mixing and freeze-trapping based on a rotating copper plate immersed in a liquid-nitrogen. A jet of mixed solution freezes on copper after traveling a variable distance.b) DNP-enhanced, DQ-filtered ¹³C solid-state NMR spectra of frozen melittin solutions with the indicated refolding times. c) DNP enhanced 2D ¹³C-¹³C spectrum of selectively ¹³C-labeled -synuclein monomers. Intra- and interresidual crosspeaks involving extended or α-helical valine residue are highlighted in blue and orange, respectively. Panel a and b adapted with permission from Jeon et al. 2019 [41]. Panel c adapted with permission from Ref. [46], copyright (2018) Elsevier.

Figure 4:

Spectral separation of static and dynamically disordered domains in solid-state NMR. Center: bottle brush model of HTTex1 fibrils as an example of a protein with a static domain (fibril core, shown in blue and orange) and a dynamic domain (Pro-rich bristles, shown in green). Left: The static fibril core can be detected with the dipolar based ¹H-¹³C CP experiment resulting in the spectrum shown in blue. Right: dynamically disordered domains can be detected with the J-based, ¹H-¹³C refocused INEPT experiment resulting in the spectrum shown in green.

Figure 5:

Examples of insoluble protein systems with dynamic disorder studied with solid-state NMR. a) ¹³C spectra of α-synuclein fibrils recorded with several methods to generate initial ¹³C magnetization as indicated. The refocused INEPT is selective to dynamically disordered domains found adjacent to the core [57]. b) ¹H-¹³C 2D INEPT HETCOR spectrum selective for the disordered domains of AFA-PLN in DMPC bilayers that are located outside the membrane. Site specific assignments are indicated. c) Structural model of AFA-PLN in DMPC bilayers illustrating the dynamic disorder of the cytoplasmic N-terminus [56]. d) ¹H-¹H TOCSY spectra detect the highly flexible regions of HET-s(218–289) fibrils. Amino acid type assignments are indicated [58]. e) AFM images of 17-mer nucleosome arrays used for the NMR spectra shown in f). f) ¹H-¹³C 2D INEPT HETCOR spectrum of 17-mer nucleosome arrays containing ¹⁵N-¹³C-labeled H3. Amino acid type assignments are indicated [61]. g) Overlay of 2D ¹³C-¹³C CP-DARR and INEPT-TOBSY spectra of mature pHP1α droplets. The CP-DARR spectra detect the more static regions in these droplets whereas the INEPT-TOBSY is selective to the dynamically disordered domains. Amino acid type assignment are indicated [62]. Panel a adapted with permission from Ref. [57]. Copyright (2005) National Academy of Sciences. Panel b and c adapted with permission from Ref. [56]. Panel d adapted with permission from Ref. [58]. Panels e and f adapted with permission from Ref. [61]. Copyright (2005, 2006, 2013) American Chemical Society. Panel g adapted with permission from Ref.[62]. Copyright (2019) John Wiley and Sons.

Figure 6:

Techniques to characterize dynamic protein disorder in the solid state. a) ¹H detection can increase S/N. HSQC spectra of HTTex1’s dynamic C-terminus. Assignments are shown and linewidths of A106 illustrated. b) Site specific assignment of IDRs using out-and-back type experiments from solution NMR. Strip plots of HNCA and HNcoCA spectra recorded of HTTex1 fibrils. Assignments are indicated. c) Site specific assignments deliver residual secondary structure via secondary chemical shifts (yellow) and degree of dynamics via peak intensity (cyan). Examples of HTTex1 fibrils are shown. d) Relaxation rates are sensitive to the timescale of dynamics. Examples of R₂ and R_1pcurves recorded on HTTex1 fibrils are shown on top and site specific R₂ rates determined by both approaches on the bottom. e) Residual dipolar couplings determine order parameters. Top: example of ¹H–¹⁵N REDOR curve recorded on HTTex1 fibrils. Bottom: site specific residual dipolar couplings. f) Diffusion measurements can confirm that signals are from an immobilized protein. Diffusion decay curves of flexible domains in HET-s(218–289) fibrils (circle) and of DSS as control (diamonds). g) PREs can detect transient long-range interactions of IDRs. PRE profiles of tau’s fuzzy coat with the spin label located in the fibril core (left). Transient interactions of the N-terminus with the fibril core result in a decrease in intensity of the spin labeled sample compared to the diamagnetic control (right). Panel a-e adapted with permission from Ref. [72]. Panel f adapted with permission from Ref. [58]. Copyright (2006, 2018) American Chemical Society. Panel g adapted with permission from Ref. [65]. Copyright (2011) John Wiley and Sons.