Resolution in cryogenic solid state NMR: Challenges and solutions

Abstract

NMR at cryogenic temperatures has the potential to provide rich site-specific details regarding biopolymer structure, function, and mechanistic intermediates. Broad spectral lines compared with room temperature NMR can sometimes present practical challenges. A number of hypotheses regarding the origins of line broadening are explored. One frequently considered explanation is the presence of inhomogeneous conformational distributions. Possibly these arise when the facile characteristic motions that occur near room temperature become dramatically slower or "frozen out" at temperatures below the solvent phase change. Recent studies of low temperature spectra harness the distributions in properties in these low temperature spectra to uncover information regarding the conformational ensembles that drive biological function.

Keywords: DNP; cryogenic NMR; dynamic nuclear polarization; magnetic resonance; protein dynamics; solid state NMR.

FIGURE 1

(a) DNP enhanced MAS NMR experiments of nucleosome arrays at 100 K enabled detection of long‐range correlations between histone residues and DNA. (b) Low temperature NMR of an 18‐residue peptide derived from the V3 loop sequence of the gp120 envelope glycoprotein of the HIV‐1 (frozen glycerol/water solution) helped to define the conformation with antibody bound.

FIGURE 2

(a) Bleaching of solution state NMR (H−N HSQC) for U − ¹⁵N,¹³C‐DHFR:H₂NADPH:TMP‐V‐T complex due to the affinity radical. A heat map is used to encode intensity ratios I _ox/I _red onto the structure, illustrating a distance dependence. (b) Low temperature SSNMR bleaching resembles the spatial patterns in solution NMR. A heat map conveys the ratio of DNP‐enhanced signal intensity for specifically isotopically enriched DHFR samples with bound TMP‐V‐T relative to samples with exogenous AMUPol/TMP ¹³C − ¹³C DARR spectra (I _TMPVT/I _AMUPol).

FIGURE 3

Specific loss or broadening of NMR signals at low temperature can result from exchange processes that interfere with magnetization transfer or decoupling.

FIGURE 4

(a) Protein unfolding is associated with increased spectral linewidths. Chemical denaturation of the 35‐residue villin headpiece subdomain (HP35) monitored at the site‐specific level by 2D solid state NMR. (b) DNP‐enhanced 2D ¹³C SSNMR spectra of Aβ40 assemblies with selective isotopic enrichment. Note reduced disorder in Aβ40 fibrils relative to the other samples. Tertiary contacts (L34–F19) seen in several samples suggest nascent structure in the monomer.

FIGURE 5

(A) Various “basins” can be identified within the broad spectral lineshape of I60 C′–I61N correlation spectra. (b) Torsion angle restraints from three “basins” within the lineshape of I60 C′'–I61N correlation spectra.

FIGURE 6

DNP NMR spectra at 105 K are compared for rapid freeze quench samples vs. with normal freezing samples. For this study, DHFR was selectively isotopically enriched with U ¹³C in four amino acids, LAPG. FWHM linewidths are indistinguishable for all sites in this case.

FIGURE 7

Panels (a‐c) Expansions from a ¹³C‐¹³C DARR spectra of DNP enhanced cryo MAS spectra of U‐¹³C,15N proline, showing diagonal and off‐diagonal peak shapes commonly observed in NMR experiments performed at cryogenic temperatures. Horizontal slices through the peaks in (b) at the indicated frequencies are shown in (c). Peak slopes are defined as the ratio, Df2/Df1, of the change in maximum intensity position in the direct dimension with respect to the position in the indirect dimension. Slopes are indicated as light green guide‐lines through each peak's maximum and are summarized in (h). The autopeak is notably narrow in slices at any position, consistent with the observation of transverse relaxation rates much longer than the inverse peak envelope width (T₂*<<T₂ indicating dominant inhomogeneous linewidth mechanism). Negative peak slope for the Ca—>Cb cross‐peak indicates that the chemical shift distributions of neighboring nuclei are anti‐correlated. Panels (d‐g) GFT‐encoded h(CC)C sum and difference spectra DNP enhanced cryo MAS spectra of U‐¹³C,¹⁵N proline, wherein two ¹³C coevolution periods separated by a mixing time make up the indirect dimension, allowing magnetization to evolve at sum and difference chemical shift distributions across the lineshape. NMR pulse sequence is shown schematically in (g). Expanded crosspeaks (e) and corresponding horizontal slices at indicated frequencies (f) indicate that the Ca shift is strongly anticorrelated to the corresponding Cb shift when comparing various microstates across the distribution. Anticorrelated chemical shifts within the distributions lead to diagonal‐like peak shapes for the Ca‐Cb—> Cb with a somewhat larger peak width for the difference frequency indirect measurement as for the sum frequency. A likely (and testable) explanation for these observations is that proline has conformational microstates when frozen, and within these conformations directly bonded carbons such as Ca and Cb have anticorrelated isotropic chemical shifts. By contrast, if the linewidth were dominated by correlated effects such as local susceptibility of dipolar fields, the difference frequency width would be expected to be notably narrower.