DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu

Abstract

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition. We found that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.

Keywords: DNP solid-state NMR; In-cell NMR; Sup35; Yeast prions.

Figure 1

Lower cryoprotectant percentages result in better DNP performance of dilute proteins in pelleted cellular lysates. A) Sup35NM (red) is diluted in a matrix composed of cryoprotectant (blue), solvent in the buffer (green) and cellular components (yellow), all of which can have different concentrations and isotope content. (B) DNP enhancement collected with a recycling delay of 7 s, ε_on/off (black) and raw signal intensity from Sup35NM (red) using zTEDOR (¹⁵N-¹³C) of the carbonyl peak are both dependent upon the percentage of glycerol in the sample. (C) The build-up time values, T_B,ON, derived from ¹H-saturation recovery experiments fit to a stretched exponential function at the carbonyl are dependent upon the percentage of glycerol in the sample. (D) The β-factor from the ¹H-saturation recovery experiments fit to stretched exponential decrease upon the percentage of glycerol for pelleted lysates (black) and complete lysates (gray). Samples in perdeuterated natural abundance lysates and buffer with 10% H₂O. Dotted lines are to guide the eye. Data from one out of three independent sets of samples are shown.

Figure 2.

DNP performance is maintained in protonated DNP matrix conditions for cellular lysate samples using AMUPol. DNP sample buffer protonation scheme is marked with 85% or 10%. DNP cellular lysate protonation scheme is marked with protonated (100%) or per-deuterated (30% proton content). Samples from the same data sets are indicated by color (pink, purple and blue). Average and standard deviations are shown in black. Black lines indicate results of paired student t-tests. (A) DNP enhancement (ε_on/off) of cellular lysate samples measured by the ratio of on/off microwaves signal of the ¹³C-CP signal at the carbonyl. (B) Build-up time values derived from ¹H-saturation recovery experiments fit to a stretched exponential function at the carbonyl are dependent upon the protonation of the buffer. Samples contain 15% d₈-glycerol. Brackets indicate results of paired two-tailed homoscedastic student’s t-tests (n.s. p > 0.05, * p < 0.05, ** p < 0.01).

Figure 3:

Calculated specificity for the isotopically enriched protein of interest diluted in natural abundance cellular milieu depends upon the concentration of the protein of interest, the selectivity of the pulse sequence, and the degeneracy of the chemical shift of the site. The percent of the signal that is derived from isotopically enriched Sup35NM rather than from the natural molecules in the cellular milieu increases as the concentration of Sup35NM added to the sample increases. When Sup35NM is present at 25 μM (vertical line), the signal from all the carbonyl carbons in Sup35NM and from the cellular milieu is calculated to be of equal magnitude (dotted black line). Selection for sites with an adjacent isotopically labeled carbon (dashed lines) or nitrogen (solid lines) decreases the contribution from the cellular milieu by two orders of magnitude. Selection for a site through two adjacent isotopically labeled sites (thick lines) decreases the contribution from the cellular milieu by four orders of magnitude. All amino acids have at least one carbonyl moiety so specific detection of a single labeled CO site requires high concentrations and/or very selective pulse sequences (blue lines). Single sites in less abundant amino acids and/or in unique chemical moieties can be specifically detected at lower concentrations. About 5% of the amino acids in yeast are Arg (green) while only 1% are Trp (orange). With the use of highly selective pulse sequences these sites can be specifically detected (>98%) at 100 nM or lower concentrations.

Figure 4:

The protein of interest and naturally abundant cellular lysates have predictable magnitudes of contribution to the spectrum. The ¹³C spectra of natural abundance lysate alone (blue) has many signals while the ¹⁵N filtered ¹³C spectra has none (purple). The ¹³C spectra of 25 μM of isotopically enriched Sup35NM diluted in natural abundance lysates (green) has many signals while the ¹⁵N filtered ¹³C spectra reports uniquely on the isotopically enriched Sup35NM (red). Subtraction of the blue spectrum from the green spectrum results in the yellow spectrum (left). All data were collected at 600 MHz with 395 GHz microwave irradiation at 104 K with a recycling delay of 7 s.

Figure 5.

Specific detection of isolated sites in Sup35NM in cellular milieu. (A) Protein sequence of Sup35NM. Lysines (purple), glycines (blue), prolines (pink), arginines (green) and histadine (yellow) are highlighted. (B) Two-dimensional ¹⁵N-¹³C correlation spectra (zTEDOR). Uniformly labeled Sup35NM in protonated, natural abundance strong [PSI⁺] yeast lysate (blue) with 85% protonated buffer and 15% d₈-glycerol with 5 mM AMUPol, n=1280, acquisition time = 23 hours, ε=72. Purified amyloid fibers (red) with 10% protonation, 60% ¹³C depleted d₈-glycerol with 10 mM AMUPol, n=128, acquisition time = 2.5 hours ε=44. Data were collected at 600 MHz with 12.5 kHz MAS at 104 K with a recycle delay of 4 s. Asterisks indicate spinning side bands. (C) Projections of regions of the zTEDOR for amino acids with unique chemical shifts for the lysate sample (colors, projected regions marked in (B) by boxes) overlayed on the purified fibril spectra (blue). The number of each amino acid in Sup35NM is indicated in paratheses. Arrows indicate the average chemical shifts from the BMRB for each site. Signal to noise ratios, integrated peak areas and centers are reported in Table S1.