https://doi.org/10.3389/fmolb.2022.1074743
“Intrinsically disordered proteins are frequent targets for functional regulation through post-translational modification due to their high accessibility to modifying enzymes and the strong influence of changes in primary structure on their chemical properties. While lysine Nε-acetylation was first observed as a common modification of histone tails, proteomic data suggest that lysine acetylation is ubiquitous among both nuclear and cytosolic proteins. However, compared with our biophysical understanding of the other common post-translational modifications, mechanistic studies to document how lysine Nε-acetyl marks are placed, utilized to transduce signals, and eliminated when signals need to be turned off, have not kept pace with proteomic discoveries. Herein we report a nuclear magnetic resonance method to monitor Nε-lysine acetylation through enzymatic installation of a13C-acetyl probe on a protein substrate, followed by detection through 13C direct-detect spectroscopy. We demonstrate the ease and utility of this method using histone H3 tail acetylation as a model. The clearest advantage to this method is that it requires no exogenous tags that would otherwise add steric bulk, change the chemical properties of the modified lysine, or generally interfere with downstream biochemical processes. The non-perturbing nature of this tagging method is beneficial for application in any system where changes to local structure and chemical properties beyond those imparted by lysine modification are unacceptable, including intrinsically disordered proteins, bromodomain containing protein complexes, and lysine deacetylase enzyme assays.”
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Nuclear magnetic resonance (NMR) is an advantageous method for studying PTMs of IDPs as it is label-free and non-destructive (Liokatis et al., 2010; Theillet et al., 2012). Conventional heteronuclear 2D NMR, such as the [1H, 15N]—HSQC, yields two observables that have been used to monitor acetylation events using 15N-labeled substrate. Firstly, whereas rapid exchange of the proton with solvent eliminates the lysine sidechain 1Hε–15Nε resonances from most spectra, this NMR resonance becomes observable upon acetylation, due to the accompanying reduced rate of solvent exchange. Secondly, small changes in backbone chemical shift are often observable upon sidechain acetylation (Liokatis et al., 2010), yielding an indirect readout. These methods have a downside of requiring at the minimum 15N enriched peptide substrate. To fully benefit from site resolution, these experiments require a full determination of backbone chemical shift assignments. Thus, available techniques are characterized by relatively high cost in materials and time and require expertise that is often beyond many laboratories that might be interested in pursuing these studies.
The [1H,15N]-HSQC experiments described above have traditionally been performed without isotopic enrichment of the acetyl group transferred to the substrate protein, yet there are major advantages to be gained by instead transferring a13C-acetyl group for direct observation. Previously, we have developed a strategy analogous to this using a13C-SAM methyl donor to provide an NMR visible signal to monitor lysine methylation events (Usher et al., 2021). Here we demonstrate enzymatic transfer of uniformly 13C-acetyl groups, either with or without concomitant isotopic enrichment of the substrate protein. As the acetyllysine moiety bears chemical similarity to the peptide backbone, we hypothesized that modifying 13C direct-detect NMR methods typically used for observation of IDP backbone residues would provide an unambiguous and easily interpreted signal corresponding to an acetylation event. Thus, we report here modifications to the 13C direct-detect CACO and CON experiments that yield optimized detection of the acetamide functional group created upon lysine Nε acetylation. This technique has the capability to dramatically advance knowledge of structure and function impacts from protein acetylation.
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2.5 NMR methods
2.5.1 Validation of semi-synthetic and enzymatic Acetyl-CoA production
1H-1D with water suppression using excitation sculpting with gradients for 12C labeled acetyl CoA (Hwang and Shaka, 1995), or 1H-1D with garp decoupling applied on the 13C channel for 13C labeled acetyl CoA were collected on a Bruker Avance AVIII 500 with TCI Cryoprobe using 65 k data points, 16 scans, a sweep width of 16 ppm, and a spectral Center of 4.69 ppm (Supplementary Figure S1).
2.5.2 General methods for the collection of acetyllysine spectra
With the exceptions noted above, all NMR experiments were conducted on a Bruker Avance NEO 600 MHz spectrometer equipped with a 5 mm TCI triple-resonance cryoprobe and a SampleJet autosampler [1H, 13C]—HSQC experiments were collected using the standard pulse program from the Bruker Topspin library, with 1,024 direct data points and 128 indirect data points, 16 scans, and a sweep width of 2.98 ppm with a Center of 2.95 ppm in the direct dimension and 70 ppm with a Center of 40 ppm in the indirect dimension. and a recycle delay of 1 s. Hard 90° pules on 1H and 13C, as well as appropriate decoupling pulses, should be calibrated as per standard protocols on user instruments. For the spectra represented here, the typical 90° 1H pulse was 8 μs and the typical 13C 90° pulse was 15 μs. All pulsed field gradients were applied for 1 m with a sine shape.
All 13C direct-detect spectra were designed around strategies similar to those found in the previously reported [13C,13C]-CACO and [13C,15N]-CON experiments (Bermel et al., 2005). In all cases, virtual decoupling in the 13C direct dimension was achieved through utilization of in-phase anti-phase (IPAP) spectra processed to yield virtual decoupling using the standard ‘splitcomb’ AU program distributed with the Bruker Topspin library (Duma et al., 2003). Protocols for implementation of the standard Bruker library versions of the [13C,13C]-CACO-IPAP and [13C,15N]-CON-IPAP are thoroughly described elsewhere (Bastidas et al., 2015). All required 1H and 15N pulses are standard hard pulses or composite pulse decoupling sequences. For the spectra represented here, the typical 90° 1H pulse was 8 μs and the typical 15N 90° pulse was 25 μs. All pulsed field gradients were applied for 1 m with a sine shape. All pulses applied on the 13C channel are shaped, frequency selective pulses. Here, where all spectra were collected at 14.0 T, 90° 13C band-selective pulses used the Q5_sebop shape (or its time-reversed equivalent as noted in timing diagrams) and the 180° band-selective pulses used the Q3_surbop shape with durations of 350 µμs and 270 µs, respectively. Adiabatic inversion during the nitrogen chemical shift labeling period was achieved through application of a 500-m CHIRP pulse with 60 Hz sweep and 25% smoothing (Bohlen and Bodenhausen, 1993). Note that for spectrometers operating at alternative magnetic field strengths, pulse timings will vary, and on older spectrometers the traditional Q5 and Q3 pulses may be required as substitution for those listed above (Emsley and Bodenhausen, 1992). The user is referred to their manufacturer documentation, or to previous protocols as appropriate (Cook et al., 2018).
We report three variants of the acetyllysine, 13Caliphatic–13Ccarbonyl selective, experiment. Each of the reported [13Cʹ,13Cali]-CaliCO-Kac variants present identical spectral information but utilize three different excitation schemes that the user may choose between depending on their specific application and needs. Inspiration for the reported pulse programs comes from the protonless CACO-IPAP (Bermel et al., 2005) and the 3D HNCOCa and 3D HCOCa (Serber et al., 2000), respectively. The Supplementary Material S1 includes timing diagrams corresponding to the protonless [13Cʹ,13Cali]-CaliCO-Kac (Supplementary Figure S2), amide-start [13Cʹ,13Cali]-CaliCO-Kac (Supplementary Figure S3), and methyl proton-start [13Cʹ,13Cali]-CaliCO-Kac (Supplementary Figure S4). All reported [13Cʹ,13Cali]-CaliCO-Kac experiments were recorded with 1,024 direct data points and 128 indirect data points, 64 scans, and a sweep width of 20 ppm with a Center of 172 ppm in the direct dimension, and a sweep width of 60 ppm with a Center of 25 ppm in the indirect dimension.
We report four variants of the acetyllysine, 13Ccarbonyl–15N selective, experiment. Each of the reported [13C,15N]-CON-Kac variants utilize different excitation schemes that the user may choose between depending on their specific application and needs. Inspiration for the reported pulse programs comes from the protonless CON-IPAP (Bermel et al., 2005), HN-flip CON-IPAP (Bermel et al., 2009), (HACA)-CON-IPAP (Bertini et al., 2011), and (HACA)-CON-ALA (Schubert et al., 1999; Sahu et al., 2014). The Supplementary Material S1 includes timing diagrams corresponding to the protonless [13C,15N]-CON-Kac (Supplementary Figure S5), amide-start [13C,15N]-CON-Kac (Supplementary Figure S6), methyl proton-start [13C,15N]-CON-Kac (Supplementary Figure S7), and triple quantum filtered methyl-selective [13C,15N]-CON-Kac (Supplementary Figure S8). All reported [13C,15N]-CON-Kac experiments were recorded with 1,024 direct data points and 256 indirect data points, 32 scans, and a sweep width of 20 ppm with a Center of 172 ppm in the direct dimension, and a sweep width of 42 ppm with a Center of 127 ppm in the indirect dimension. For 13Cmethyl-selective pulses, the offset from the carrier position should be set to Center the pulses on 25 ppm. The 1JCCO in the acetamide functional group was measured to be 41.6 Hz, resulting in a refocusing delay of 4.9 m for this coupling. For the triple quantum selective experiment, which suppresses resonances from the backbone in uniformly 13C,15N-enriched proteins, the delay for 13Cali–13Cʹ was further shortened to reduce the intensity of artifacts resulting from long-range coupling into the backbone, resulting in a delay of 5.0 m, as annotated in the Supplementary Figure S8 legend.
2.5.3 H3 binding to Gcn5 BRD
NMR experiments to monitor Gcn5 bromodomain binding to H3 were conducted on a 11.7 T Bruker Avance-3 spectrometer equipped with a TCI-cryoprobe. 1H-methyl-start [13C,13C] CaliCO-Kac experiments were collected with 1,024 direct data points and 128 indirect data points, 16 scans, and a sweep width of 20 ppm with a Center of 172 ppm in the direct dimension and 80 ppm with a Center of 172 ppm in the indirect dimension, and a recycle delay of 1 s.
2.5.4 Processing and analysis
On-instrument processing of all acetyllysine spectra was performed using Bruker Topspin V 4.0.5. Additionally, 3D spectra were acquired to assign the backbone of H3 using our standard suite of 13C direct-detect experiments (Sahu et al., 2014), with on-instrument processing using Bruker Topspin V 3.2.6. Viewing and processing for image generation of all spectra were carried out in NMRFAM-SPARKY (Lee et al., 2015).
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