Alpha protons as NMR probes in deuterated proteins

Abstract

We describe a new labeling method that allows for full protonation at the backbone Hα position, maintaining protein side chains with a high level of deuteration. We refer to the method as alpha proton exchange by transamination (α-PET) since it relies on transaminase activity demonstrated here using Escherichia coli expression. We show that α-PET labeling is particularly useful in improving structural characterization of solid proteins by introduction of an additional proton reporter, while eliminating many strong dipolar couplings. The approach benefits from the high sensitivity associated with 1.3 mm samples, more abundant information including Hα resonances, and the narrow proton linewidths encountered for highly deuterated proteins. The labeling strategy solves amide proton exchange problems commonly encountered for membrane proteins when using perdeuteration and backexchange protocols, allowing access to alpha and all amide protons including those in exchange-protected regions. The incorporation of Hα protons provides new insights, as the close Hα-Hα and Hα-H^N contacts present in β-sheets become accessible, improving the chance to determine the protein structure as compared with H^N-H^N contacts alone. Protonation of the Hα position higher than 90% is achieved for Ile, Leu, Phe, Tyr, Met, Val, Ala, Gln, Asn, Thr, Ser, Glu, Asp even though LAAO is only active at this degree for Ile, Leu, Phe, Tyr, Trp, Met. Additionally, the glycine methylene carbon is labeled preferentially with a single deuteron, allowing stereospecific assignment of glycine alpha protons. In solution, we show that the high deuteration level dramatically reduces R₂ relaxation rates, which is beneficial for the study of large proteins and protein dynamics. We demonstrate the method using two model systems, as well as a 32 kDa membrane protein, hVDAC1, showing the applicability of the method to study membrane proteins.

Keywords: Isotopic labeling; L-Amino acid oxidase; Membrane proteins; NMR; Structural restraints; Transamination.

Fig. 1

Amino acid metabolic pathways and the different enzymatic stages of the α-PET labeling method. The metabolic pathways of the TCA cycle are shown in a. Inb, the transamination reaction is shown, which is the main route for Hα incorporation. In c, the generation of α-keto acids from amino acids by the enzyme LAAO is shown. d Shows the main biosynthesis pathways of glycine with the observed stereospecific labeling

Fig. 2

Effective incorporation of Hα protons in a ubiquitin sample, while suppressing many side-chain signals. The solution¹³C-HSQC of uniformly labelled ubiquitin (blue) is compared with α-PET ubiquitin (red). Selected slices show the intensity at backbone and sidechain sites. Intensities are not corrected for differences in T₂

Fig. 3

Residue-specific characterization of labeling from¹H–¹⁵N TOCSY-HSQC spectra of 1 mM ubiquitin using 75 ms MLEV-17 mixing. α-PET ubiquitin (red) is compared with¹⁵N,¹³C-ubiquitin (black)

Fig. 4

Cross-polarization based carbon-proton correlation spectra, hCH, of microcrystalline SH3 either uniformly α-PET labeled (red) and ¹³C,¹⁵N-labled (black) crystalized from a protonated buffer. Spectra were recorded at a magnetic field of 800 MHz and 30 °C, 55 kHz MAS. 1D slices from the spectrum indicate the improvement in linewidths for G28 (top left) and A55 (bottom right). The glycine peak intensities show stereospecific labeling with preference for R (α3 protonated) over S (α2) configuration. At the bottom right, the backbone and side-chain protons are indicated on the solution NMR structure (pdb: 1aey) for α-PET SH3 (red ribbon) and¹³C,¹⁵N SH3 (black ribbon)

Fig. 5

Long-range distance information is highlighted in a 3D H(H)CH spectrum of α-PET SH3 (pdb: 1aey) in D₂O (blue) and in H₂O (red). a Shows a contact between L33 Hα and V44 Hα. Inb, the contact between T32 Hα and L8 Hα is readily observed in D₂O (in blue) while it is much weaker in the presence of additional protons in H₂O (in red). Recorded in a 800 MHz Bruker spectrometer at 30 °C and 55 kHz MAS

Fig. 6

Identification of a cross beta strand contact (F99–I114 Hα) in the beta barrel membrane protein VDAC in lipid bilayers. a Shows, the comparison of the (H)CH spectrum at 105 kHz on a 950 MHz spectrometer (black) and at 55 kHz on an 800 MHz spectrometer (red). b Shows a¹³C–¹⁵N projection of a (H)NCAHA spectrum. F99 Hα is assigned from the strip comparing (H)NCAHA (green) and (H)N(CO)CAHA (brown). Inc and e, the contact is shown on the X-ray structure of mouse VDAC (pdb: 2jk4). d Shows the F99–I114 cross-peak in the carbon–carbon 2D plane of the (H)C(HH)CH spectrum at the proton frequency of F99, 4.72 ppm

Fig. 7

Selected residues showing the reduction in proton (Hα) R₂ relaxation rates with α-PET labeling (red) as compared with full protonation (black). The correlation plot (right) shows a reduction for all residues. The data is from ubiquitin samples exchanged in 100% D₂O at 277 K and measured at a 600 MHz spectrometer