Abstract
We describe a new labeling method that allows for full protonation atthe backbone Hα position, maintaining protein side chains with a high level ofdeuteration. 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-PETlabeling is particularly useful in improving structural characterization of solidproteins by introduction of an additional proton reporter, while eliminating manystrong dipolar couplings. The approach benefits from the high sensitivity associatedwith 1.3 mm samples, more abundant information including Hα resonances, and thenarrow proton linewidths encountered for highly deuterated proteins. The labelingstrategy solves amide proton exchange problems commonly encountered for membraneproteins when using perdeuteration and backexchange protocols, allowing access toalpha and all amide protons including those in exchange-protected regions. Theincorporation of Hα protons provides new insights, as the close Hα–Hα andHα–HN contacts present in β-sheets become accessible,improving the chance to determine the protein structure as compared withHN–HN 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 thisdegree for Ile, Leu, Phe, Tyr, Trp, Met. Additionally, the glycine methylene carbonis labeled preferentially with a single deuteron, allowing stereospecific assignmentof glycine alpha protons. In solution, we show that the high deuteration leveldramatically reduces R2 relaxation rates, which is beneficialfor the study of large proteins and protein dynamics. We demonstrate the methodusing two model systems, as well as a 32 kDa membrane protein, hVDAC1, showing theapplicability of the method to study membrane proteins.
Highlights
The study of proteins by nuclear magnetic resonance (NMR) has been continuously evolving to improve sensitivity in order to resolve signals in multidimensional spectra, which serve as the basis for studies of structure and dynamics
Many applications of proton detected magic-angle spinning (MAS) NMR are applied at about 60 kHz with 1.3 mm rotors, a spinning frequency that for fully protonated samples is not enough to average the strong network of 1H–1H dipolar couplings
We found that efficient transamination occurs when E. coli is grown primarily on amino acids
Summary
The study of proteins by nuclear magnetic resonance (NMR) has been continuously evolving to improve sensitivity in order to resolve signals in multidimensional spectra, which serve as the basis for studies of structure and dynamics. Many applications of proton detected MAS NMR are applied at about 60 kHz with 1.3 mm rotors, a spinning frequency that for fully protonated samples is not enough to average the strong network of 1H–1H dipolar couplings. This results in proton line broadening and about 200–300 Hz proton linewidths (Andreas et al 2015). The advantage of this spinning frequency is that narrow lines are observed at high sensitivity when selected sites are labeled to 100% incorporation of protons, while others are deuterated
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