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–HN contacts present in β-sheets become accessible, improving the chance to determine the protein structure as compared with HN–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 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.

Proton Detected Solid-State NMR of Membrane Proteins at 28 Tesla (1.2 GHz) and 100 kHz Magic-Angle Spinning

“…The high cost of SAIL can be offset by the use of smaller rotors [ 40 ]. For alpha proton labeling, alpha proton exchange via transamination (αPET) leads to improved linewidths at 55–100 kHz, albeit with scrambling of the label for certain residue types [ 41 ].…”

Heteronuclear and Homonuclear Finite Pulse Radio Frequency Driven Recoupling

“…The evolutions of the operators, however, are completely different for heteronuclear and homonuclear cases between these points. For the experimental demonstrations we perform heteronuclear 1D 1 H-{ 13 C} and 1 H-{ 15 N} fp-RFDR experiments using -PET (Movellan et al 2019) labeled SH3 at 100 kHz MAS.…”

“…Sample preparation: 100% back bone protonated Transamination (-PET SH3) was prepared by Movellan with the protocol described in Ref. [(Movellan et al 2019)].…”

Heteronuclear and Homonuclear Radio Frequency Driven Recoupling

“…Sample preparation: Microcrystalline chicken alpha spectrin SH3 protein was used for acquisition of all experimental data. The samples were labeled with 100% protonation at exchangeable sites and either with alpha proton exchange by transamination (-PET) or with uniform 13 C and 15 N labeling with the protocol described in (Movellan et al, 2019). Simulations: HET-RFDR and RFDR simulations were performed with in-house MATLAB scripts using numerical solution of the equation of motion (Nimerovsky and Goldbourt, 2012).…”

“…In this article we investigate spin dynamics under HET-RFDR, in which RFDR π-pulses are applied simultaneously on two channels (Figure 1). We demonstrate simultaneous heteronuclear and homonuclear transfers using HET-RFDR applied to -PET (Movellan et al, 2019) labeled SH3 at 10 kHz and 55.555 kHz MAS.…”