Enhancing Protein NMR for Drug Discovery – The Role of Deuteration
STRUCTURAL BIOLOGY IN DRUG DISCOVERY
In drug discovery, understanding the structure and dynamics of target proteins is essential for rational design and optimisation of therapeutic compounds. Structural biology provides the tools to achieve this, offering atomic-level insights into biomolecular interactions. Currently, X-ray crystallography, cryo-electron microscopy (cryo-EM), and nuclear magnetic resonance (NMR) are the only techniques capable of achieving such resolution. It is important to emphasise that none of these methods is inherently superior; rather, they are complementary, as we have discussed in this blog.
However, sample preparation is critical for all structural biology techniques. In this article, we focus on the importance of protein deuteration in enhancing data quality for NMR studies.
NMRs ROLE IN DRUG DISCOVERY
Among these techniques, NMR offers unique advantages for drug discovery – particularly in studying proteins in solution, fragment-based screening, and characterising dynamic interactions. The quality of NMR data, however, is highly dependent on sample preparation. In this blog, we highlight the importance of protein deuteration in improving NMR data quality, especially for large protein targets.
ISOTOPIC LABELLING
NMR is a powerful and flexible technique that enables the study of biomolecules in solution, including small molecules, peptides, proteins, nucleic acids, carbohydrates, and lipids. Its ability to probe molecular dynamics and binding events in near-physiological conditions makes it especially valuable in early-stage drug discovery. While there is no strict size limit for NMR, larger proteins often present challenges due to signal overlap and reduced signal-to-noise (S/N) ratios.
To overcome these limitations, isotopic labelling with 15N and/or 13C and partial deuteration is commonly used. These isotopes must be incorporated through de novo synthesis, either in prokaryotic or eukaryotic expression systems. Once labelled, proteins can be analysed using 2D-NMR, where a single well-resolved signal is observed per every 1H/15N and/or 1H/13C correlation.
HOW DEUTERATION IMPROVES NMR DATA
For proteins larger than ~25 kDa—common among drug targets—NMR spectra often suffer from broadened signals and poor S/N due to increased relaxation rates caused by ¹H–¹H dipolar coupling. One effective strategy to address this is deuteration: replacing non-exchangeable protons with deuterons (2H) in CH, CH2, and CH3 groups.
This is achieved by expressing the protein in partially or fully deuterated media — a service we offer as part of our protein expression platform. Deuteration reduces dipolar interactions, increases relaxation times, and significantly enhances the S/N ratio of 1H/15N resonances. As a result, researchers can obtain more complete and higher-quality datasets—not only for hydrogen and nitrogen atoms, but also for 1H/13C resonances when 13C labelling is included.
CASE STUDY: IMPROVING NMR WITH 80% DEUTERATION
As an example, we present a case study from one of our internal development projects. We successfully replaced approximately 80% of the aliphatic protons with deuterons, as shown in the 1H NMR spectrum (Figure. 1, region -0.5 to 4.5 ppm).
Figure 1. 1H 1D-NMR spectra of our case study protein in fully protonated (red) and ~80% deuterated (black) forms. Deuteration significantly reduces the number of non-exchangeable protons, as seen by comparing the black and red spectra in the -0.5 to 4.5 ppm region.
This 80% deuteration had a substantial impact on the quality of the 2D 1H/15N NMR spectra. As shown in Figure 2, several HN resonances exhibited marked improvements in S/N ratio, enabling more confident peak assignment and structural interpretation.
Figure 2. 2D 1H/15N NMR spectra of the case study protein after 80% deuteration (black). Resonances B–F show varying degrees of S/N improvement, demonstrating the benefit of deuteration for high-quality NMR data.
SUMMARY
Deuteration is a powerful strategy to enhance NMR data quality, particularly for larger proteins that are often key targets in drug discovery. By improving signal resolution and completeness of datasets, deuteration enables more accurate structural insights, better fragment screening, and more informed decision-making in early-stage drug development.
GET IN TOUCH
If you’re working on challenging protein targets and would like to explore how NMR might benefit your project, our team is here to help. We offer expert support in protein expression, isotopic labelling, deuteration, and structural analysis. Contact us at info@peakproteins.com
