Thesis presented September 07, 2023

Abstract:
aDynamic nuclear polarization (DNP) has emerged as a promising hyperpolarization technique to significantly enhance the sensitivity of solid-state nuclear magnetic resonance (NMR) experiments under magic angle spinning (MAS). However, its use for biomolecular systems is often limited by the cryogenic temperature required for DNP. Freezing of the dynamics present in such systems leads to conformational heterogeneity and results in line broadening and loss of resolution. To tackle this limitation, our group has introduced a new methodology called selective DNP (SelDNP), which allows the recovery of highly resolved DNP spectra specific to protein binding sites. SelDNP relies on the uniform polarization obtained with conventional DNP on a first sample and the additional localized bleaching produced by introducing a paramagnetic tag on the protein in a second sample. The difference between the two spectra results in highly resolved sub-spectra containing only resonances from the protein site targeted by the tag. In addition to providing a complete assignment of the targeted region, SelDNP spectra convey distance-dependent information from the paramagnetic tag due to the selectivity factor we use to weight the difference spectroscopy. The SelDNP proof-of-concept was originally demonstrated on the β-D-galactose binding site of the lectin LecA. Using ß-D-galactose functionalized with the DNP polarizing agent allowed to simultaneously achieve both effects of selective bleaching of the binding site and uniform hyperpolarization of the rest of the protein. Although successful, this strategy is limited by the specific synthesis of the functionalized ligand and the impossibility of independently optimizing site selectivity and sensitivity.
In my PhD, we addressed these limitations by proposing various alternatives capable of focusing on almost any region of the protein, using exclusively generic tags. First, we considered the use of iodoacetamide tags, which can react on a cysteine residue in a site-directed spin labeling (SDSL) approach, allowing the study of any exposed region of the protein. As for iodoacetamide tags, we considered the bis-nitroxide DNP polarizing agent AsymPol to provide both the site selectivity and the DNP sensitivity, or TEMPO, which produces only site specificity through targeted bleaching. In the latter case, the addition of the DNP polarizing agent can be optimized independently. Both tags provided highly resolved SelDNP spectra by focusing on the region around the cysteine.
Relying on the fact that hyperpolarization and bleaching can be handled separately, we extended the SelDNP methodology to metalloproteins, where diamagnetic metal ions can be replaced by equivalent paramagnetic ions for specific bleaching of the metal binding site. As with the SDSL approach using TEMPO, the use of the paramagnetic ion allowed us to separately optimize the DNP sensitivity. Since the newly developed SelDNP strategies were only demonstrated on the model system LecA, we aimed to exploit the most suitable strategy to decipher a more complicated lectin-carbohydrate interaction, and selected PilA. In addition to the biological relevance of PilA, its interaction with glycosphingolipid carbohydrates (GSL) has not yet been fully described at the atomic level by any of the conventional structural biology techniques.
In order to address this question by SelDNP, the strategy consisted in establishing the protocol for the production of the labelled protein, obtaining evidence for the interaction with GSLs by one of the contemporary techniques, and then functionalizing the binding carbohydrate with TEMPO to perform SelDNP. Despite the obstacles encountered in producing the protein, we obtained a sufficient amount to test the putative interaction using a wide range of techniques. Unfortunately, this affinity could not be demonstrated, which prevented us from performing SelDNP on this system.

Keywords:
Dynamic nuclear polarization (DNP), Binding sites, Proteins, Carbohydrates, Lectins, NMR