Targetable Tetrazine‐Based Dynamic Nuclear Polarization Agents for Biological Systems

Abstract

Dynamic nuclear polarization (DNP) has shown great promise as a tool to enhance the nuclear magnetic resonance signals of proteins in the cellular environment. As sensitivity increases, the ability to select and efficiently polarize a specific macromolecule over the cellular background has become desirable. Herein, we address this need and present a tetrazine‐based DNP agent that can be targeted selectively to proteins containing the unnatural amino acid (UAA) norbornene‐lysine. This UAA can be introduced efficiently into the cellular milieu by genetic means. Our approach is bio‐orthogonal and easily adaptable to any protein of interest. We illustrate the scope of our methodology and investigate the DNP transfer mechanisms in several biological systems. Our results shed light on the complex polarization‐transfer pathways in targeted DNP and ultimately pave the way to selective DNP‐enhanced NMR spectroscopy in both bacterial and mammalian cells.

Emerging Contributions of Solid-State NMR Spectroscopy to Chromatin Structural Biology

“…The major drawback of amber suppression for MAS NMR studies of chromatin is that it results in much lower yields of the desired protein, a situation that can be severely exacerbated under isotopic labeling conditions. Nevertheless, we have successfully used this technique to introduce UAAs in different isotopically labeled proteins, including some that are relevant for chromatin studies ( Lim et al, 2020 ).…”

Targeted DNP for biomolecular solid-state NMR

“… 87,88 In the context of DNP experiments, Debelouchina and coworkers have reported the first successful example of targetable tetrazine-based PAs for biological systems. 75 In their work, the unnatural amino acid norbornene-lysine is introduced in the protein sequence through amber suppression. 85 DNP spin labeling is then obtained through biorthogonal conjugation of a TOTAPOL-tetrazine radical (TTz) as illustrated in Fig.…”

“…More recently, the Debelouchina group demonstrated the first use of bioorthogonal chemistry in the context of t DNP experiments as described above. 75 Their approach was first tested on three purified mutant proteins (ubiquitin, heterochromatin protein HP1α, and the structural maintenance of chromosomes protein SMC) at concentrations of only tens of μM. As a proof of concept, t DNP using the tetrazine-based PA was then applied to isotopically labeled bacterial lysates containing overexpressed ubiquitin mutants without purification.…”

“…10b ). 75 Both approaches show clear promises for future in-cell t DNP applications, if sufficient spectral resolution can be obtained.…”

When Small becomes Too Big: Expanding the Use of In‐Cell Solid‐State NMR Spectroscopy

“…Hence, advancements in radical development could further reduce intracellular protein concentrations than can be studied by DNP‐ssNMR methods. In a similar vein, taggable radicals that would irreversibly conjugate to the proteins of interest harboring an inert unnatural amino acid have recently been developed . In complex and heterogeneous biological environments, it has already been shown that tagging the DNP agents directly to the protein of interest is advantageous in obtaining molecule‐specific DNP enhancements …”