Highly Efficient Polarizing Agents for MAS-DNP of Proton-Dense Molecular Solids.

Efficiently hyperpolarizing proton-dense molecular solids through dynamic nuclear polarization (DNP) solid-state NMR is still an unmet challenge. Polarizing agents (PAs) developed so far do not perform well on proton-rich systems, such as organic microcrystals and biomolecular assemblies. Herein we introduce a new PA, cAsymPol-POK, and report outstanding hyperpolarization efficiency on 12.76 kDa U-13 C,15 N-labeled LecA protein and pharmaceutical drugs at high magnetic fields (up to 18.8 T) and fast magic angle spinning (MAS) frequencies (up to 40 kHz). The performance of cAsymPol-POK is rationalized by MAS-DNP simulations combined with electron paramagnetic resonance (EPR), density functional theory (DFT) and molecular dynamics (MD). This work shows that this new biradical is compatible with challenging biomolecular applications and unlocks the rapid acquisition of 13 C-13 C and 15 N-13 C correlations of pharmaceutical drugs at natural isotopic abundance, which are key experiments for structure determination.

Targeted DNP for biomolecular solid-state NMR.

High-field dynamic nuclear polarization is revolutionizing the scope of solid-state NMR with new applications in surface chemistry, materials science and structural biology. In this perspective article, we focus on a specific DNP approach, called targeted DNP, in which the paramagnets introduced to polarize are not uniformly distributed in the sample but site-specifically located on the biomolecular system. After reviewing the various targeting strategies reported to date, including a bio-orthogonal chemistry-based approach, we discuss the potential of targeted DNP to improve the overall NMR sensitivity while avoiding the use of glass-forming DNP matrix. This is especially relevant to the study of diluted biomolecular systems such as, for instance, membrane proteins within their lipidic environment. We also discuss routes towards extracting structural information from paramagnetic relaxation enhancement (PRE) induced by targeted DNP at cryogenic temperature, and the possibility to recover site-specific information in the vicinity of the paramagnetic moieties using high-resolution selective DNP spectra. Finally, we review the potential of targeted DNP for in-cell NMR studies and how it can be used to extract a given protein NMR signal from a complex cellular background.

Structural basis of client specificity in mitochondrial membrane-protein chaperones.

Chaperones are essential for assisting protein folding and for transferring poorly soluble proteins to their functional locations within cells. Hydrophobic interactions drive promiscuous chaperone-client binding, but our understanding of how additional interactions enable client specificity is sparse. Here, we decipher what determines binding of two chaperones (TIM8·13 and TIM9·10) to different integral membrane proteins, the all-transmembrane mitochondrial carrier Ggc1 and Tim23, which has an additional disordered hydrophilic domain. Combining NMR, SAXS, and molecular dynamics simulations, we determine the structures of Tim23/TIM8·13 and Tim23/TIM9·10 complexes. TIM8·13 uses transient salt bridges to interact with the hydrophilic part of its client, but its interactions to the transmembrane part are weaker than in TIM9·10. Consequently, TIM9·10 outcompetes TIM8·13 in binding hydrophobic clients, while TIM8·13 is tuned to few clients with both hydrophilic and hydrophobic parts. Our study exemplifies how chaperones fine-tune the balance of promiscuity versus specificity.