In‐cell NMR spectroscopy is a powerful tool to investigate protein behavior in physiologically relevant environments. Although proven valuable for disordered proteins, we show that in commonly used 1H‐15N HSQC spectra of globular proteins, interactions with cellular components often broaden resonances beyond detection. This contrasts 19F spectra in mammalian cells, in which signals are readily observed. Using several proteins, we demonstrate that surface charges and interaction with cellular binding partners modulate linewidths and resonance frequencies. Importantly, we establish that 19F paramagnetic relaxation enhancements using stable, rigid Ln(III) chelate pendants, attached via non‐reducible thioether bonds, provide an effective means to obtain accurate distances for assessing protein conformations in the cellular milieu.

Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron‐electron resonance (DEER) technique can track proteins’ conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII‐19F Mims electron‐nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in‐cell ENDOR measurements, complemented with room temperature solution and in‐cell GdIII‐19F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid GdIII tags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived GdIII‐19F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIII and 19F regions in the cell.

In-cell NMR spectroscopy is a powerful tool to investigate protein behavior in physiologically relevant environments. Although proven valuable for disordered proteins, we show that in commonly used 1 H-15 N HSQC spectra of globular proteins, interactions with cellular components often broaden resonances beyond detection. This contrasts 19 F spectra in mammalian cells, in which signals are readily observed. Using several proteins, we demonstrate that surface charges and interaction with cellular binding partners modulate linewidths and resonance frequencies. Importantly, we establish that 19 F paramagnetic relaxation enhancements using stable, rigid Ln(III) chelate pendants, attached via non-reducible thioether bonds, provide an effective means to obtain accurate distances for assessing protein conformations in the cellular milieu.Structure and dynamics investigations of biological macromolecules are commonly performed in vitro and, as such, employ a reductionist approach that involves removing a molecule from its native milieu, the cell, thereby ignoring environmental influences that may affect protein folding, [1] local conformation and overall structure, [2] enzymatic activities [3] and protein-protein/ligand interactions. [4] Although this traditional divide-and-conquer strategy has provided indispensable information, recent efforts are focused on developing biophysical and structural methods to directly investigate biomolecules inside living cells. In addition to spectacular advances in cryo-ET for evaluating cellular systems in situ, [5] NMR spectroscopy is now emerging as another method for studying structure, dynamics, interactions and conformations of biomolecules in cells. [2,3,6] NMR, like any spectroscopic method, relies on intrinsic probes as reporters. For biological macromolecules, these probes are 1 H, 13 C, 15 N and 31 P nuclei, and enrichment with 15

In structural studies by NMR, pseudocontact shifts (PCSs) provide both angular and distance information. For proteins, incorporation of a di-histidine (diHis) motif, coordinated to Co2+, has emerged as an important tool to measure PCS. Here, we show that using different Co­(II)-chelating ligands, such as nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA), resolves the isosurface ambiguity of Co2+-diHis and yields orthogonal PCS data sets with different Δχ-tensors for the same diHis-bearing protein. Importantly, such capping ligands effectively eliminate undesired intermolecular interactions, which can be detrimental to PCS studies. Devising and employing ligand-capping strategies afford versatile and powerful means to obtain multiple orthogonal PCS data sets, significantly extending the use of the diHis motif for structural studies by NMR.

Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron‐electron resonance (DEER) technique can track proteins’ conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII‐19F Mims electron‐nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in‐cell ENDOR measurements, complemented with room temperature solution and in‐cell GdIII‐19F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid GdIII tags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived GdIII‐19F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIII and 19F regions in the cell.