A Two‐Armed Probe for In‐Cell DEER Measurements on Proteins**

Abstract

The application of double electron-electron resonance (DEER) with site-directedspin labeling (SDSL)t om easure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed Gd III complexes,G d III-CLaNP13a/b/cw ere synthesized. Rather than the disulfide linkageo fm ost other CLaNP molecules, at hioether linkagew as used to avoid re-ductive dissociation of the linker.T he doubly Gd III labeled N55C/V57C/K147C/T151C variants of T4Lysozymew ere measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent withr esults from paramagnetic NMR. An arrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites.

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…The most appealing application of in-cell EPR is its capacity to characterize in-cell distance distribution between two spin-labels in the range of 2.5–6 nm using DEER/PELDOR or RIDME techniques. ,− They report for dipolar interactions, i.e., the sum of all the interlabel distances to the power of −3 (∑ d ij –3 ). The corresponding measurements are usually executed (i) during ∼12 h, (ii) at cryogenic temperatures (from 10 to 60 K, a recent trityl spin-label permitted DEER acquisition at 150 K), (iii) using Q-band (34 GHz) or even the less common W-band (94 GHz) spectrometers, (iv) on cellular samples of ∼50 μL (10–20 million mammalian cells are enough) containing down to submicromolar concentrations of doubly spin-labeled proteins/nucleic acids, preferably in deuterated buffers. − ,,− Hence, the measured distance distribution reports the ensemble of frozen conformations, independently of the size and location of the studied objects. The distance extraction resolves an ill-posed problem from noisy data, which can bias the distribution shape. ,,− The global accuracy depends also on the size and rigidity of the two paramagnetic tags. ,− …”

“…In-cell EPR techniques rely on the covalent attachment of spin labels, which have to be stable in the reducing cellular environment. Resistant nitroxide labels have been designed both for proteins and nucleic acids over the last 10 years. ,,,− In-cell distance measurements can also be performed between two trityl- ,,,, or Gd 3+ -cages. ,,,,,,,− The attachment of spin-labels has been mostly performed on purified proteins in vitro , which were later delivered in cells . These paramagnetic tags have also been directly bound on exposed loops of overexpressed membrane proteins in live cells. ,,− In-cell EPR measurements were also carried out using genetically encodable lanthanide-binding tags or noncanonical amino acids via amber codon recoding. ,,, Spin-labeled nanobodies might be useful in the future, too .…”

Paramagnetic Chemical Probes for Studying Biological Macromolecules

“…Recently, the double-anchored probe CLaNP-13 (Figure , 85 ) was designed for DEER studies in living cells . By virtue of two maleimide groups, CLaNP-13 forms thioether bonds with cysteine residues, which in a cellular environment are more stable than disulfide bonds.…”

“…Another standard reactive functionality is the maleimide group, which has a high reactivity and selectivity for cysteine residues around neutral pH. , Maleimide probes react with cysteines via Michael addition, forming a new stereocenter ,, and thus giving rise to different stereoisomers which complicate PCS assignments. The use of a maleimide functionality to attach a probe for NMR experiments is therefore not recommended.…”

“…Except for a recent study on domain motions in diubiquitin, the use of other double-arm probes, such as T1/T2 (Figure , 82 ) and CLaNP-13 (Figure , 85 ) has only been explored for DEER distance measurements between two Gd­(III) ions, with the aim to obtain narrow distance distributions by restricting the flexibility of the tags. In either case, the widths of the distance distributions obtained was not narrower than those obtained with some of the singly linked probes.…”