In nature, most biomolecules perform their function in a cellular environment. It is therefore important to study their structure–dynamics–function relationships in cells and establish methods that can achieve this. Here, the suitability of pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy (PDS) for such studies is tested in combination with different label and cell types. The model protein GB1 was labeled with either a trityl- or gadolinium-based spin label and transfected via electroporation into two types of mammalian cells, i.e., HeLa and HEK293, and one type of bacterial cells, i.e., E. coli. The in-cell Q-band PDS measurements revealed that both labels remain intact in all three cell types, whereas unspecific binding to the tested cell types was observed for the trityl-labeled protein when used in high concentrations, no such behavior was detectable for the gadolinium label due to its significantly lower sensitivity at Q-band. Interestingly, the modulation depth decreased for both labels with increasing cell recovery time, potentially indicating intracellular protein degradation or aggregation processes. In contrast, E. coli enabled substantially shorter recovery times and yielded PDS data with preserved modulation depth and distance information. Thus, in-cell PDS studies on spin-labeled proteins are particularly useful when combined with E. coli cells.
Hierarchical mesoporous ZSM-5 zeolites were prepared via alkaline desilication and pillaring, and characterized by a combination of XRD, N2 adsorption, 29Si MAS NMR, SEM, and low-field 1D and 2D (T 1-T 2 correlation) 1H NMR relaxation of confined water. Alkaline treatment resulted in a progressive desilication, increased mesopore volume, and the formation of surface defects, whereas pillaring produced more ordered mesoporosity. The T 1-T 2 correlation maps revealed distinct relaxation regimes reflecting the strength of the water-surface interactions. The samples with different mesopore organization exhibit very different surface relaxivities and T 1/T 2: as compared with the parent microporous zeolite, the samples with disordered mesoporosity introduced via the alkaline treatment show higher surface relaxivities but lower T 1/T 2, whereas the pillared sample, oppositely, exhibits surface relaxivities close to that of the parent sample and essentially higher T 1/T 2 ratio. The decoupling of T 1 and T 2 points out on a complex water dynamics in the pillared zeolite with ordered micro- and mesoporosity.
MucR is a conserved global regulator in alpha-proteobacteria, essential for virulence in Brucella and symbiotic adaptation in rhizobia. It primarily acts as a xenogeneic silencer, repressing AT-rich horizontally acquired genes. Structurally, MucR comprises an N-terminal oligomerization domain, a linker region, and a C-terminal zinc finger DNA-binding domain. Here, we report the optimization of NMR experimental condition and NMR resonance assignments for the complex formed by the C-terminal DNA-binding domain of MucR and 3A3T-DNA. Nearly complete assignments are obtained for 1H, 13C, and 15N resonances of the protein and 1H resonances of 3A3T-DNA. Histidine tautomeric state analysis confirms that the two zinc-coordinating histidine residues adopt N delta 1-H tautomeric state. Additional NMR-based analyses provided structural insights into the MucRCTD and DNA complex formation. The NMR assignments provide a good basis for further structural study of the DNA binding and recognition mechanism of MucR.
It has been shown that a high rate of spin exchange in bimolecular collisions of radicals in dilute solutions and alkali metal atoms in gases is a necessary, but still insufficient, condition for the observation of the effect of exchange narrowing of the initially inhomogeneously broadened EPR spectrum. For the manifestation of exchange narrowing, when fast spin exchange practically ceases to contribute to the exchange broadening of the observed homogeneously broadened EPR line, spin exchange must be equivalent. This means that when calculating the effective collision radii for spin dephasing (spin decoherence) and for the effect of quantum coherence transfer from the interaction partner (coherence transfer), conditions must be met that allow only the large Heisenberg exchange interaction to be considered at the moment of collision, while all other interactions such as spin–orbit, dipole–dipole spin–spin and hyperfine interactions can be neglected.
