Biochemie und Chemie
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1H-detected solid-state NMR experiments feasible at fast magic-angle spinning (MAS) frequencies allow accessing 1H chemical shifts of proteins in solids, which enables their interpretation in terms of secondary structure. Here we present 1H and 13C-detected NMR spectra of the RNA polymerase subunit Rpo7 in complex with unlabeled Rpo4 and use the 13C, 15N, and 1H chemical-shift values deduced from them to study the secondary structure of the protein in comparison to a known crystal structure. We applied the automated resonance assignment approach FLYA including 1H-detected solid-state NMR spectra and show its success in comparison to manual spectral assignment. Our results show that reasonably reliable secondary-structure information can be obtained from 1H secondary chemical shifts (SCS) alone by using the sum of 1Hα and 1HN SCS rather than by TALOS. The confidence, especially at the boundaries of the observed secondary structure elements, is found to increase when evaluating 13C chemical shifts, here either by using TALOS or in terms of 13C SCS.
A novel thiazol‐based ratiometric dye for the detection of local pH values is synthesized, and its properties are characterized by a combination of optical spectroscopy, solid‐state NMR and DNP (dynamic nuclear polarization)‐enhanced solid‐state NMR. This novel dye covers a completely different sensitivity range with its acidic pKa value of 3.5 compared to other established dyes for ratiometric pH detection, such as SNARF. The dye is grafted to the surfaces of mesoporous silica materials, which enables, for the first time, direct in situ measurements of the local pH values in silica mesopores by a simple UV‐vis spectroscopy method. The obtained results, which are in good agreement with previous indirect techniques, indicate a background electrolyte‐dependent pKa shift of at least one pH unit under nanoconfined conditions compared to the pKa of the dye in bulk solution.
Light‐induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid‐state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS‐NMR conditions. Uncaging of NPE‐ATP or of lipid substrate NPE‐DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real‐time 31P‐MAS NMR. This proof‐of‐concept illustrates that combining MAS‐NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers.