Internal and Global Protein Motion Assessed with a Fusion Construct and In‐Cell NMR Spectroscopy

In-cell NMR spectroscopy with the H,N heteronuclear single quantum coherence (HSQC) yields high-quality spectra when applied to intrinsically disordered proteins in Escherichia coli. For globular proteins, however, protein signals from inside the cell are not observed. 3] Here, we show in a simple and direct way that protein dynamics determines the quality of the in-cell H,N HSQC spectrum by fusing the globular protein ubiquitin to the disordered protein a-synuclein. Most knowledge about protein structure and dynamics has been gleaned from experiments in dilute solution; however, the native intracellular environment of proteins where macromolecular concentrations can exceed 300 g L 1[4] presents a different set of conditions. Furthermore, it is known that crowding can have an impact on protein stability and dynamics. 6] The H,N HSQC experiment is commonly used to characterize proteins in dilute solution. However, the high-resolution spectra obtained from overexpressed, N-enriched proteins in cells are of mixed quality. Disordered proteins, such as a-synuclein and FlgM, give high-quality spectra inside cells. Globular proteins, on the other hand, fail to produce useful in-cell spectra. 3] Here, we present in a single experiment, compelling evidence that this difference in detectability is caused by their different rotational dynamics. We produced a histidine-tagged fusion protein from the globular protein ubiquitin and the disordered protein a-synuclein (Figure 1). Ligation-independent cloning resulted in a structural gene comprising a N-terminal six-histidine (H6) seg-