1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

¹H detection can significantly improve solid‐state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for ¹H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water‐inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high‐resolution ¹H‐detected solid‐state NMR (ssNMR) studies of water‐inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K⁺ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K⁺ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.     

Design Parameters of Tissue‐Engineering Scaffolds at the Atomic Scale

Stem‐cell behavior is regulated by the material properties of the surrounding extracellular matrix, which has important implications for the design of tissue‐engineering scaffolds. However, our understanding of the material properties of stem‐cell scaffolds is limited to nanoscopic‐to‐macroscopic length scales. Herein, a solid‐state NMR approach is presented that provides atomic‐scale information on complex stem‐cell substrates at near physiological conditions and at natural isotope abundance. Using self‐assembled peptidic scaffolds designed for nervous‐tissue regeneration, we show at atomic scale how scaffold‐assembly degree, mechanics, and homogeneity correlate with favorable stem cell behavior. Integration of solid‐state NMR data with molecular dynamics simulations reveals a highly ordered fibrillar structure as the most favorable stem‐cell scaffold. This could improve the design of tissue‐engineering scaffolds and other self‐assembled biomaterials.