Solid-state NMR

Methodological developments

Microsecond Timescale Protein Dynamics: a Combined Solid‐State NMR Approach

Petra Rovó, Rasmus Linser

Conformational exchange in proteins is a major determinant in protein functionality. In particular, the μs–ms timescale is associated with enzymatic activity and interactions between biological molecules. We show here that a comprehensive data set of R1r relaxation dispersion profiles employing multiple effective fields and tilt angles can be easily obtained in perdeuterated, partly back‐exchanged proteins at fast magic‐angle spinning and further complemented with chemical‐exchange saturation transfer NMR experiments. The approach exploits complementary sources of information and enables the extraction of multiple exchange parameters for μs–ms timescale conformational exchange, most notably including the sign of the chemical shift differences between the ground and excited states.

Proton Transverse Relaxation as a Sensitive Probe for Structure Determination in Solid Proteins

Petra Rovó, Kristof Grohe, Karin Giller, Stefan Becker and Rasmus Linser

Solid‐state nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to elucidate the atomic‐resolution structures of insoluble proteins. The major bottleneck is the difficulty to obtain valuable long‐distance structural information. Here, we propose the use of distance restraints as long as 32 Å, obtained from the quantification of transverse proton relaxation induced by a methanethiosulfonate spin label (MTSL). Combined with dipolar proton–proton distance restraints, this method allows us to obtain protein structures with excellent precision from single spin‐labeled 1 mg protein samples using fast magic angle spinning.

Studied systems

Chicken alpha-spectrin SH3 domain

SH3 domains are ~60 amino acid long sequences prevalent in diverse signaling and cytoskeletal multi-domain proteins of eukaryotes. Among the different polyproline-recognition modules SH3 domains are the most abundant and most studied ones. Their functions are associated with cell growth, differentiation, compartimentalization, apoptosis, splicing, transcription, and enzymatic regulation and thus they have a major influence on diseases such as leukemia, Alzheimer disease, osteoporosis, inflammation, bacterial and viral infections.

Human carbonic anhydrase II

The family of carbonic anhydrases catalyzes the conversion between dissolved CO2 and bicarbonate. They belong to the fastest enzymes known, reaching up to 1 × 106 turnovers per second for the human variant hCAII. Despite good structural understanding, the enzyme’s dynamics is poorly understood. Because of the presence of the His64 side chain in two different conformations, a flip is assumed to be essential for enzymatic activity. It is, however, unclear whether general distortions of the cavity are possible, which would facilitate the accommodation of the very different binding partners CO2 and HCO3-.