ATP-hydrolysis in the ATPase SmsC studied by a solid-state Nuclear Magnetic Resonance spectroscopic toolbox

• Applicant: Professor Dr. Thomas Wiegand
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry ; Physical Chemistry of Solids and Surfaces, Material Characterisation
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 455240421

Protein-nucleotide interactions play a fundamental role in biology and control a variety of processes, such as ATP hydrolysis, DNA replication, transcription, and DNA/RNA repair, all of which rely on very specific molecular interactions for adequate functionality. Noncovalent interactions (NCIs, i.e., hydrogen bonds, dispersion interactions) are at the center of protein-nucleotide molecular-recognition events and are responsible for the high selectivity in such processes. NMR spectroscopy plays an important role in probing such interactions, e.g. effected by molecular dynamics. In this continuation request, we plan to expand and apply our solid-state NMR toolbox to probe protein-nucleotide interactions using the example of the ATPase SmsC that belongs to the Sms-system responsible for the formation of Fe-S clusters. From a methodological perspective, we aim at introducing 19F-detected solid-state NMR at fast magic-angle spinning frequencies (>100 kHz) into our toolbox allowing us to derive distance restraints between 19F-labelled nucleotides and the protein. Such studies will be complemented by proton-detected experiments enabling the positioning of the nucleotide phosphate backbone in the nucleotide-binding domain. The phosphate backbone geometry will be accessed by homonuclear 31P-31P distance measurements complemented with computational modelling. This extended toolbox (“NONCOV 2.0”) will subsequently be applied to a challenging biological system, namely the ATPase SmsC, for which we aim at deriving a detailed understanding of the mechanism of ATP-hydrolysis. Initial studies have shown that SmsC even hydrolyses several ATP-analogues considered as “non-hydrolysable”, making this protein an excellent candidate for expanding our toolbox by real-time solid-state NMR studies, since such mimics slow down the hydrolysis reaction sufficiently. Proton-detected fast MAS experiments combined with 31P MAS NMR will enable us to follow at amino acid resolution conformational and dynamic changes in the protein during the hydrolysis of such ATP-mimics. We will compare the ATP-hydrolysis properties, e.g., the kinetics, of SmsC with the ones of the SmsCB complex allowing us to better understand the influence of SmsB on SmsC in the Sms-system. Besides the biologically relevant insights into the Sms-system, this proposal aims at providing a ubiquitous solid-state NMR toolbox to probe and quantify NCIs in protein-nucleotide complexes.

• DFG Programme: Research Grants