A solid-state NMR tool box for the investigation of ATP-fueled protein engines

Abstract

Motor proteins are involved in a variety of cellular processes. Their main purpose is to convert the chemical energy released during adenosine triphosphate (ATP) hydrolysis into mechanical work. In this work, solid-state Nuclear Magnetic Resonance (NMR) approaches are discussed allowing to study structures, conformational events and dynamic features of motor proteins during a variety of enzymatic reactions. Solid-state NMR benefits from the straightforward sample preparation based on sedimentation of the proteins directly into the Magic-Angle Spinning (MAS) rotor. Protein resonance assignment is the crucial and often limiting step to interpret the wealth of information encoded in the NMR spectra. Herein, potentials, challenges and limitations in resonance assignment for large motor proteins are presented, focussing on both, biochemical and spectroscopic approaches. This work highlights NMR tools to study the action of the motor domain, and its coupling to functional processes as well as to identify protein-nucleotide interactions during events such as DNA replication. Arrested protein states of reaction coordinates such as ATP hydrolysis can be trapped for NMR studies by using stable, non-hydrolysable ATP analogues which mimic the physiological relevant states as accurate as possible. Recent advances in solid-state NMR techniques ranging from Dynamic Nuclear Polarization (DNP), 31P-based heteronuclear correlation experiments, 1H-detected spectra at fast MAS frequencies > 100 kHz to paramagnetic NMR are summarized and its applications to the bacterial DnaB helicase from Helicobacter pylori are discussed.