Elucidating Time Course of Structural Changes Leading to Receptor-Ligand Complex Formation with Transient-EPR

Abstract

Protein functions rely on protein motions. Protein structures, obtained with X-ray diffraction or NMR, reveal mostly static pictures of structure-function relation. To connect structural changes of proteins with function, we need to look at events happening in real time, i.e. the dynamics of the processes.We have chosen mlCNBD as a model protein to study the dynamics of receptor-ligand complex formation. mlCNBD is a cytosolic cyclic nucleotide-binding domain of a bacterial potassium channel (MloK1). mlCNBD binds to cyclic adenosine monophosphate (cAMP) and undergoes conformational changes as evident from the NMR and X-ray structures of the apo and holo conformational states of the protein. Recent kinetic and NMR studies indicate that these structural transitions follow the induced-fit mechanism, i.e. these are a direct consequence of ligand binding. However, the detailed mechanism of these structural rearrangements leading to receptor activation remains elusive.We use transient Electron Paramagnetic Resonance (tr-EPR) spectroscopy in conjunction with Site- Directed Spin Labelling (SDSL) to resolve the dynamics of mlCNBD-cAMP complex formation. We introduce single cysteine residues at different sites in the protein. The mutants are labelled with Methane Thio Sulphonate Spin Label (MTSSL). Binding of cAMP to the mutants is rapidly initiated either via a caged-cAMP approach or through a micro-mixer. The time-resolved EPR data reveals the progression of structural changes taking place at a particular site on millisecond time scale. Collating data across the whole protein will enable us to reconstruct the steps from the apo to the holo state of the protein. It will also provide the answer to the question whether the induced-fit mechanism follows a concerted (single step) or sequential (multi-step) path from the apo to the holo conformation.