Despite their importance in protein functions, the conformational states of proteins and their changes are often poorly understood, mainly because of the lack of an efficient strategy. A higher magnitude of conformational dynamics and variations are expected, especially for multi-domain proteins consisting of multiple structural domains. Because multi-domain proteins often undergo drastic conformational changes during their functional processes, information about their dynamic properties is essential to understand the mechanisms. Although high-resolution structural information can be obtained by X-ray crystallography, cryo-electron microscopy, nuclear magnetic resonance, and structure prediction tools, they are often insufficient for grasping the actual states of proteins in solution. In our study, we exploited a paramagnetic lanthanide ion, Gd3+, in electron paramagnetic resonance (EPR) experiments and measured the inter-gadolinium distance distribution that reflects the conformational states of the protein.
The cover image for the 120/15 issue of Biophysical Journal conceptually shows the investigation of the conformational variation of a multidomain protein, MurD, based on the inter-gadolinium distance measurements. The three domains of MurD are colored green, purple, and pink, respectively. The gadolinium ions attached to the two domains of MurD are represented as red spheres. The trajectory of the inter-gadolinium distance as predicted by molecular dynamics (MD) simulation is shown at the bottom, depicting drastic conformational changes of MurD.
Our study demonstrated that gadolinium tagging of specific positions on the protein enables EPR distance measurements for probing the conformational ensemble of the protein. The distance measurements are coupled with the MD simulation to visualize the structural variations of the protein. The data show that the protein enzyme MurD exists in a wide variety of the conformational states that were eliminated by ligand binding. We also investigated the conformational dynamics of the same protein by X-ray scattering, neutron scattering, and MD simulation. Different biophysical, experimental, and computational methods shed light on various aspects of MurD, unveiling the highly dynamic structures of MurD in solution.
- Tomohide Saio, Soya Hiramatsu, Mizue Asada, Hiroshi Nakagawa, Kazumi Shimizu, Hiroyuki Kumeta, Toshikazu Nakamura, Koichiro Ishimori