Abstract:
Current structure determination techniques for bio-macromolecules including X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR) and cryo-electron microscopy, focus on obtaining snapshots of the most stable conformer in the free-energy landscape. It is, however, imperative to fathom the conformations of other occupants of energy landscape as well that are transient in existence but are often the functionally active structures. NMR spectroscopy is a versatile technique that, in addition to providing static snapshots, has ability to measure various motional modes of a macromolecule. These motional modes connect distinct local minima in the free-energy landscape and help decipher additional conformational states that are accessible to a macromolecule. This book chapter gives a glimpse of the concept of existence of excited states for nucleic acids on free-energy landscape. Subsequently, this chapter focuses on using R1ρ relaxation dispersion NMR experiments to characterize the transient structures of nucleic acids that are otherwise not possible with standard structure determination techniques. A step-by-step guide and basic principles—including NMR data acquisition, data fitting and statistical analysis, model building and mutational approaches to test the proposed models—will be discussed to first hypothesize and then confirm the existence of such unique transient structures in nucleic acids.