Abstract
Background: Membrane proteins are crucial for cellular sensory cascades and metabolite transport, and hence are key pharmacological targets. Structural studies by traditional highresolution techniques are limited by the requirements for high purity and stability when handled in high concentration and nonnative buffers. Hence, there is a growing requirement for the use of alternate methods in a complementary but orthogonal approach to study the dynamic and functional aspects of membrane proteins in physiologically relevant conditions. In recent years, significant progress has been made in the field of X-ray radiolytic labeling in combination with mass spectroscopy, commonly known as X-ray Footprinting and Mass Spectrometry (XFMS), which provide residue-specific information on the solvent accessibility of proteins. In combination with both lowresolution biophysical methods and high-resolution structural data, XFMS is capable of providing valuable insights into structure and dynamics of membrane proteins, which have been difficult to obtain by standalone high-resolution structural techniques. The XFMS method has also demonstrated a unique capability for identification of structural waters and their dynamics in protein cavities at both a high degree of spatial and temporal resolution, and thus capable of identifying conformational hot-spots in transmembrane proteins.
Conclusion: We provide a perspective on the place of XFMS amongst other structural biology methods and showcase some of the latest developments in its usage for studying conformational changes in membrane proteins.
Keywords: Hydroxyl-radical footprinting, oxidative labeling, mass spectrometry, ion channels, transporters, radiolysis.
Graphical Abstract
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