Using X-ray Footprinting and Mass Spectrometry to Study the Structure and Function of Membrane Proteins

Background: Membrane proteins are crucial for cellular sensory cascades and metabolite transport, and hence are key pharmacological targets. Structural studies by traditional high-resolution 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 low-resolution 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.