Metal ions play an important role in a large variety of biochemical and cellular events, are present at the active sites of many catalytic processes that are at the core of modern chemistry, and are the key constituents of many new versatile materials. As such, they have a tremendous impact on many fields within life sciences, environment, energy, and industry. About one third of the proteins purified to date contain at least a metal ion as a cofactor, and approximately 20 to 30 percent are membrane proteins. Integral membrane metalloproteins are involved in the transport and homeostasis of metal ions across membranes, as well as in key redox reactions involving e.g. energy storage and conversion, gas processing, and cofactors synthesis.
In the case of integral membrane systems, single crystals large enough for X-ray diffraction cannot be easily obtained, and the problem of structure elucidation is largely unsolved. Presently, although crystallization and cryo-EM methods have made progress in the area of membrane proteins, there is still a paucity of solved transmembrane protein structures, which occupy less than one percent of the protein data bank despite their high occurrence in the biological world. Even when high-resolution crystal structures are available, often the nature of the metal ion, its oxidation state, or its coordination geometry are not determined. As a result, the details of many essential biochemical processes are thus still unknown, highlighting a need for a reliable and efficient method for the structure determination of metal centers inside membrane-bound metalloenzymes and transporters. Light in this area will enable a leap forward in the biological understanding, and will simultaneously suggest new solutions to the foremost problems in environmental and synthetic chemistry today.
In this project we have developed solid-state Magic-Angle Spinning Nuclear Magnetic Resonance (MAS-NMR) spectroscopy to allow complete characterization of the structure of integral membrane metalloproteins.
The project has capitalized on these critical areas of expertise of the PI, on new concepts, and on the availability of new state-of-the-art equipment to develop paramagnetic MAS-NMR spectroscopy through a series of new advances to address the following key challenges:
· To increase the size limit of integral membrane proteins which can be fully characterized with high resolution and sensitivity by MAS-NMR in lipid membrane environments;
· To develop new methodology to remove the current barriers to spectral acquisition from paramagnetic nuclei and to extend the amount of information that can be extracted from them;
· To determine structure-activity-property relationships in integral membrane proteins, specifically developing methods capable of determining global structure and dynamics and methods for the determination of the electronic features of metal ions.
This research project has yielded a broadly applicable method for the structural characterization of essential chemical and cellular processes and thereby has provided a powerful tool to solve challenges at the forefront of molecular and chemical sciences today.
