Ashish Tamhankar, a Ph.D. student in the Department of Inorganic Spectroscopy, recently published his first first-author paper in the Journal of Physical Chemistry Letters. His work was featured along with a supplementary cover art and he also had the opportunity to present this work at the LUCIA a 20 ans workshop in SOLEIL synchrotron in France as an oral presentation. The work was a collaborative effort between the Max Planck Institute for Chemical Energy Conversion, Max Planck Institute for Multidisciplinary Sciences, IIT Roorkee and SOLEIL synchrotron.
The work was centered around a recent, novel covalent post-translational linkage between amino acids was discovered in the enzyme transaldolase from Neisseria gonorrhoeae, the gonorrhea-causing pathogen. The intramolecular linkage is a Lys–NOS–Cys bridge formed by oxidizing the amine side group of a lysine and the thiol of a cysteine residue and acts as an allosteric redox switch. The proteins’ oxidized and reduced X-ray crystallographic structures suggested a loaded-spring mechanism with a structural relaxation upon redox activation that propagates from the regulatory allosteric lysine–cysteine redox switch site at the protein surface to the active site at the protein interior. Further studies have identified the NOS switch in crystal structures across a variety of systems and organisms including SARS-CoV-2, potentially playing an important role in regulation, cellular defense, and replication. Even though the NOS bridge’s chemical identity was clearly shown by protein crystallography at sub-ångstrom resolution, its presence in solution has so far never been directly demonstrated.
Solid-state techniques (X-ray diffraction and X-ray crystallography) are highly limited in analyzing dynamic systems like proteins. X-ray crystallography may trap proteins in specific conformations, potentially masking their dynamic behavior. To avoid structural changes in the protein and bypass the limitations of solid-state techniques, the scientists approached this problem on the complete and natively folded protein to detect the NOS bridge.
In this study, the scientists present the first in-solution detection of the NOS bridge using Sulfur K-edge XAS. The spectral shifts due to changes in the local sulfur environment provide direct experimental evidence for the presence of the NOS bridge in oxidized state in solution, which had previously only been established crystallographically. These experimental observations are supported by a systematic time-dependent density functional theory (TDDFT) study and computational analyses of the Sulfur K-edge XAS. The in-solution detection of the NOS redox switch presented herein relies on an element specific technique not requiring crystallization, thus enabling higher throughput analysis of enzyme activity regulation under physiologically relevant conditions which could facilitate novel avenues in drug discovery, biocatalytic applications, and protein design, with further biological implications in the context of redox signaling, oxidative stress, and many human disease states.
Original paper :
Ashish Tamhankar, Marie Wensien, Sergio A. V. Jannuzzi, Sayanti Chatterjee, Benedikt Lassalle-Kaiser, Kai Tittmann, and Serena DeBeer
The Journal of Physical Chemistry Letters 2024 15 (16), 4263-4267
DOI: 10.1021/acs.jpclett.4c00484