The Stelling Lab uses solution state bioanalytical methods, with a focus on vibrational spectroscopy, to probe molecular interactions in proteins and nucleic acids.

Exploiting the Dynamic Potential of S-adenosylmethionine (SAM)

S-adenosyl-L-methionine (SAM) is an abundant small biomolecule used by methyltransferases, a class of important drug targets, to regulate a wide range of essential
cellular processes such as gene expression, cell signaling, protein functions, etc. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off-target effects . Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between a number of states under equilibrium conditions when it is free in solution . If multiple
conformers of SAM are populated when it is bound within enzyme active sites, as it appears to be when free in solution, these alternate conformers of SAM might provide promising, unique structural leads that can be exploited to increase an inhibitor’s specificity for a particular methyltransferase; provided different binding sites each promotes structurally unique conformers specific to their active sites.

Vibrations of SAM, which can be detected with infrared spectroscopy, can be used as probes to detect the potentially broad number of geometrically reasonable conformations the molecule can adopt when it is free in solution and serve to further validate the structural models that were found to be consistent with the solution state NMR evidence. These internal atomic motions canalso serve as useful probes of the location and magnitude of interactions (e.g., hydrogen bonds) to the molecule once the detected frequencies are assigned to specific atoms in SAM’s
structure

Probing the thermodynamics and confirmation of an individual A-T base pair in DNA duplexes.

Individual hydrogen bonds (H-bonds) between base pairs are important for essential processes, such as DNA replication DNA mismatch repair, and DNA-protein recognition. In DNA replication, the hydrogen bonding pattern in Watson-Crick base pairing is required for DNA polymerase to accurately replicate the genetic information contained in the sequences. These H-bonds are required to be strong enough to preserve duplex structure but weak enough to allow opening of the strands for transcription, replication, and protein recognition. Recent studies on resonance assistance have been proposed to be important in determining the binding energies of the weak interactions, H-bonds, that are formed between the fundamental chemical units of genetic information, the DNA base pairs. The complementary H-bonds formed by adenine-thymine (A-T) and guanine-cytosine (G-C) base pairs in duplex DNA are of critical importance to both its structure; and the ability of proteins and drugs to achieve sequence-specific recognition. Here, we followed up on our previous works using isotope editing on T 13C2=O to extract T C2=O signals from thymine bases in DNA duplexes with various chemically modified A sites and perform hydrogen-deuterium exchange experiments to pinpoint the possible resonance stabilization (enol-keto) forms of T base.