S-adenosyl-L-methionine (SAM), is one of the most abundant cofactors in cells and plays key roles in epigenetic regulation and in the posttranslational modification of proteins. Recent surveys of methyltransferase crystal structures in the Protein Data Bank reveal a number of unusual noncovalent interactions between the SAM cofactor and active site residues, solvent, and small molecules, which, if they are found to exist in solution, can be exploited in structure based drug design and can provide key insights into the S<sub>N</sub>2 methyl transfer reaction they catalyze.<br/> <br/>However, detecting noncovalent interactions to SAM in active sites is challenging for many molecularly resolved analytical methods. X-ray crystallography faces challenges due both to the fact that hydrogen atoms do not scatter well and statistical disorder of the cofactor that is likely due to the presence of multiple bound conformations. Meanwhile, although sensitive to these atomic-level interactions, NMR signal can be difficult to obtain due to the size of these enzymes. Thus new solution state detection methods are required to reveal any alternate conformations that may be present within active sites. Vibrational spectroscopic methods are immune to size and can be used to directly sense both the SAM conformation as well as any molecular interactions it engages in when bound. Here, we set the stage for examining proteins bound to SAM with vibrational spectroscopy by using a series of small molecule mimics and isotope labeling to assign Raman and IR signals to specific functionals groups of SAM and their conformations in the solution state. Our results are consistent with NMR evidence that SAM when free in solution exists in multiple conformeric states, some of which were not detectable with either NMR or crystallography. We propose several specific conformations of this flexible cofactor that are consistent with our data.

Assignment of vibrations sensitive to conformation and environment in the Raman and IR spectrum of S-adenosyl-L-methionine with small molecule mimics and isotope labeling

ACS Fall 2021

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