Isotopically Discriminated NMR Spectroscopy: A Tool for Investigating Complex Protein Interactions in Vitro

A new NMR approach is presented for observing in vitro multicomponent protein−protein−ligand(s) interactions, which should help to understand how cellular networks of protein interactions operate on\na molecular level and how they can be controlled with drugs. The method uniquely allows at least two\npolypeptide components of the mixture to be simultaneously closely monitored in a single sample, without\nincreased signal overlap, and can be used to study complex (e.g., sequential, competitive, cooperative,\nallosteric, induced, etc.) binding events, witnessed by two polypeptides independently. One polypeptide is\nuniformly labeled with ¹⁵N and another with ¹⁵N and ¹³C. The ¹H−¹⁵N correlation spectra are recorded for\neach of these molecules separately, discriminated on the basis of the type of ¹³C‘/¹²C‘ atom attached to\nthe amide group nitrogen. Any changes to the state of the two differently isotopically labeled molecules will\nbe reported individually by fingerprint signals from amide groups, e.g., as unlabeled ligands are added. To\nour knowledge, no other technique currently exists which can monitor complex binding events in similar\ndetail. The proposed method can be combined easily with traditional protein NMR techniques and\nincorporated in a variety of applications.