Structure-based assignment of methyl groups by FLAMEnGO 2.0 (Fuzzy Logic Assignment of MEthyl GrOups): Application to protein kinase A

Methyl-TROSY spectroscopy is a powerful technique that extends the solution-state NMR regime to probe the structures and conformational dynamics of large biomolecular systems (1, 2). However, the main drawback of the application of this technique is the difficulty of assigning the resonances to methyl groups in a given protein. Several different approaches have been proposed to tackle this problem. Traditional through-bond approaches, such as a combination of triple resonance and methyl out-and-back experiments (3), rely on back-bond assignments and suffer from low sensitivity. The brutal force method, using mutagenesis, is time-consuming, and the changes in chemical shifts due to mutagenesis can cause ambiguity when assigning the resonances (4). Recently, inspired by a method by Matthews and co-workers (5), we develop a new algorithm FLAMEnGO to assign methyl resonances by combining the crystal structure and sparse and ambiguous NMR restraints (6), which illuminates a new approach to assign methyl resonances.