Probing non-specific interactions of Ca2+-calmodulin in E. coli lysate

The biological environment in which a protein performs its function is a crowded milieu containing millions of molecules that can potentially lead to a great many transient, non-specific interactions. NMR spectroscopy is especially well suited to study these weak molecular contacts. Here, non-specific interactions between the Ca2+-bound form of calmodulin (CaM) and non-cognate proteins in Escherichia coli lysate are explored using Ile, Leu, Val and Met methyl probes. Changes in CaM methyl chemical shifts as a function of added E. coli lysate are measured to determine a minimum 'average' dissociation constant for interactions between Ca2+-CaM and E. coli lysate proteins. 2H R2 and 13C R1 spin relaxation rates report on the binding reaction as well. Our results further highlight the power of methyl containing side-chains for characterizing biomolecular interactions, even in complex in-cell like environments.