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 Ca(2+)-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 Ca(2+)-CaM and E. coli lysate proteins. (2)H R 2 and (13)C R 1 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.