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 R 2 and 13C 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.
Bibliographical noteFunding Information:
Acknowledgments The authors thank Professor Mistu Ikura (Ontario Cancer Institute) for the gift of a CaM plasmid and assignments for Ca2?-CaM and Dr. John Rubinstein (Hospital for Sick Children) for providing laboratory space. M. P. L. acknowledges support in the form of post-doctoral fellowships from the National Science Foundation (OISE-0853108) and the Canadian Institutes of Health Research (CIHR) Training Grant in Protein Folding and Disease. This work was supported by a grant from the CIHR. L. E. K. holds a Canada Research Chair in Biochemistry.
- CHD methyl isotopomers
- Chemical shift titration
- Deuterium relaxation
- In "cell-like"
- Methyl side-chain dynamics