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PLoS One. 2012;7(10):e48226. doi: 10.1371/journal.pone.0048226. Epub 2012 Oct 30.

Is buffer a good proxy for a crowded cell-like environment? A comparative NMR study of calmodulin side-chain dynamics in buffer and E. coli lysate.

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1
Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, Ontario, Canada. latham@pound.med.utoronto.ca

Abstract

Biophysical studies of protein structure and dynamics are typically performed in a highly controlled manner involving only the protein(s) of interest. Comparatively fewer such studies have been carried out in the context of a cellular environment that typically involves many biomolecules, ions and metabolites. Recently, solution NMR spectroscopy, focusing primarily on backbone amide groups as reporters, has emerged as a powerful technique for investigating protein structure and dynamics in vivo and in crowded "cell-like" environments. Here we extend these studies through a comparative analysis of Ile, Leu, Val and Met methyl side-chain motions in apo, Ca(2+)-bound and Ca(2+), peptide-bound calmodulin dissolved in aqueous buffer or in E. coli lysate. Deuterium spin relaxation experiments, sensitive to pico- to nano-second time-scale processes and Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, reporting on millisecond dynamics, have been recorded. Both similarities and differences in motional properties are noted for calmodulin dissolved in buffer or in lysate. These results emphasize that while significant insights can be obtained through detailed "test-tube" studies, experiments performed under conditions that are "cell-like" are critical for obtaining a comprehensive understanding of protein motion in vivo and therefore for elucidating the relation between motion and function.

PMID:
23118958
PMCID:
PMC3484071
DOI:
10.1371/journal.pone.0048226
[Indexed for MEDLINE]
Free PMC Article
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