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 Michael P. Latham 1 *, Lewis E. Kay 1,2 * 1 Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, Ontario, Canada, 2 Hospital for Sick Children, Program in Molecular Structure and Function, Toronto, Ontario, Canada 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. Citation: Latham MP, Kay LE (2012) 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. PLoS ONE 7(10): e48226. doi:10.1371/journal.pone.0048226 Editor: Daniel S. Sem, Concordia University Wisconsin, United States of America Received July 13, 2012; Accepted September 24, 2012; Published October 30, 2012 Copyright: ß 2012 Latham, Kay. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: 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. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: latham@pound.med.utoronto.ca; kay@pound.med.utoronto.ca Introduction A clear link has been established between protein structure, dynamics and function through studies using a wide-range of structural, biophysical and biochemical techniques [1–5]. For the most part, however, these studies are performed in vitro, using isolated proteins that have been purified to homogeneity so that the effects of the ‘‘natural biological environment’’ are removed. The environment in which a protein functions is a complicated heterogeneous mixture containing as many as 100 million metabolites and 2.4 million proteins per cell [6,7]. This complex milieu can have both stabilizing and destabilizing effects on the constituent proteins. For example, the high concentrations of macromolecules can lead to increased folding propensity due to excluded volume effects, whereas, non-specific associations between macromolecules can potentially result in unfolding [8– 15]. Additionally, the kinetics and thermodynamics of interactions can be significantly changed relative to the pristine environment of a test-tube [16–18]. It is clear that an in-depth picture of a protein’s structure, dynamics and function must take into account the unique properties that are found in-cell. Nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful technique for studying proteins within the biological context of the cellular environment [14,15,19–28]. While the vast majority of in vivo or in ‘‘cell-like’’ NMR studies have utilized backbone amide groups as probes of structure and dynamics, hydrophobic methyl containing side-chains could potentially also serve as valuable reporters [26,29]. For example, NMR spectra of methyl groups have signal-to-noise ratios that are significantly higher than amide data sets [26]. Methyl containing side-chains are often localized to protein-protein interfaces, ligand binding pockets, enzyme active sites and the hydrophobic cores of globular proteins and thus provide complementary information to amide groups [30]. The importance of methyl containing residues in facilitating non-specific interactions in the cellular environment has been demonstrated by mutation of a surface hydrophobic patch on the protein ubiquitin, resulting in a significant improvement in in vivo NMR spectral quality [21]. This presum- ably reflects the elimination of transient contacts with one or more native proteins in the cell. Here we have prepared a series of U-[ 2 H], Ile-d1[ 13 CHD 2 ]-, Leu,Val-[ 13 CHD 2 , 13 CHD 2 ]-, Met[ 13 CHD 2 ]-labeled calmodulin (CaM) samples to study methyl side-chain dynamics in a ‘‘cell-like’’ environment comprising 100 g/L (native protein concentration) E. coli lysate (referred to as lysate CaM). 2 H-based spin relaxation experiments [31,32] were recorded on Ca 2+ -free CaM (apoCaM in what follows) as well as Ca 2+ -loaded CaM in complex with a substrate peptide from smooth muscle myosin light chain kinase, Ca 2+ -CaM-smMLCK(p). These measurements establish that the amplitude and time-scale of fast (picosecond, ps –nanosecond, ns) PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e48226