The Low-Temperature Inflection Observed in Neutron Scattering Measurements of Proteins Is Due to Methyl Rotation: Direct Evidence Using Isotope Labeling and Molecular Dynamics Simulations Kathleen Wood,* ,†,⊥ Douglas J. Tobias,* ,‡ Brigitte Kessler, § Frank Gabel, ¶ Dieter Oesterhelt, § Frans A. A. Mulder, | Giuseppe Zaccai, † and Martin Weik ¶ Institut Laue LangeVin, Grenoble Cedex 9, France, Australian Nuclear Science and Technology Organisation, Menai NSW Australia, Department of Chemistry, UniVersity of California, IrVine, California, Max Planck Institute of Biochemistry, Martinsried, Germany, CEA, IBS, Laboratoire de Biophysique Moleculaire, F-38054 Grenoble, France, CNRS, UMR5075, F-38027, Grenoble, France, UniVersite Joseph Fourier, F-38000, Grenoble, France, Groningen Biomolecular Sciences and Biotechnology Institute, UniVersity of Groningen, The Netherlands Received December 13, 2009; E-mail: kwo@ansto.gov.au; dtobias@uci.edu Biological macromolecules are animated by motions occurring over a wide range of time and length scales, and an understanding of the correlation between their dynamics and functions remains a challenge. A first step is to characterize protein dynamical complex- ity and identify the types of motions contributing to measurements. Several papers have underlined the important contribution of methyl groups 1–7 to measured protein dynamics. Protein dynamics is a signature of its underlying energy landscape, typically explored through analysis of temperature- dependent data. 8 The so-called protein dynamical transition between 180 and 250 K, observed as a deviation from linear behavior in atomic mean square displacements (MSD) as a function of temperature, has been discussed extensively in this context. 9,10 It is present only in samples hydrated to a sufficient degree. 9,11,12 A second break from linear behavior, present in both dry and hydrated samples, was then observed in high-resolution neutron scattering data 13–15 at 120-150 K, which was also reproduced by molecular dynamics simulations. 16 The low-temperature inflection was found to be absent in both tRNA 17 and hydration water 18–20 and was attributed to the onset of methyl-group rotations through analysis of molecular dynamics (MD) simulations. 2,21 Elastic incoherent neutron scattering (EINS) studies on homomeric polypeptide model systems provided experimental evidence that the low-temperature inflection is related to methyl rotation. 22 Others have linked it to the onset of interfacial water rotational motions. 23 Here, we investigated the contribution of methyl groups to purple membrane (PM) dynamics as measured by EINS, by using isotope labeling and MD simulations, and comparison to NMR results. PM is formed predominantly of a single membrane protein, bacterio- rhodopsin (BR), and various lipid species. EINS is sensitive to hydrogen atoms and generally provides information averaged over the whole system. Using hydrogen-deuterium isotope labeling it is possible to focus on one part of a complex sample. In previous work, where deuterated PM samples were produced with either hydrogenated leucine or isoleucine residues, both methyl-containing side chains, an inflection was observed at 120-130 K 24 (see Figure S1, Supporting Information). In the present study we therefore measured the dynamics of a non-methyl-containing side chain, lysine, in BR by studying a completely deuterated PM in which the lysine residues are hydrogenated and compared these dynamics to the dynamics of a natural abundance control PM sample. We take the lysine side chain as representative of non-methyl-containing moieties in the protein. Natural abundance PM (H-PM) and deuterated PM with hydro- genated lysine residues (Lys-PM) were purified and hydrated to 86% relative humidity. Neutron scattering experiments were performed on the IN16 backscattering spectrometer at the Institut Laue Langevin, Grenoble, with an energy resolution of 0.9 μeV and an accessible wavevector (Q) range of 0.02-1.9 Å -1 . The elastically scattered neutrons were recorded on heating from 20 to 300 K and MSD were extracted. Figure 1A represents atomic MSD on the nanosecond time scale extracted from neutron scattering measurements for the H-PM and † Institut Laue Langevin. ⊥ Australian Nuclear Science and Technology Organisation. ‡ University of California. § Max Planck Institute of Biochemistry. ¶ CEA, IBS, Laboratoire de Biophysique Moleculaire; CNRS, UMR5075; and Universite Joseph Fourier. | University of Groningen. Figure 1. MSD from neutron scattering measurements (A) and molecular dynamics simulations (B). In A, natural abundance PM is shown as open diamonds, and deuterated PM with hydrogenated lysine residues is shown as filled diamonds. In B, analysis of the simulations is performed at each temperature for all nonexchangeable PM hydrogen atoms (open diamonds) and for lysine residues only (full diamonds). Published on Web 03/19/2010 10.1021/ja910502g  2010 American Chemical Society 4990 9 J. AM. CHEM. SOC. 2010, 132, 4990–4991