Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR

Abstract

Detailed descriptions of atomic coordinates and motions are required for an understanding of protein dynamics and their relation to molecular recognition, catalytic function, and allostery. Historically, NMR relaxation measurements have played a dominant role in the determination of the amplitudes and timescales (picosecond-nanosecond) of bond vector fluctuations, whereas high-resolution X-ray diffraction experiments can reveal the presence of and provide atomic coordinates for multiple, weakly populated substates in the protein conformational ensemble. Here we report a hybrid NMR and X-ray crystallography analysis that provides a more complete dynamic picture and a more quantitative description of the timescale and amplitude of fluctuations in atomic coordinates than is obtainable from the individual methods alone. Order parameters (S 2 ) were calculated from single-conformer and multiconformer models fitted to room temperature and cryogenic X-ray diffraction data for dihydrofolate reductase. Backbone and side-chain order parameters derived from NMR relaxation experiments are in excellent agreement with those calculated from the room-temperature single-conformer and multiconformer models, showing that the picosecond timescale motions observed in solution occur also in the crystalline state. These motions are quenched in the crystal at cryogenic temperatures. The combination of NMR and X-ray crystallography in iterative refinement promises to provide an atomic resolution description of the alternate conformational substates that are sampled through picosecond to nanosecond timescale fluctuations of the protein structure. The method also provides insights into the structural heterogeneity of nonmethyl side chains, aromatic residues, and ligands, which are less commonly analyzed by NMR relaxation measurements.B factor | nuclear magnetic resonance | atomic displacement parameters | Lipari-Szabo T he conformational dynamics of proteins play an important role in their functional mechanisms, influencing such diverse processes as conformational selection in molecular recognition (1), catalytic function (2, 3), and allosteric regulation (4). To understand the atomic motions that underlie protein dynamics and configurational entropy, it has become increasingly important to study both the timescales of motions and how those motions are manifest in the structure as coordinate changes. Two complementary methods are high-resolution X-ray crystallography and NMR spectroscopy, which provide coordinate precision and probe motions within well-defined timescales, respectively. The combination of X-ray and NMR data has thus far been limited to coordinate refinement because no rigorous framework exists to connect the coordinates and motional timescales (5-7).Proteins are not rigid and constantly sample distinct conformational substates (8). The coordinates derived from X-ray diffraction are accompanied by atomic displacement parameters or B factors that are related to the displacements of the atoms from their mean positions (9). B ...

Application of helium ion microscopy to nanostructured polymer materials

“…This has been notoriously difficult for extremely large proteins or transmembrane proteins [144,145]. Current NMR techniques require the protein or other biological molecule to be small and soluble [146]. The other level of inquiry regarding biological structure is at the cellular level.…”

Geometric analysis characterizes molecular rigidity in generic and non-generic protein configurations

“…While X-ray crystallography experiments mostly yield a single, low-energy ground state of the molecule, nuclear magnetic resonance relaxation dispersion experiments can provide insight into functionally relevant excited states, but lack a structural basis for collective motions. Computationally integrating these data sources has proved challenging [8, 9]. Molecular dynamics simulations can yield atomically detailed trajectories, but rely on imperfect force-fields and often demand specialized hardware [17] and algorithms to examine long, biologically relevant time scales or larger molecules [25].…”

Correlated motions are a fundamental property of β-sheets

“…In a complementary fashion, theoretical methods have also provided insights into the mechanistic details of how interresidue interactions can lead to correlated motions spanning long distances 18 . Finally, hybrid experimental/computational approaches have also been used to study this phenomenon [19][20][21][22][23] .…”