Michael Härtlein

Coincidence of dynamical transitions in a soluble protein and its hydration water: direct measurements by neutron scattering and MD simulations.

The coupling between protein dynamics and hydration-water dynamics was assessed by perdeuteration, temperature-dependent neutron scattering, and molecular dynamics simulations. Mean square displacements of water and protein motions both show a broad transition at 220 K and are thus coupled. In particular, the protein dynamical transition appears to be driven by the onset of hydration-water translational motion.

Translational diffusion of hydration water correlates with functional motions in folded and intrinsically disordered proteins

Hydration water is the natural matrix of biological macromolecules and is essential for their activity in cells. The coupling between water and protein dynamics has been intensively studied, yet it remains controversial. Here we combine protein perdeuteration, neutron scattering and molecular dynamics simulations to explore the nature of hydration water motions at temperatures between 200 and 300 K, across the so-called protein dynamical transition, in the intrinsically disordered human protein tau and the globular maltose binding protein. Quasi-elastic broadening is fitted with a model of translating, rotating and immobile water molecules. In both experiment and simulation, the translational component markedly increases at the protein dynamical transition (around 240 K), regardless of whether the protein is intrinsically disordered or folded. Thus, we generalize the notion that the translational diffusion of water molecules on a protein surface promotes the large-amplitude motions of proteins that are required for their biological activity.

Hydration water mobility is enhanced around tau amyloid fibers

Significance Protein aggregation into amyloid fibers and oligomers is observed in a variety of neurodegenerative diseases. The fibers formed by the intrinsically disordered human protein tau, for instance, are one of the hallmarks of Alzheimer disease. In this work, we report on the dynamic behavior of tau hydration water, which we found to be more mobile in tau fibers than in nonaggregated tau. This increase in mobility could promote fiber formation through an increase in hydration water entropy. That hydration water is more mobile around the pathological form of tau corroborates that methodologies sensitive to the diffusion of water, such as diffusion magnetic resonance imaging, could be used to diagnose Alzheimer patients in an early stage of the disease. The paired helical filaments (PHF) formed by the intrinsically disordered human protein tau are one of the pathological hallmarks of Alzheimer disease. PHF are fibers of amyloid nature that are composed of a rigid core and an unstructured fuzzy coat. The mechanisms of fiber formation, in particular the role that hydration water might play, remain poorly understood. We combined protein deuteration, neutron scattering, and all-atom molecular dynamics simulations to study the dynamics of hydration water at the surface of fibers formed by the full-length human protein htau40. In comparison with monomeric tau, hydration water on the surface of tau fibers is more mobile, as evidenced by an increased fraction of translationally diffusing water molecules, a higher diffusion coefficient, and increased mean-squared displacements in neutron scattering experiments. Fibers formed by the hexapeptide 306VQIVYK311 were taken as a model for the tau fiber core and studied by molecular dynamics simulations, revealing that hydration water dynamics around the core domain is significantly reduced after fiber formation. Thus, an increase in water dynamics around the fuzzy coat is proposed to be at the origin of the experimentally observed increase in hydration water dynamics around the entire tau fiber. The observed increase in hydration water dynamics is suggested to promote fiber formation through entropic effects. Detection of the enhanced hydration water mobility around tau fibers is conjectured to potentially contribute to the early diagnosis of Alzheimer patients by diffusion MRI.

A comparison of refined X-ray structures of hydrogenated and perdeuterated rat γE-crystallin in H2O and D2O

Rat gammaE-crystallin was overexpressed, purified under different labelling conditions and crystallized and X-ray data were collected at resolutions between 1.71 and 1.36 A. The structures were determined by molecular replacement. In these structures, the cd loop of the Greek-key motif 3, which is the major structural key motif of the two phase-transition groups of gamma-crystallins, presents a double conformation. The influence of the perdeuteration on the protein structure was determined by comparison of the atomic positions and temperature factors of the different models. The perdeuterated proteins have a similar structure to their hydrogenated counterparts, but partial or full deuteration may have some effect on the atomic B-factor values.

Small angle neutron scattering for the study of solubilised membrane proteins.

Small angle neutron scattering (SANS) is a powerful technique for investigating association states and conformational changes of biological macromolecules in solution. SANS is of particular interest for the study of the multi-component systems, as membrane protein complexes, for which in vitro characterisation and structure determination are often difficult. This article details the important physical properties of surfactants in view of small angle neutron scattering studies and the interest to deuterate membrane proteins for contrast variation studies. We present strategies for the production of deuterated membrane proteins and methods for quality control. We then review some studies on membrane proteins, and focus on the strategies to overcome the intrinsic difficulty to eliminate homogeneously the detergent or surfactant signal for solubilised membrane proteins, or that of lipids for membrane proteins inserted in liposomes.Graphical abstract

X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB.

The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB(321)) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB(321) consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB(321) in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB(321) binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB(321). These results call into question whether receptor dimerization is the basic underlying event in InlB(321)-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB(321) bind and activate the Met receptor.

A polymer surfactant corona dynamically replaces water in solvent-free protein liquids and ensures macromolecular flexibility and activity.

The observation of biological activity in solvent-free protein-polymer surfactant hybrids challenges the view of aqueous and nonaqueous solvents being unique promoters of protein dynamics linked to function. Here, we combine elastic incoherent neutron scattering and specific deuterium labeling to separately study protein and polymer motions in solvent-free hybrids. Myoglobin motions within the hybrid are found to closely resemble those of a hydrated protein, and motions of the polymer surfactant coating are similar to those of the hydration water, leading to the conclusion that the polymer surfactant coating plasticizes protein structures in a way similar to hydration water.

Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase.

The present work illustrates that small-angle neutron scattering, deuteration and contrast variation, combined with in vitro particle reconstruction, constitutes a very efficient approach to determine subunit architectures in large, symmetric protein complexes. In the case of the 468 kDa heterododecameric TET peptidase machine, it was demonstrated that the assembly of the 12 subunits is a highly controlled process and represents a way to optimize the catalytic efficiency of the enzyme.

Investigation into the Relaxation Dynamics of Polymer-Protein Conjugates Reveals Surprising Role of Polymer Solvation on Inherent Protein Flexibility.

Fully biodegradable protein-polymer conjugates, namely, MBP-PMeEP (maltose binding protein-poly methyl-ethylene phosphonate), have been investigated in order to understand the role of polymer solvation on protein flexibility. Using elastic and quasi-elastic incoherent neutron scattering, in combination with partially deuterated conjugate systems, we are able to disentangle the polymer dynamics from the protein dynamics and meaningfully address the coupling between both components. We highlight that, in the dry state, the protein-polymer conjugates lack any dynamical transition in accordance with the generally observed behavior for dry proteins. In addition, we observe a larger flexibility of the conjugated protein, compared to the native protein, as well as a lack of polymer-glass transition. Only upon water hydration does the conjugate recover its dynamical transition, leading to the conclusion that exclusive polymer solvation is insufficient to unfreeze fluctuations on the picosecond-nanosecond time scale in biomolecules. Our results also confirm the established coupling between polymer and protein dynamics in the conjugate.

Determinants of ligand binding and catalytic activity in the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase.

2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) is an enzyme highly abundant in the central nervous system myelin of terrestrial vertebrates. The catalytic domain of CNPase belongs to the 2H phosphoesterase superfamily and catalyzes the hydrolysis of nucleoside 2′,3′-cyclic monophosphates to nucleoside 2′-monophosphates. The detailed reaction mechanism and the essential catalytic amino acids involved have been described earlier, but the roles of many amino acids in the vicinity of the active site have remained unknown. Here, several CNPase catalytic domain mutants were studied using enzyme kinetics assays, thermal stability experiments, and X-ray crystallography. Additionally, the crystal structure of a perdeuterated CNPase catalytic domain was refined at atomic resolution to obtain a detailed view of the active site and the catalytic mechanism. The results specify determinants of ligand binding and novel essential residues required for CNPase catalysis. For example, the aromatic side chains of Phe235 and Tyr168 are crucial for substrate binding, and Arg307 may affect active site electrostatics and regulate loop dynamics. The β5-α7 loop, unique for CNPase in the 2H phosphoesterase family, appears to have various functions in the CNPase reaction mechanism, from coordinating the nucleophilic water molecule to providing a binding pocket for the product and being involved in product release.