Martin Weik

Protein dynamics studied by neutron scattering

This review of protein dynamics studied by neutron scattering focuses on data collected in the last 10 years. After an introduction to thermal neutron scattering and instrumental aspects, theoretical models that have been used to interpret the data are presented and discussed. Experiments are described according to sample type, protein powders, solutions and membranes. Neutron-scattering results are compared to those obtained from other techniques. The biological relevance of the experimental results is discussed. The major conclusion of the last decade concerns the strong dependence of internal dynamics on the macromolecular environment.

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.

Structural insights into substrate traffic and inhibition in acetylcholinesterase

Acetylcholinesterase (AChE) terminates nerve‐impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active‐site gorge.

Coupling of protein and hydration-water dynamics in biological membranes

The dynamical coupling between proteins and their hydration water is important for the understanding of macromolecular function in a cellular context. In the case of membrane proteins, the environment is heterogeneous, composed of lipids and hydration water, and the dynamical coupling might be more complex than in the case of the extensively studied soluble proteins. Here, we examine the dynamical coupling between a biological membrane, the purple membrane (PM), and its hydration water by a combination of elastic incoherent neutron scattering, specific deuteration, and molecular dynamics simulations. Examining completely deuterated PM, hydrated in H2O, allowed the direct experimental exploration of water dynamics. The study of natural abundance PM in D2O focused on membrane dynamics. The temperature-dependence of atomic mean-square displacements shows inflections at 120 K and 260 K for the membrane and at 200 K and 260 K for the hydration water. Because transition temperatures are different for PM and hydration water, we conclude that ps–ns hydration water dynamics are not directly coupled to membrane motions on the same time scale at temperatures <260 K. Molecular-dynamics simulations of hydrated PM in the temperature range from 100 to 296 K revealed an onset of hydration-water translational diffusion at ≈200 K, but no transition in the PM at the same temperature. Our results suggest that, in contrast to soluble proteins, the dynamics of the membrane protein is not controlled by that of hydration water at temperatures <260 K. Lipid dynamics may have a stronger impact on membrane protein dynamics than hydration water.

Snapshots of Enzymatic Baeyer-Villiger Catalysis: Oxygen Activation and Intermediate Stabilization.

Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP+ ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and “Criegee-stabilizing” catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.

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.

Mechanisms of cholinesterase inhibition by inorganic mercury

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X‐ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo‐first‐order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury‐induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.

Localization of glycolipids in membranes by in vivo labeling and neutron diffraction.

Evidence is accumulating for the lateral organization of cell membrane lipids and proteins in the context of sorting or intracellular signaling. So far, however, information has been lacking on the details of protein-lipid interactions in such aggregates. Purple membranes are patches made up of lipids and the protein bacteriorhodopsin in the plasma membrane of certain Archaea. Naturally crystalline, they provide a unique opportunity to study the structure of a natural membrane at submolecular resolution by diffraction methods. We present a direct structural determination of the glycolipids with respect to bacteriorhodopsin in these membranes. Deuterium labels incorporated in vivo into the sugar moieties of the major glycolipid were localized by neutron diffraction. The data suggest a role for specific aromatic residue-carbohydrate stacking interactions in the formation of the purple membrane crystalline patches.

Flexibility of Aromatic Residues in the Active-Site Gorge of Acetylcholinesterase: X-ray versus Molecular Dynamics

The high aromatic content of the deep and narrow active-site gorge of acetylcholinesterase (AChE) is a remarkable feature of this enzyme. Here, we analyze conformational flexibility of the side chains of the 14 conserved aromatic residues in the active-site gorge of Torpedo californica AChE based on the 47 three-dimensional crystal structures available for the native enzyme, and for its complexes and conjugates, and on a 20-ns molecular dynamics (MD) trajectory of the native enzyme. The degree of flexibility of these 14 aromatic side chains is diverse. Although the side-chain conformations of F330 and W279 are both very flexible, the side-chain conformations of F120, W233, W432, Y70, Y121, F288, F290 and F331 appear to be fixed. Residues located on, or adjacent to, the Omega-loop (C67-C94), namely W84, Y130, Y442, and Y334, display different flexibilities in the MD simulations and in the crystal structures. An important outcome of our study is that the majority of the side-chain conformations observed in the 47 Torpedo californica AChE crystal structures are faithfully reproduced by the MD simulation on the native enzyme. Thus, the protein can assume these conformations even in the absence of the ligand that permitted their experimental detection. These observations are pertinent to structure-based drug design.

Colouring cryo-cooled crystals: online microspectrophotometry

A portable and readily aligned online microspectrophotometer that can be easily installed on macromolecular crystallography beamlines is described. It allows measurement of the spectral characteristics of macromolecular crystals prior, during, and after the X-ray diffraction experiment.