A Consensus-Based Set of Measures for Oral Health Care.

Increasingly more responsive and accountable health care systems are demanded, which is characterized by transparency and explicit demonstration of competence by health care providers and the systems in which they work. This study aimed to establish measures of oral health for transparent and explicit reporting of routine data to facilitate more patient-centered and prevention-oriented oral health care. To accomplish this, an intermediate objective was to develop a comprehensive list of topics that a range of stakeholders would perceive as valid, important, and relevant for describing oral health and oral health care. A 4-stage approach was used to develop the list of topics: 1) scoping of literature and its appraisal, 2) a meeting of experts, 3) a 2-stage Delphi process (online), and 4) a World Café discussion. The aim was to create consensus through structured conversations via a range of stakeholders (general dental practitioners, patients, insurers, and policy makers) from the Netherlands, Germany, the United Kingdom, Ireland, Hungary, and Denmark. The study was part of the ADVOCATE project, and it resulted in a list of 48 topics grouped into 6 clusters: 1) access to dental care, 2) symptoms and diagnosis, 3) health behaviors, 4) oral treatments, 5) oral prevention, and 6) patient perception. All topics can be measured, as they all have a data source with defined numerators and denominators. This study is the first to establish a comprehensive and multiple-stakeholder consented topic list designed for guiding the implementation of transparent and explicit measurement of routine data of oral health and oral health care. Successful measurement within oral health care systems is essential to facilitate learning from variation in practice and outcomes within and among systems, and it potentiates improvement toward more patient-centered and prevention-oriented oral health care.

Oligomeric states along the folding pathways of ß2-microglobulin: kinetics, thermodynamics, and structure.

The transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein ß2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of ß2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration.

Dynamical coupling of intrinsically disordered proteins and their hydration water: comparison with folded soluble and membrane proteins.

Hydration water is vital for various macromolecular biological activities, such as specific ligand recognition, enzyme activity, response to receptor binding, and energy transduction. Without hydration water, proteins would not fold correctly and would lack the conformational flexibility that animates their three-dimensional structures. Motions in globular, soluble proteins are thought to be governed to a certain extent by hydration-water dynamics, yet it is not known whether this relationship holds true for other protein classes in general and whether, in turn, the structural nature of a protein also influences water motions. Here, we provide insight into the coupling between hydration-water dynamics and atomic motions in intrinsically disordered proteins (IDP), a largely unexplored class of proteins that, in contrast to folded proteins, lack a well-defined three-dimensional structure. We investigated the human IDP tau, which is involved in the pathogenic processes accompanying Alzheimer disease. Combining neutron scattering and protein perdeuteration, we found similar atomic mean-square displacements over a large temperature range for the tau protein and its hydration water, indicating intimate coupling between them. This is in contrast to the behavior of folded proteins of similar molecular weight, such as the globular, soluble maltose-binding protein and the membrane protein bacteriorhodopsin, which display moderate to weak coupling, respectively. The extracted mean square displacements also reveal a greater motional flexibility of IDP compared with globular, folded proteins and more restricted water motions on the IDP surface. The results provide evidence that protein and hydration-water motions mutually affect and shape each other, and that there is a gradient of coupling across different protein classes that may play a functional role in macromolecular activity in a cellular context.

Amphipols from A to Z.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

Structural characterization of HBXIP: the protein that interacts with the anti-apoptotic protein survivin and the oncogenic viral protein HBx.

Hepatitis B X-interacting protein (HBXIP) is a ubiquitous protein that was originally identified as a binding partner of the hepatitis B viral protein HBx. HBXIP is also thought to serve as an anti-apoptotic cofactor of survivin, promoting the suppression of pro-caspase-9 activation. Here were port the crystal structure of the shortest isoform of HBXIP (91 aa long,∼11 kDa) at 1.5 Å resolution. HBXIP crystal shows a monomer per asymmetric unit, with a profilin-like fold which is common to a super family of proteins, the Roadblock/LC7 domain family involved in protein-protein interactions. Based on this fold, we propose that HBXIP can form a dimer that can indeed be found in the crystal when symmetric molecules are generated around the asymmetric unit. This dimer shows an extended ß-sheet area formed by 10 anti-parallel ß-strands from both subunits. Another interesting aspect of the proposed HBXIP dimer interface is the presence of a small leucine zipper between the two α2 helices of each monomer. In solution, the scattering curve obtained by small-angle X-ray scattering for the sample used for crystallization indicates that the protein is dimeric form in solution. The fit between the experimental small angle X-ray scattering curve and the back calculated curves for two potential crystal dimers shows a significant preference for the Roadblock/LC7 fold dimer model. Moreover, the HBXIP crystal structure represents a step towards understanding the cellular role of HBXIP.

Direct correlation between molecular dynamics and enzymatic stability: a comparative neutron scattering study of native human butyrylcholinesterase and its "aged" soman conjugate.

An incoherent elastic neutron scattering study of the molecular dynamics of native human butyrylcholinesterase and its "aged" soman-inhibited conjugate revealed a significant change in molecular flexibility on an angstrom-nanosecond scale as a function of temperature. The results were related to the stability of each state as established previously by differential scanning calorimetry. A striking relationship was found between the denaturation behavior and the molecular flexibility of the native and inhibited enzymes as a function of temperature. This was reflected in a quantitative correlation between the atomic mean-square displacements on an angstrom-nanosecond scale determined by neutron spectroscopy and the calorimetric specific heat. By the application of a simple two-state model that describes the transition from a folded to a denatured state, the denaturation temperatures of the native and the inhibited enzyme were correctly extracted from the atomic mean-square displacements. Furthermore, the transition entropy and enthalpy extracted from the model fit of the neutron data were, within the experimental accuracy, compatible with the values determined by differential scanning calorimetry.

From shell to cell: neutron scattering studies of biological water dynamics and coupling to activity.

An integrated picture of hydration shell dynamics and of its coupling to functional macromolecular motions is proposed from studies on a soluble protein, on a membrane protein in its natural lipid environment, and on the intracellular environment in bacteria and red blood cells. Water dynamics in multimolar salt solutions was also examined, in the context of the very slow water component previously discovered in the cytoplasm of extreme halophilic archaea. The data were obtained from neutron scattering by using deuterium labelling to focus on the dynamics of different parts of the complex systems examined.

A structure refinement protocol combining NMR residual dipolar couplings and small angle scattering restraints.

We present the implementation of a target function based on Small Angle Scattering data (Gabel et al. Eur Biophys J 35(4):313-327, 2006) into the Crystallography and NMR Systems (CNS) and demonstrate its utility in NMR structure calculations by simultaneous application of small angle scattering (SAS) and residual dipolar coupling (RDC) restraints. The efficiency and stability of the approach are demonstrated by reconstructing the structure of a two domain region of the 31 kDa nuclear export factor TAP (TIP-associated protein). Starting with the high resolution X-ray structures of the two individual TAP domains, the translational and orientational domain arrangement is refined simultaneously. We tested the stability of the protocol against variations of the SAS target parameters and the number of RDCs and their uncertainties. The activation of SAS restraints results in an improved translational clustering of the domain positions and lifts part of the fourfold degeneracy of their orientations (associated with a single alignment tensor). The resulting ensemble of structures reflects the conformational space that is consistent with the experimental SAS and RDC data. The SAS target function is computationally very efficient. SAS restraints can be activated at different levels of precision and only a limited SAS angular range is required. When combined with additional data from chemical shift perturbation, paramagnetic relaxation enhancement or mutational analysis the SAS refinement is an efficient approach for defining the topology of multi-domain and/or multimeric biomolecular complexes in solution based on available high resolution structures (NMR or X-ray) of the individual domains.

Dynamics of hydration water in deuterated purple membranes explored by neutron scattering.

The function and dynamics of proteins depend on their direct environment, and much evidence has pointed to a strong coupling between water and protein motions. Recently however, neutron scattering measurements on deuterated and natural-abundance purple membrane (PM), hydrated in H2O and D2O, respectively, revealed that membrane and water motions on the ns-ps time scale are not directly coupled below 260 K (Wood et al. in Proc Natl Acad Sci USA 104:18049-18054, 2007). In the initial study, samples with a high level of hydration were measured. Here, we have measured the dynamics of PM and water separately, at a low-hydration level corresponding to the first layer of hydration water only. As in the case of the higher hydration samples previously studied, the dynamics of PM and water display different temperature dependencies, with a transition in the hydration water at 200 K not triggering a transition in the membrane at the same temperature. Furthermore, neutron diffraction experiments were carried out to monitor the lamellar spacing of a flash-cooled deuterated PM stack hydrated in H2O as a function of temperature. At 200 K, a sudden decrease in lamellar spacing indicated the onset of long-range translational water diffusion in the second hydration layer as has already been observed on flash-cooled natural-abundance PM stacks hydrated in D2O (Weik et al. in J Mol Biol 275:632-634, 2005), excluding thus a notable isotope effect. Our results reinforce the notion that membrane-protein dynamics may be less strongly coupled to hydration water motions than the dynamics of soluble proteins.

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 H(2)O, allowed the direct experimental exploration of water dynamics. The study of natural abundance PM in D(2)O 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 approximately 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.

Effects of soman inhibition and of structural differences on cholinesterase molecular dynamics: a neutron scattering study.

Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.

The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study.

A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.