Time change in the distribution of physical activity and its correlates among retired older Swedish adults: a repeated cross-sectional study from a national survey.

BACKGROUND: Understanding how older adults spend time in moderate- to vigorous-intensity physical activity (MVPA) is crucial to understanding healthy ageing. This study connects 24-h time-use diary records of the daily activities of a sample of Swedish older adults to energy intensities. The aim was to: i) estimate the prevalence of Swedish older adults (aged 65-84 years) who achieved recommended daily levels of physical activity; ii) identify what domains of everyday life contribute to MVPA; and iii) explore socio-demographic factors affecting rates of active living. METHODS: We draw on two Swedish nationally representative samples of time-use diary data from 2000/2001 and 2010/2011. Data covering the duration of all activities performed over two days were combined with activity-intensity information (metabolic equivalent of task [MET] values) to estimate the energy expenditure (MET min) originating from MVPA. RESULTS: Results indicate that 94.1% of Swedish older adults achieved the WHO-recommended minimum level of daily MVPA in 2010/2011; the share remained unchanged over the period. MVPA performed in natural environments (24.2%), during housework (22.8%), and on everyday walks in one's local area (18.1%) were dominant domains contributing to energy expenditure. Home maintenance and repairs (8.8%), active transport (9.9%), and physical exercise (8.2%) contributed to a lesser extent. In 2000/2001, total MVPA energy expenditure was associated with gender, housing, living region, and disability; in 2010/2011, except for disability, these associations were no longer significant. CONCLUSIONS: The high proportion of older adults who achieved the recommended level of MVPA, their allocation of MVPA time to diverse domains, and the reduced social distribution over time suggest that elderly people increasingly find their own paths to everyday physical activity. This indicates a need to promote MVPA not only in established ways, such as prescribed training programmes. The importance of active physical activities in natural environments, and of regular walks in the vicinity of home, indicates a need to incorporate healthy ageing considerations in wider urban and regional planning, for example, to increase access to natural environments and urban walkability. Also, older adults' involvement in household chores, maintenance and repairs, and active transport extends responsibility to new policy areas.

Is ageing becoming more active? Exploring cohort-wise changes in everyday time use among the older population in Sweden.

The time older people spend on various daily activities is critical for their health and well-being. New generations of older adults are increasingly expected to participate in 'active' activities. We explore shifts in active time use among upcoming cohorts of older people in Sweden. Recognizing the diverging meanings associated with the active ageing concept, we develop a classification model comprising the spheres of work, social engagement, and active leisure. We observe differences in time use of the 'older middle-aged' (pre-retirement), 'young old', and 'older old' observed in 2000/2001 and 2010/2011. We draw on two cross sections of Swedish time-use survey data covering 120 activities related to people's everyday lives. We measure between-cohort differences in mean time use and employ covariate analysis to control for the influence of group-wise changes in socio-demographics. Linear regression is used to explore social differentiation, e.g. the influence of gender. Comparisons between new and previous generations indicate substantial increases in overall active ageing activity: increases by 7 h per week among the older old and 3.5 h among the young old and older middle-aged. New generations spend more time on work, paid or unpaid, and leisure digital interaction; for some, this is counteracted by less free time spent on social engagement. The new generation of the older old group spends more time on outdoor activity and exercise. These time-use patterns are gendered and dependent on education, mainly due to changes in cohort composition.

Benefits and constrains of covalency: the role of loop length in protein stability and ligand binding.

Protein folding is governed by non-covalent interactions under the benefits and constraints of the covalent linkage of the backbone chain. In the current work we investigate the influence of loop length variation on the free energies of folding and ligand binding in a small globular single-domain protein containing two EF-hand subdomains-calbindin D9k. We introduce a linker extension between the subdomains and vary its length between 1 to 16 glycine residues. We find a close to linear relationship between the linker length and the free energy of folding of the Ca2+-free protein. In contrast, the linker length has only a marginal effect on the Ca2+ affinity and cooperativity. The variant with a single-glycine extension displays slightly increased Ca2+ affinity, suggesting that the slightly extended linker allows optimized packing of the Ca2+-bound state. For the extreme case of disconnected subdomains, Ca2+ binding becomes coupled to folding and assembly. Still, a high affinity between the EF-hands causes the non-covalent pair to retain a relatively high apparent Ca2+ affinity. Our results imply that loop length variation could be an evolutionary option for modulating properties such as protein stability and turnover without compromising the energetics of the specific function of the protein.

On the role of sidechain size and charge in the aggregation of Aß42 with familial mutations.

The aggregation of the amyloid-ß (Aß) peptide is linked to the pathogenesis of Alzheimer's disease (AD). In particular, some point mutations within Aß are associated with early-onset familial Alzheimer's disease. Here we set out to explore how the physical properties of the altered side chains, including their sizes and charges, affect the molecular mechanisms of aggregation. We focus on Aß42 with familial mutations-A21G (Flemish), E22K (Italian), E22G (Arctic), E22Q (Dutch), and D23N (Iowa)-which lead to similar or identical pathology with sporadic AD or severe cerebral amyloid angiopathy. Through global kinetic analysis, we find that for the E22K, E22G, E22Q, and D23N mutations, the acceleration of the overall aggregation originates primarily from the modulation of the nucleation processes, in particular secondary nucleation on the surface of existing fibrils, whereas the elongation process is not significantly affected. Remarkably, the D23 position appears to be responsible for most of the charge effects during nucleation, while the size of the side chain at the E22 position plays a more significant role than its charge. Thus, we have developed a kinetic approach to determine the nature and the magnitude of the contribution of specific residues to the rate of individual steps of the aggregation reaction, through targeted mutations and variations in ionic strength. This strategy can help rationalize the effect of some disease-related mutations as well as yield insights into the mechanism of aggregation and the transition states of the wild-type protein.

N-Terminal Extensions Retard Aß42 Fibril Formation but Allow Cross-Seeding and Coaggregation with Aß42.

Amyloid ß-protein (Aß) sequence length variants with varying aggregation propensity coexist in vivo, where coaggregation and cross-catalysis phenomena may affect the aggregation process. Until recently, naturally occurring amyloid ß-protein (Aß) variants were believed to begin at or after the canonical ß-secretase cleavage site within the amyloid ß-protein precursor. However, N-terminally extended forms of Aß (NTE-Aß) were recently discovered and may contribute to Alzheimer's disease. Here, we have used thioflavin T fluorescence to study the aggregation kinetics of Aß42 variants with N-terminal extensions of 5-40 residues, and transmission electron microscopy to analyze the end states. We find that all variants form amyloid fibrils of similar morphology as Aß42, but the half-time of aggregation (t1/2) increases exponentially with extension length. Monte Carlo simulations of model peptides suggest that the retardation is due to an underlying general physicochemical effect involving reduced frequency of productive molecular encounters. Indeed, global kinetic analyses reveal that NTE-Aß42s form fibrils via the same mechanism as Aß42, but all microscopic rate constants (primary and secondary nucleation, elongation) are reduced for the N-terminally extended variants. Still, Aß42 and NTE-Aß42 coaggregate to form mixed fibrils and fibrils of either Aß42 or NTE-Aß42 catalyze aggregation of all monomers. NTE-Aß42 monomers display reduced aggregation rate with all kinds of seeds implying that extended termini interfere with the ability of monomers to nucleate or elongate. Cross-seeding or coaggregation may therefore represent an important contribution in the in vivo formation of assemblies believed to be important in disease.

Förster resonance energy transfer studies of calmodulin produced by native protein ligation reveal inter-domain electrostatic repulsion.

This study explores the influence of long-range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium-free protein. At low salt, calmodulin has a relatively extended conformation and the distance between the domains is further increased by denaturation, by heat or by non-ionic denaturants. The repulsion between domains is screened by salt and is also diminished by calcium binding, which changes the protein net charge from -23 to -15. Compared with the calcium-free form at low salt, the FRET efficiency for the calcium-bound form has, on average, increased 10-fold. The conformation of the calcium form is insensitive to salt screening. These results imply that when the two globular domains of calmodulin interact with target, there is no significant free energy penalty due to electrostatic interactions.

Role of aromatic side chains in amyloid ß-protein aggregation.

Aggregation of the amyloid ß-protein (Aß) is believed to be involved in Alzheimer's disease pathogenesis. Here we have investigated the importance of the aromatic rings at positions 19 and 20 for the aggregation rate and mechanism by substituting phenylalanine with leucine. Aggregation kinetics were monitored as a function of time and peptide concentration by thioflavin T (ThT) fluorescence, the aggregation equilibrium by sedimentation assay, structural changes using circular dichroism spectroscopy and the presence of fibrillar material was detected with cryo-transmission electron microscopy. All peptides convert from monomer to amyloid fibrils in a concentration-dependent manner. Substituting F19 with leucine results in a peptide that aggregates significantly slower than the wild type, while substitution of F20 produces a peptide that aggregates faster. The effects of the two substitutions are additive, since simultaneous substitution of F19 and F20 produces a peptide with aggregation kinetics intermediate between F19L and F20L. Our results suggest that the aromatic side-chain of F19 favors nucleation of the aggregation process and may be an important target for therapeutic intervention.

Molecular determinants of S100B oligomer formation.

BACKGROUND: S100B is a dimeric protein that can form tetramers, hexamers and higher order oligomers. These forms have been suggested to play a role in RAGE activation. METHODOLOGY/PRINCIPAL FINDINGS: Oligomerization was found to require a low molecular weight trigger/cofactor and could not be detected for highly pure dimer, irrespective of handling. Imidazol was identified as a substance that can serve this role. Oligomerization is dependent on both the imidazol concentration and pH, with optima around 90 mM imidazol and pH 7, respectively. No oligomerization was observed above pH 8, thus the protonated form of imidazol is the active species in promoting assembly of dimers to higher species. However, disulfide bonds are not involved and the process is independent of redox potential. The process was also found to be independent of whether Ca(2+) is bound to the protein or not. Tetramers that are purified from dimers and imidazol by gel filtration are kinetically stable, but dissociate into dimers upon heating. Dimers do not revert to tetramer and higher oligomer unless imidazol is again added. Both tetramers and hexamers bind the target peptide from p53 with retained stoichiometry of one peptide per S100B monomer, and with high affinity (lgK = 7.3±0.2 and 7.2±0.2, respectively in 10 mM BisTris, 5 mM CaCl(2), pH 7.0), which is less than one order of magnitude reduced compared to dimer under the same buffer conditions. CONCLUSION/SIGNIFICANCE: S100B oligomerization requires protonated imidazol as a trigger/cofactor. Oligomers are kinetically stable after imidazol is removed but revert back to dimer if heated. The results underscore the importance of kinetic versus thermodynamic control of S100B protein aggregation.

Interactions in the native state of monellin, which play a protective role against aggregation.

A series of recent studies have provided initial evidence about the role of specific intra-molecular interactions in maintaining proteins in their soluble state and in protecting them from aggregation. Here we show that the amino acid sequence of the protein monellin contains two aggregation-prone regions that are prevented from initiating aggregation by multiple non-covalent interactions that favor their burial within the folded state of the protein. By investigating the behavior of single-chain monellin and a series of five of its mutational variants using a variety of biochemical, biophysical and computational techniques, we found that weakening of the non-covalent interaction that stabilizes the native state of the protein leads to an enhanced aggregation propensity. The lag time for fibrillation was found to correlate with the apparent midpoint of thermal denaturation for the series of mutational variants, thus showing that a reduced thermal stability is associated with an increased aggregation tendency. We rationalize these findings by showing that the increase in the aggregation propensity upon mutation can be predicted in a quantitative manner through the increase in the exposure to solvent of the amyloidogenic regions of the sequence caused by the destabilization of the native state. Our findings, which are further discussed in terms of the structure of monellin and the perturbation by the amino acid substitutions of the contact surface between the two subdomains that compose the folded state of monellin, provide a detailed description of the specific intra-molecular interactions that prevent aggregation by stabilizing the native state of a protein.

100% complete assignment of non-labile (1)H, (13)C, and (15)N signals for calcium-loaded Calbindin D(9k) P43G.

Here we present the 100% complete assignment chemical shift of non-labile (1)H, (15)N and (13)C nuclei of Calbindin D(9k) P43G. The assignment includes all non-exchangeable side chain nuclei, including ones that are rarely reported, such as LysNζ as well as the termini. NMR experiments required to achieve truly complete assignments are discussed. To the best of our knowledge our assignments for Calbindin D(9k) extend beyond previous studies reaching near-completeness (Vis et al. in Biochem 33:14858-14870, 1994; Yamazaki et al. in J Am Chem Soc 116:6464-6465, 1994; Yamazaki et al. in Biochem 32:5656-5669, 1993b).

Green fluorescence induced by EF-hand assembly in a split GFP system.

The affinity between the 1-157 and 158-238 fragments of green fluorescent protein (GFP) is too low for spontaneous in vivo reassembly of the protein upon co-expression of the two fragments. This prevents chromophore maturation and the cells lack GFP fluorescence. We have utilized the very high affinity between the two EF-hands of calbindin D(9k) to facilitate GFP assembly from its fragments and to introduce a calcium dependent molecular switch. In GFPN-EF1, residues 1-157 of GFP are fused to residues 1-43 of calbindin, and in EF2-GFPC, residues 44-75 of calbindin are fused to residues 158-238 of GFP. When co-expressed, GFPN-EF1 and EF2-GFPC associate spontaneously and rapidly resulting in a folded reconstituted protein with bright GFP fluorescence. The high affinity of GFPN-EF1 for EF2-GFPC leads to brighter fluorescence of the cells compared to cells with a control constructs carrying leucine zippers (Wilson et al., Nature Methods 2004;3:255). The complex of GFPN-EF1 and EF2-GFPC was purified from cells using metal-ion chelate chromatography and the temperature dependence of GFP fluorescence was found to be calcium dependent. The GFPN-EF1 and EF2-GFPC fragments were separated by ion exchange chromatography. The assembly of the fragments was found to be reversible and the complex was regained upon mixing, as evidenced by surface plasmon resonance (SPR) data. The affinity between GFPN-EF1 and EF2-GFPC as well as rates of association and dissociation were found to be Ca(2+)-dependent.

A facile method for expression and purification of the Alzheimer's disease-associated amyloid beta-peptide.

We report the development of a high-level bacterial expression system for the Alzheimer's disease-associated amyloid beta-peptide (Abeta), together with a scaleable and inexpensive purification procedure. Abeta(1-40) and Abeta(1-42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-Abeta(1-40) and Met-Abeta(1-42), referred to as Abeta(M1-40) and Abeta(M1-42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure Abeta peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized Abeta1-40 and Abeta1-42. Formation of fibrils by all peptides was concentration-dependent, and exhibited kinetics typical of a nucleation-dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss.

Role of protein surface charge in monellin sweetness.

A small number of proteins have the unusual property of tasting intensely sweet. Despite many studies aimed at identifying their sweet taste determinants, the molecular basis of protein sweetness is not fully understood. Recent mutational studies of monellin have implicated positively charged residues in sweetness. In the present work, the effect of overall net charge was investigated using the complementary approach of negative charge alterations. Multiple substitutions of Asp/Asn and Glu/Gln residues radically altered the surface charge of single-chain monellin by removing six negative charges or adding four negative charges. Biophysical characterization using circular dichroism, fluorescence, and two-dimensional NMR demonstrates that the native fold of monellin is preserved in the variant proteins under physiological solution conditions although their stability toward chemical denaturation is altered. A human taste test was employed to determine the sweetness detection threshold of the variants. Removal of negative charges preserves monellin sweetness, whereas added negative charge has a large negative impact on sweetness. Meta-analysis of published charge variants of monellin and other sweet proteins reveals a general trend toward increasing sweetness with increasing positive net charge. Structural mapping of monellin variants identifies a hydrophobic surface predicted to face the receptor where introduced positive or negative charge reduces sweetness, and a polar surface where charges modulate long-range electrostatic complementarity.

Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles.

Copolymeric NiPAM:BAM nanoparticles of varying hydrophobicity were found to retard fibrillation of the Alzheimer's disease-associated amyloid beta protein (Abeta). We found that these nanoparticles affect mainly the nucleation step of Abeta fibrillation. The elongation step is largely unaffected by the particles, and once the Abeta is nucleated, the fibrillation process occurs with the same rate as in the absence of nanoparticles. The extension of the lag phase for fibrillation of Abeta is strongly dependent on both the amount and surface character of the nanoparticles. Surface plasmon resonance studies show that Abeta binds to the nanoparticles and provide rate and equilibrium constants for the interaction. Numerical analysis of the kinetic data for fibrillation suggests that binding of monomeric Abeta and prefibrillar oligomers to the nanoparticles prevents fibrillation. Moreover, we find that fibrillation of Abeta initiated in the absence of nanoparticles can be reversed by addition of nanoparticles up to a particular time point before mature fibrils appear.

Zn2+ binding to human calbindin D(28k) and the role of histidine residues.

We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn2+. The binding of Zn2+ is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.

Nucleation of protein fibrillation by nanoparticles.

Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrillation by decreasing the lag time for nucleation. Protein fibrillation is involved in many human diseases, including Alzheimer's, Creutzfeld-Jacob disease, and dialysis-related amyloidosis. Fibril formation occurs by nucleation-dependent kinetics, wherein formation of a critical nucleus is the key rate-determining step, after which fibrillation proceeds rapidly. We show that nanoparticles (copolymer particles, cerium oxide particles, quantum dots, and carbon nanotubes) enhance the probability of appearance of a critical nucleus for nucleation of protein fibrils from human beta(2)-microglobulin. The observed shorter lag (nucleation) phase depends on the amount and nature of particle surface. There is an exchange of protein between solution and nanoparticle surface, and beta(2)-microglobulin forms multiple layers on the particle surface, providing a locally increased protein concentration promoting oligomer formation. This and the shortened lag phase suggest a mechanism involving surface-assisted nucleation that may increase the risk for toxic cluster and amyloid formation. It also opens the door to new routes for the controlled self-assembly of proteins and peptides into novel nanomaterials.

Systematic investigation of the thermodynamics of HSA adsorption to N-iso-propylacrylamide/N-tert-butylacrylamide copolymer nanoparticles. Effects of particle size and hydrophobicity.

Nanoparticles in biological fluids almost invariably become coated with proteins that may confer nanomedical and nanotoxicological effects. Understanding these effects requires quantitative measurements using simple systems. Adsorption of HSA to copolymer nanoparticles of varying hydrophobicity and curvature was studied using ITC, yielding stoichiometry, affinity, and enthalpy changes upon binding. The hydrophobicity was controlled via the co-monomer ratio, N-iso-propylacrylamide/N-tert-butylacrylamide. The most hydrophobic particles become fully covered with a single layer of protein, except at high curvature.

Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles.

Due to their small size, nanoparticles have distinct properties compared with the bulk form of the same materials. These properties are rapidly revolutionizing many areas of medicine and technology. Despite the remarkable speed of development of nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid, proteins associate with nanoparticles, and the amount and presentation of the proteins on the surface of the particles leads to an in vivo response. Proteins compete for the nanoparticle "surface," leading to a protein "corona" that largely defines the biological identity of the particle. Thus, knowledge of rates, affinities, and stoichiometries of protein association with, and dissociation from, nanoparticles is important for understanding the nature of the particle surface seen by the functional machinery of cells. Here we develop approaches to study these parameters and apply them to plasma and simple model systems, albumin and fibrinogen. A series of copolymer nanoparticles are used with variation of size and composition (hydrophobicity). We show that isothermal titration calorimetry is suitable for studying the affinity and stoichiometry of protein binding to nanoparticles. We determine the rates of protein association and dissociation using surface plasmon resonance technology with nanoparticles that are thiol-linked to gold, and through size exclusion chromatography of protein-nanoparticle mixtures. This method is less perturbing than centrifugation, and is developed into a systematic methodology to isolate nanoparticle-associated proteins. The kinetic and equilibrium binding properties depend on protein identity as well as particle surface characteristics and size.

Intra- versus intermolecular interactions in monellin: contribution of surface charges to protein assembly.

The relative significance of weak non-covalent interactions in biological context has been much debated. Here, we have addressed the contribution of Coulombic interactions to protein stability and assembly experimentally. The sweet protein monellin, a non-covalently linked heterodimeric protein, was chosen for this study because of its ability to spontaneously reconstitute from separated fragments. The reconstitution of monellin mutants containing large surface charge perturbations was compared to the thermostability of structurally equivalent single-chain monellin containing the same sets of mutations under varying salt concentrations. The affinity between monellin fragments is found to correlate with the thermostability of single chain monellin, indicating the involvement of the same underlying Coulombic interactions. This confirms that there are no principal differences in the interactions involved in folding and binding. Based on comparison with a previous mutational study involving hydrophobic core residues, the relative contribution of Coulombic interactions to stability and affinity is modest. However, the Coulombic perturbations only affect the association rates of reconstitution in contrast to perturbations involving hydrophobic residues, which affect primarily the dissociation rates. These results indicate that Coulombic interactions are likely to be of main importance for the association of protein assembly, relevant for functions of proteins.