Relating molecular flexibility to function: a case study of tubulin

Microtubules (MT), along with a variety of associated motor proteins, are involved in a range of cellular functions including vesicle movement, chromosome segregation, and cell motility. MTs are assemblies of heterodimeric proteins, alpha beta-tubulins, the structure of which has been determined by electron crystallography of zinc-induced, pacilitaxel-stabilized tubulin sheets. These data provide a basis for examining relationships between structural features and protein function. Here, we study the fluctuation dynamics of the tubulin dimer with the aim of elucidating its functional motions relevant to substrate binding, polymerization/depolymerization and MT assembly. A coarse-grained model, harmonically constrained according to the crystal structure, is used to explore the global dynamics of the dimer. Our results identify six regions of collective motion, comprised of structurally close but discontinuous sequence fragments, observed only in the dimeric form, dimerization being a prerequisite for domain identification. Boundaries between regions of collective motions appear to act as linkages, found primarily within secondary-structure elements that lack sequence conservation, but are located at minima in the fluctuation curve, at positions of hydrophobic residues. Residue fluctuations within these domains identify the most mobile regions as loops involved in recognition of the adjacent regions. The least mobile regions are associated with nucleotide binding sites where lethal mutations occur. The functional coupling of motions between and within regions identifies three global motions: torsional and wobbling movements, en bloc, between the alpha- and beta-tubulin monomers, and stretching longitudinally. Further analysis finds the antitumor drug pacilitaxel (TaxotereR) to reduce flexibility in the M loop of the beta-tubulin monomer; an effect that may contribute to tightening lateral interactions between protofilaments assembled into MTs. Our analysis provides insights into relationships between intramolecular tubulin movements of MT organization and function.     

Mapping molecular flexibility of proteins with site-directed spin labeling: a case study of myoglobin

Site-directed spin labeling (SDSL) has potential for mapping protein flexibility under physiological conditions. The purpose of the present study was to explore this potential using 38 singly spin-labeled mutants of myoglobin distributed throughout the sequence. Correlation of the EPR spectra with protein structure provides new evidence that the site-dependent variation in line shape, and hence motion of the spin label, is due largely to differences in mobility of the helical backbone in the ns time range. Fluctuations between conformational substates, typically in the μs-ms time range, are slow on the EPR time scale, and the spectra provide a snapshot of conformational equilibria frozen in time as revealed by multiple components in the spectra. A recent study showed that osmolyte perturbation can positively identify conformational exchange as the origin of multicomponent spectra ( López et al. ( 2009 ) , Protein Sci. 18 , 1637 ). In the present study, this new strategy is employed in combination with line shape analysis and pulsed-EPR interspin distance measurements to investigate the conformation and flexibility of myoglobin in three folded and partially folded states. The regions identified to be in conformational exchange in the three forms agree remarkably well with those assigned by NMR, but the faster time scale of EPR allows characterization of localized states not detected in NMR. Collectively, the results suggest that SDSL-EPR and osmolyte perturbation provide a facile means for mapping the amplitude of fast backbone fluctuations and for detecting sequences in slow conformational exchange in folded and partially folded protein sequences.     

Measuring performances and footprint of blockchains with BCTMark: a case study on Ethereum smart contracts energy consumption

Cluster Computing - A rich ecosystem of blockchain-based projects has emerged since the introduction of Bitcoin in 2008. New protocols seek to improve the performances of blockchain systems. In...     

Social flexibility and social evolution in mammals: a case study of the African striped mouse (Rhabdomys pumilio)

Environmental change poses challenges to many organisms. The resilience of a species to such change depends on its ability to respond adaptively. Social flexibility is such an adaptive response, whereby individuals of both sexes change their reproductive tactics facultatively in response to fluctuating environmental conditions, leading to changes in the social system. Social flexibility focuses on individual flexibility, and provides a unique opportunity to study both the ultimate and proximate causes of sociality by comparing between solitary and group-living individuals of the same population: why do animals form groups and how is group-living regulated by the environment and the neuro-endocrine system? These key questions have been studied for the past ten years in the striped mouse Rhabdomys pumilio. High population density favours philopatry and group-living, while reproductive competition favours dispersal and solitary-living. Studies of genetic parentage reveal that relative fitness of alternative reproductive tactics depends on the prevailing environment. Tactics have different fitness under constrained ecological conditions, when competitive ability is important. Under conditions with relaxed ecological constraints, alternative tactics can yield equal fitness. Both male and female striped mice display alternative reproductive tactics based on a single strategy, i.e. all individuals follow the same decision rules. These changes are regulated by endocrine mechanisms. Social flexibility is regarded as an adaptation to unpredictably changing environments, selecting for high phenotypic flexibility based on a broad reaction norm, not on genetic polymorphism for specific tactics.     

Relative flexibility of DNA and RNA: a molecular dynamics study

State of the art molecular dynamics simulations are used to study the structure, dynamics, molecular interaction properties and flexibility of DNA and RNA duplexes in aqueous solution. Special attention is paid to the deformability of both types of structures, revisiting concepts on the relative flexibility of DNA and RNA duplexes. Our simulations strongly suggest that the concepts of flexibility, rigidity and deformability are much more complex than usually believed, and that it is not always true that DNA is more flexible than RNA.     

Interdependence of backbone flexibility,residue conservation,and enzyme function: a case study on beta1,4-galactosyltransferase-I

Beta1,4-galactosyltransferase-I (beta4Gal-T1) catalyzes the transfer of a galactose from UDP-galactose to N-acetylglucosamine. A recent crystal structure determination of the substrate-bound enzyme reveals a large conformational change, which creates binding sites for the oligosaccharide and alpha-lactalbumin, when compared to the ligand-free structure. The conformational changes take place in a 21-residue-long loop (I345-H365) and in a smaller loop containing a tryptophan residue (W314) flanked by glycines (Y311-G316; Trp loop). A series of molecular dynamics simulations carried out with an implicit solvent model and with explicit water successfully identify flexibility in the two loops and in another interacting loop. These observations are confirmed by limited proteolysis experiments that reveal an intrinsic flexibility of the long loop. The multiple simulation runs starting with the substrate-free structure show that the long loop moves toward its conformation in the ligand-bound structure; however, it gets stabilized in an intermediate position. The Trp loop moves in the opposite direction to that of the long loop, making contacts with residues in the long loop. Remarkably, when the Trp loop is restrained in its starting conformation, no large conformational change takes place in the long loop, indicating residue communication of flexibility. Sequence and structural analysis of the beta4Gal-T1 family with 37 known sequences reveals that in contrast to the unconserved long loop, which undergoes a much larger conformational change, the Trp loop including the glycines is highly conserved. These observations lead us to propose a new functional mechanism that may be conserved by evolution to perform a variety of functions.     

Chemical purity and mutagenicity: case study of a drug in development.

During a routine Ames assay of a potential antipsychotic drug candidate, the compound appeared to be a frameshift mutagen in Salmonella typhimurium strains TA98 and TA1538. Additional testing indicated the mutagenic activity was due to one or more contaminants incurred during synthesis. While the compound was initially shown to be greater than 98% pure by high-performance liquid chromatography, the presence of small amounts (0.01-0.1%) of a highly mutagenic impurity produced positive mutagenicity results. The need to assess for chemical purity before discontinuing development of drug candidates found positive in the Ames assay is discussed.     

Restoration programmes and the development of a natural diet: a case study of captive-bred European mink

As part of a conservation initiative, we released captive-bred individuals of European mink (Mustela lutreola) onto a Baltic island ‘sanctuary’, Hiiumaa (Estonia), and investigated the development of their diet in the wild. Fifty-four animals out of the 172 released were equipped with radio collars and tracked in 2000–2003 intensively after release. Based on the analysis of the contents of 564 collected scats, we monitored how the diet of released individuals changed after release and how this was affected by habitat and by season. Diet changed as they adapted to the wild: some prey consumed immediately after release were later substituted with prey more typical of wild European mink elsewhere. The mink’s tendency to take typical prey increased (crayfish, 3; fish, 1.5; and small mammals, 2 times), while the proportion of atypical prey decreased more than five times in 60 days after release. Once established in the wild, the composition of the diet and its variation between seasons, habitats or weather conditions were similar to that reported elsewhere for wild European mink. There is no indication therefore that the components of the diet provided in captivity persisted in the wild after the adaptation period. We suggest that the adaptation of released carnivores to natural prey merits more attention in reintroduction projects.     

Balancing views on community monitoring: the case of REDD+

In response to the commentary by Jens Friis Lund we suggest that the problem lies not in community monitoring but in the way the reward systems are designed. For the case of REDD+ we propose that if payments are made for inputs rather than for outputs, the problem can largely be resolved.     

Assessing wetland services for improved development decision-making: a case study of mangroves in coastal Bangladesh

Mangroves provide valuable ecosystem services for the wellbeing of coastal communities. Assessment and valuation of these mangroves services are increasingly advocated in development and conservation decision-making. Translating the values of services into more explicit monetary terms requires understanding of stakeholder activities, socio-economic context and local organizational structure to effectively support decision-making. Based on a survey of 100 households of three villages of Sundarban in Bangladesh, mangroves services to local communities were identified and their economic values estimated. The households perceived 18 mangroves services, of which capture fisheries, fuel energy, storm protection, habitat for fish breeding and nursery grounds and aesthetic enjoyment were ranked the most important. For provisioning services, households obtained important monetary benefits annually from capture fishery (US$ 976 per ha), fuel energy (US$ 80 per ha), honey (US$ 53 per ha) and fodder (US$ 26 per ha). The average annual willingness to pay for storm protection, erosion control and habitat for fish breeding and nursery services were estimated, respectively, as US$ 13 per ha, US$ 2 per ha and US$ 9 per ha. However, unsustainable exploitation and salinity intrusion impacted the services provided by mangroves. This study provides an important insight into the services and values of mangroves for local welfare, and thus can inform policy for protection and better use of mangrove resources.     

Conformational flexibility of aqueous monomeric and dimeric insulin: a molecular dynamics study

A series of molecular dynamics simulations have been used to investigate the nature of monomeric and dimeric insulin in aqueous solution. It is shown that in the absence of crystal contacts both monomeric and dimeric insulin have a high degree of intrinsic flexibility. Neither of the two monomer conformations of 2Zn crystalline insulin appears to be favored in solution nor is the asymmetry of the crystal dimer reduced in the absence of crystal contacts. A shift is observed in the relative positions of molecules 1 and 2 in the dimer compared with that found in the crystal, which may have consequences for the prediction of the effects of mutants in the monomer-monomer interface designed to alter the self-association properties of insulin.     

Production and comprehensive quality control of recombinant human Interleukin-1beta: a case study for a process development strategy

We describe an efficient strategy to produce high-quality proteins by using a single large IMAC chromatography column and enzymatic His-tag removal via the TAGZyme system in pilot scale. Numerous quality assays demonstrated a high purity of the final product, the human cytokine Interleukin-1beta (IL-1beta). The protein preparation was apparently free of host cell proteins, endotoxins, protease, and aggregates. The N-terminal amino acid sequence of IL-1beta was in full agreement with the natural mature form of IL-1beta. The homogeneity of the product was further shown by X-ray structure determination which confirmed the previously solved structure of the protein. We propose the applied workflow as a strategy for industrial production of protein-based biopharmaceuticals.     

Urban-rural differences in the relationship between women's employment and fertility: a preliminary study

A statistical study of data from the Egyptian census for 1960 points up the influence of the employment of women on their fertility. Results show that the child/woman ratio rises with the level of female employment in the rural areas, but falls as the level of employment rises in the urban areas. Subject to confirmation by more detailed methods, it seems that, in urban areas, provision of opportunities for women to work is of major importance in attempting to reduce national fertility levels, but this will have the opposite effect to what is intended in rural areas. Rural and urban fertility levels appear to be similar at a low level of economic activity; however, when this economic activity surpasses 10%, a noticeable fertility differential appears. Industrial or commercial work opportunities change a woman's pattern of living, and are essential to the permanent lowering of fertility to replacement levels.     

Balancing sampling and specialization: an adaptationist model of incremental development

Development is typically a constructive process, in which phenotypes incrementally adapt to local ecologies. Here, we present a novel model in which natural selection shapes developmental systems based on the evolutionary ecology, and these systems adaptively guide phenotypic development. We assume that phenotypic construction is incremental and trades off with sampling cues to the environmental state. We computed the optimal developmental programmes across a range of evolutionary ecological conditions. Using these programmes, we simulated distributions of mature phenotypes. Our results show that organisms sample the environment most extensively when cues are moderately, not highly, informative. When the developmental programme relies heavily on sampling, individuals transition from sampling to specialization at different times in ontogeny, depending on the consistency of their sampled cue set; this finding suggests that stochastic sampling may result in individual differences in plasticity itself. In addition, we find that different selection pressures may favour similar developmental mechanisms, and that organisms may incorrectly calibrate development despite stable ontogenetic environments. We hope our model will stimulate adaptationist research on the constructive processes guiding development.     

A quantitative and qualitative study of craniofacial development in the rat embryo aged 302 hours to 374 hours (days 12.5 to 15.5)

One-hour mated Sprague-Dawley rats underwent a normal pregnancy, which was terminated at hours 302, 326, 350, and 374. After appropriate processing and scanning electron microscopy of the embryos, absolute measurements of the craniofacial area, especially of the snout, maxillary process, eye, lower face, and brain were taken in the 302- and 326-hr embryos. Additionally, growth ratios were established in all stages. The greatest growth and developmental changes occurred between 302 and 326 hr, when 1) the snout developed from the nasal anlagen and the maxillary process, 2) the eye lens replaced the eyepit, and 3) the hindlimb developed. Between 326 and 350 hr, the optic pit developed. The results of the present study can markedly enhance precision in interpretation of results of teratological studies in the rat. The detailed quantitative data established allow reporting of subtle departures from the normal hitherto impossible.     

Evolution and development of budding by stem cells: Ascidian coloniality as a case study

The evolution of budding in metazoans is not well understood on a mechanistic level, but is an important developmental process. We examine the evolution of coloniality in ascidians, contrasting the life histories of solitary and colonial forms with a focus on the cellular and developmental basis of the evolution of budding. Tunicates are an excellent group to study colonial transitions, as all solitary larvae develop with determinant and invariant cleavage patterns, but colonial species show robust developmental flexibility during larval development. We propose that acquiring new stem cell lineages in the larvae may be a preadaptation necessary for the evolution of budding. Brooding in colonial ascidians allows increased egg size, which in turn allows greater flexibility in the specification of cells and cell numbers in late embryonic and pre-metamorphic larval stages. We review hypotheses for changes in stem cell lineages in colonial species, describe what the current data suggest about the evolution of budding, and discuss where we believe further studies will be most fruitful.     

Setting priorities for research and development in the NHS: a case study on the interface between primary and secondary care.

Since 1991 the NHS has attempted to identify and prioritise its needs for research and development in a systematic manner. This has not been done before and there is little evidence on which to draw. Multidisciplinary expert groups have identified priorities in different topics using explicit criteria and after widespread consultation within the NHS and research community to identify pressing problems and opportunities for research. This paper focuses on a review completed in 1993 to identify research and development priorities for the NHS in relation to the interface between primary and secondary care. The review covered several recent developments which require evaluation. The authors describe the process used to identify research and development priorities in this complex subject and examine the strengths and weaknesses of the approach. This case study should help to stimulate a wider debate on methods of identifying priorities, particularly those using participatory approaches, in research and non-research contexts.