Ionic effects on subunit interactions in a cold-active alkaline phosphatase from the marine bacterium Vibrio splendidus

Protein quaternary structures are fundament for life. Proteins have evolved towards large assemblies of subunits needed for the execution of complex chemistry, for building structural components or for compartmentalization. Most oligomeric enzymes exist as homodimers, having two subunits with identical active sites. It is largely unknown why enzymes have evolved in so many cases towards the homodimer structure (and larger oligomeric structures). Alkaline phosphatase (AP) is an excellent model enzyme for the large family of homodimeric hydrolases. Its activity is dependent on dimerization and correct metal occupancy of the three metal ions in the active site. The reason why the enzyme does not function in the monomeric form is still unknown. The focus of this Ph.D. work was on the AP ortholog from Vibrio splendidus (VAP), a cold-adapted marine bacterium. This enzyme is thermally unstable above room-temperature, but also one of the most active variants of its kind at low temperatures. These characteristics are very dependent on the solution conditions. Stability is only maintained at high ionic strength and pH. Here, the focus was on studying the importance of the dimeric structural state for the function, in particular the role of the metal ions for catalysis. The results suggested that the enzyme inactivates irreversibly to a dimeric intermediate state. This dimeric intermediate state was shown to be more dynamic than the native state, defined as measurable changes in the range of conformations of the ensemble of enzyme molecules, yet fully metalated. Furthermore, the enzyme lost its putative half-of-sites reactivity which is dependent on subunit interactions and communication between active sites. The effect of ionic strength on the activity and stability was studied and found to be positive for both features. It is proposed that a deprotonation step causes a conformational change in VAP, based on pH specific effects on physical parameters. Furthermore, the large interface loop, which characterizes VAP, was shown to be mostly important for stability but also needed for local structural rigidity close to the catalytically active residues. This thesis promotes the idea that the role of the homodimeric state is to provide a conformational energy for both stabilization and catalysis of the enzyme, through the interface, not available in the monomeric state.Fjórða stigs bygging próteina er grunnur alls lífs á jörðinni eins og við þekkjum það. Prótein hafa þróast í átt að stórum komplexum til að hvata flókin efnahvörf, til margvíslegra byggingahlutverka, eða hólfunar. Flest ensím eru virk sem einþátta tvíliður með tvær eins hvarfstöðvar. Hvers vegna svo mörg ensím finnast sem einþátta tvíliður er ekki vel rannsakað. Alkalískur fosfatasi (AP) er módel ensím fyrir hýdrólasa sem eru einþátta tvíliður. Virkni AP er háð tvíliðumyndun og málmjónum í hvarfstöð. Hvers vegna hvarfstöðvarnar hafa ekki hvarfgetu í einliðuforminu er ekki vitað. Rannsóknarefni þessarar doktorsritgerðar var AP úr Vibrio splendidus (VAP) kaldsjávarbakteríu. VAP er eitt hitaóstöðugasta ensím sem þekkist en einnig eitt það virkasta við lág hitastig miðað við sambærileg ensím. Áhersla var lögð á að skýra hlutverk tvíliðumyndunar hjá VAP og áhrif jóna á hvötun og stöðugleika, en einnig að útskýra hvers vegna AP hafa ekki virkar einliður. Þessir þættir eru mjög háðir lausnaaðstæðum. Niðurstöður bentu til þess að við afvirkjun ensímsins verði óafturkræf myndbreyting sem leiðir til óvirkrar tvíliðu. Óvirka tvíliðan var lausbundnari, innihélt allar þrjár málmjónirnar í hvarfstöð en hafði mögulega misst eiginleikann til þess að hvata með svokölluðum “half-of-sites” hvarfgangi. Áhrif jónastyrks voru mikil á virkni og stöðugleika og voru áhrifin háð pH, þar sem afprótónering óþekkts sýruhóp veldur myndbreytingu. Að auki var kannað hlutverk löngu yfirborðlykkjunnar sem einkennir VAP frá flestum AP. Meginhlutverk lykkjunna er að stuðla að stöðugleika hvarfstöðvar en einnig í að halda ákveðinni stífni í lykkjum nálægt virkum hliðarkeðjum. Niðurstaða vinnu þessarar ritgerðar bendir til þess að meginhlutverk tvíliðunnar sé það tillegg byggingarfríorku sem verður til á snertiflötum einliðanna sem stuðlar bæði að stöðugleika og hvötun, sem ekki eru til staðar í einliðunum.RANNÍ

Chloride promotes refolding of active Vibrio alkaline phosphatase through an inactive dimeric intermediate with an altered interface

Publisher's version (útgefin grein)Most enzymes are homodimers or higher order multimers. Cold‐active alkaline phosphatase from Vibrio splendidus (VAP) transitions into a dimer with very low activity under mild denaturation conditions. The desire to understand why this dimer fails to efficiently catalyse phosphomonoester hydrolysis led us to investigate interfacial communication between subunits. Here, we studied in detail the unfolding mechanism at two pH values and in the presence or absence of sodium chloride. At pH 8.0, the denaturation model had to include an inactive dimer intermediate and follow the pathway: N2 → I2 → 2U. At pH 10.5, the model was of a two‐state nature. Enzyme activity was not recovered under several examined refolding conditions. However, in the presence of 0.5 m NaCl, the enzyme was nearly fully reactivated after urea treatment. Thermal inactivation experiments were biphasic where the inactivation could be detected using CD spectroscopy at 190–200 nm. By incorporating a bimane fluorescence probe at the dimer interface, we could monitor inactivation/denaturation at two distinct sites at the dimer interface. A change in bimane fluorescence at both sites was observed during inactivation, but prior to the global unfolding event. Furthermore, the rate of change in bimane fluorescence correlated with inactivation rates at 40 °C. These results indicate and support the hypothesis that the subunits of VAP are only functional in the dimeric state due to the cooperative nature of the reaction mechanism when proper crosstalk between subunits is facilitated.Icelandic Research Fund. Grant Number: 141619‐051Peer reviewe

MacImage: a user-friendly image-analysis system for the accurate mensuration of marine organisms

Changes in abundance, dimensions, and biovolume of manne microorganisms are important parameters used in the estimation of a wide range of biological processes, including cell activity, growth rate, predation, and nutrient cycling. Routine measurement of these parameters has been hampered by the tedium of counting and measuring large numbers of microorganisms, the subjechvity of visual measurement using the eyepiece micrometer, cell shrinkage during preparahon for electron microscopy, and the small size of marine bacteria and some protists. Image-analysed microscopy overcomes these problems, but extant equipment and programs are cumbersome. MacImage, an image-analysis system that interfaces an Artek 810V image-analysis computer and a Macintosh computer, has been developed using the language Forth to improve operator-machine control and versatility. By using a combination of a more sensitive camera tube, additional magnification lenses. and a software system for the Macintosh computer specifically tailored for the measurement of marine microorganisms, we are now able to accurately and rapidly determine the abundance, size, shape, volume, and surface area of microorganisms ranging from marine bactena to fish larvae. Data collected by this system can be transferred directly to other Macintosh spreadsheet and graphics programs. To dustrate the versawity of MacImage, we present data on size, biovolume, biosurface area, and abundance of a natural sample of picoplankton from Narragansett Bay, Rhode Island; size and biovolume data on a cultured clone of Synechococcus; and size, shape and area on the larvae of the winter flounder Pseudopleuronectes amencanus

pH-Dependent Binding of Chloride to a Marine Alkaline Phosphatase Affects the Catalysis, Active Site Stability, and Dimer Equilibrium

Post-print (lokagerð höfundar)The effect of ionic strength on enzyme activity and stability varies considerably between enzymes. Ionic strength is known to affect the catalytic activity of some alkaline phosphatases (APs), such as Escherichia coli AP, but how ions affect APs is debated. Here, we studied the effect of various ions on a cold-adapted AP from Vibrio splendidus (VAP). Previously, we have found that the active form of VAP is extremely unstable at low ionic strengths. Here we show that NaCl increased the activity and stability of VAP and that the effect was pH-dependent in the range of pH 7–10. The activity profile as a function of pH formed two maxima, indicating a possible conformational change. Bringing the pH from the neutral to the alkaline range was accompanied by a large increase in both the Ki for inorganic phosphate (product inhibition) and the KM for p-nitrophenyl phosphate. The activity transitions observed as the pH was varied correlated with structural changes as monitored by tryptophan fluorescence. Thermal and urea-induced inactivation was shown to be accompanied by neither dissociation of the active site metal ions nor dimer dissociation. This would suggest that the inactivation involved subtle changes in active site conformation. Furthermore, the VAP dimer equilibrium was studied for the first time and shown to highly favor dimerization, which was dependent on pH and NaCl concentration. Taken together, the data support a model in which anions bind to some specific acceptor in the active site of VAP, resulting in great stabilization and catalytic rate enhancement, presumably through a different mechanism.Icelandic Research Fund 141619Peer reviewe

Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations

Post-print (lokagerð höfundar)Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn2+ and Mg2+ are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg2+ in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp → Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15 Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition.Financial support from the Icelandic Research Fund (project 141619-051) and the Science Institute of the University of Iceland is gratefully acknowledged. The authors also extend their gratitude to Tinna Pálmadóttir for performing the experiment of Fig. 2B.Peer reviewe

Century-long cod otolith biochronology reveals individual growth plasticity in response to temperature

Otolith biochronologies combine growth records from individual fish to produce long-term growth sequences, which can help to disentangle individual from population-level responses to environmental variability. This study assessed individual thermal plasticity of Atlantic cod (Gadus morhua) growth in Icelandic waters based on measurements of otolith increments. We applied linear mixed-effects models and developed a century-long growth biochronology (1908-2014). We demonstrated interannual and cohort-specific changes in the growth of Icelandic cod over the last century which were mainly driven by temperature variation. Temperature had contrasting relationships with growth-positive for the fish during the youngest ages and negative during the oldest ages. We decomposed the effects of temperature on growth observed at the population level into within-individual effects and among-individual effects and detected significant individual variation in the thermal plasticity of growth. Variance in the individual plasticity differed across cohorts and may be related to the mean environmental conditions experienced by the group. Our results underscore the complexity of the relationships between climatic conditions and the growth of fish at both the population and individual level, and highlight the need to distinguish between average population responses and growth plasticity of the individuals for accurate growth predictions

Comparative metabologenomics analysis of polar actinomycetes.

Biosynthetic and chemical datasets are the two major pillars for microbial drug discovery in the omics era. Despite the advancement of analysis tools and platforms for multi-strain metabolomics and genomics, linking these information sources remains a considerable bottleneck in strain prioritisation and natural product discovery. In this study, molecular networking of the 100 metabolite extracts derived from applying the OSMAC approach to 25 Polar bacterial strains, showed growth media specificity and potential chemical novelty was suggested. Moreover, the metabolite extracts were screened for antibacterial activity and promising selective bioactivity against drug-persistent pathogens such as Klebsiella pneumoniae and Acinetobacter baumannii was observed. Genome sequencing data were combined with metabolomics experiments in the recently developed computational approach, NPLinker, which was used to link BGC and molecular features to prioritise strains for further investigation based on biosynthetic and chemical information. Herein, we putatively identified the known metabolites ectoine and chrloramphenicol which, through NPLinker, were linked to their associated BGCs. The metabologenomics approach followed in this study can potentially be applied to any large microbial datasets for accelerating the discovery of new (bioactive) specialised metabolites

Mechanisms and consequences of life cycle diversity of beaked redfish, Sebastes mentella

Recent genetic research, supported by life history information, indicates that there are three biological stocks of S. mentella in the Irminger Sea and adjacent waters: a ‘Deep Pelagic’ stock (>500m), a ‘Shallow Pelagic’ stock (<500m), and an ‘Icelandic Slope’ stock. Throughout their range, Sebastes species are adapted to a diversity of ecological niches, with overlapping spatial distributions of different species that have little or no morphological differences. Divergence of behavioral groups into depth-defined adult habitats has led to reproductive isolation, adaptive radiation and speciation of several Sebastes species. Congruent differences in fatty acid composition and parasites suggests that the three genetically distinct populations of S. mentella are adapted to disparate trophic habitats in pelagic waters (shallower and deeper than the deep-scattering layer), and in demersal habitats on the continental slope. Patterns of morphology are also consistent with adaptation to different habitats, because pelagic forms are more streamlined. Although genetic differences and evidence for reproductive isolation are clear, these populations appear to share common nursery habitats on the Greenlandic Shelf. Spatial overlap at early life stages and depth-defined adult populations present challenges for stock identification and fishery management. Effective resource monitoring, conservation and fishery management requires that the spatial definition of management units reflects biological stock structure. We describe a proposal for a redefinition of practical management units that are based on geographic proxies for biological stocks which minimizes mixed-stock catches according to spatial patterns of the recent fishery

Recovering the self: a manifesto for primary care.

Huge political, ideological and organisational changes are engulfing primary care, placing intense pressures on the sense of self for both patient and doctor within the consultation.A recent Health Foundation report urges us to develop care practices rooted in a philosophy of people as ‘purposeful, thinking, feeling, emotional, reflective, relational, responsive beings’.1 GPs are encouraged to work collaboratively with patients, fostering shared decision-making and promoting self-management. This assumes that patients (and doctors) have agency and capacity, the ability to make their own choices and decisions and the power to take action in a given situation. But these assumptions are problematic when you are running 15 minutes late during a morning surgery with 18 patients, most of whom are unknown to you, and your QOF screen pop-up urges you to update the patient’s CVD risk assessment score and take action to reduce their HbA1c levels.We wish to give clinicians ‘permission’ to do person-centred care by offering a language of self that they can use to describe and defend their practice. Our principal motivations in establishing the centrality of the self in primary care are to offer hope to those entering the field, encourage those jaded by their current experience in practice, and provide vital underpinning to the generalist cause

Growth portfolios buffer climate-linked environmental change in marine systems

Large-scale, climate-induced synchrony in the productivity of fish populations is becoming more pronounced in the world's oceans. As synchrony increases, a population's “portfolio” of responses can be diminished, in turn reducing its resilience to strong perturbation. Here we argue that the costs and benefits of trait synchronization, such as the expression of growth rate, are context dependent. Contrary to prevailing views, synchrony among individuals could actually be beneficial for populations if growth synchrony increases during favorable conditions, and then declines under poor conditions when a broader portfolio of responses could be useful. Importantly, growth synchrony among individuals within populations has seldom been measured, despite well-documented evidence of synchrony across populations. Here, we used century-scale time series of annual otolith growth to test for changes in growth synchronization among individuals within multiple populations of a marine keystone species (Atlantic cod, Gadus morhua). On the basis of 74,662 annual growth increments recorded in 13,749 otoliths, we detected a rising conformity in long-term growth rates within five northeast Atlantic cod populations in response to both favorable growth conditions and a large-scale, multidecadal mode of climate variability similar to the East Atlantic Pattern. The within-population synchrony was distinct from the across-population synchrony commonly reported for large-scale environmental drivers. Climate-linked, among-individual growth synchrony was also identified in other Northeast Atlantic pelagic, deep-sea and bivalve species. We hypothesize that growth synchrony in good years and growth asynchrony in poorer years reflects adaptive trait optimization and bet hedging, respectively, that could confer an unexpected, but pervasive and stabilizing, impact on marine population productivity in response to large-scale environmental change.publishedVersio