Extensive structural change of the envelope protein of dengue virus induced by a tuned ionic strength: conformational and energetic analyses

The Dengue has become a global public health threat, with over 100 million infections annually; to date there is no specific vaccine or any antiviral drug. The structures of the envelope (E) proteins of the four known serotype of the dengue virus (DENV) are already known, but there are insufficient molecular details of their structural behavior in solution in the distinct environmental conditions in which the DENVs are submitted, from the digestive tract of the mosquito up to its replication inside the host cell. Such detailed knowledge becomes important because of the multifunctional character of the E protein: it mediates the early events in cell entry, via receptor endocytosis and, as a class II protein, participates determinately in the process of membrane fusion. The proposed infection mechanism asserts that once in the endosome, at low pH, the E homodimers dissociate and insert into the endosomal lipid membrane, after an extensive conformational change, mainly on the relative arrangement of its three domains. In this work we employ all-atom explicit solvent Molecular Dynamics simulations to specify the thermodynamic conditions in that the E proteins are induced to experience extensive structural changes, such as during the process of reducing pH. We study the structural behavior of the E protein monomer at acid pH solution of distinct ionic strength. Extensive simulations are carried out with all the histidine residues in its full protonated form at four distinct ionic strengths. The results are analyzed in detail from structural and energetic perspectives, and the virtual protein movements are described by means of the principal component analyses. As the main result, we found that at acid pH and physiological ionic strength, the E protein suffers a major structural change; for lower or higher ionic strengths, the crystal structure is essentially maintained along of all extensive simulations. On the other hand, at basic pH, when all histidine residues are in the unprotonated form, the protein structure is very stable for ionic strengths ranging from 0 to 225 mM. Therefore, our findings support the hypothesis that the histidines constitute the hot points that induce configurational changes of E protein in acid pH, and give extra motivation to the development of new ideas for antivirus compound design.

三峡库区巴东组泥灰岩溶蚀作用与岩体力学性质弱化研究

Abstract The karsrt erosion engineering geology became a highlight problem in recent years, in particularly, the karst erosion of marlite of Badong formation made the rock mechanics weaken in Three Gorges Reservoir area, which reduces the safety of slope. During the immigrant construction, many high slopes have been formed, whose instabilities problems pose serious threats to the safety of the people and properties. The accidents of the slope failure take place now and then. By testing, it has been found that the karst erosion pattern and dissolution rate of marlite are not weaker than that of the pure limestone. Furthermore, owning to the weathering and unloading, the karst erosion of the marlite will reach certain depth of the slope, which is named infiltrated karst erosion. The karst erosion made the rock mass quality of slope or foundation worse in a large scale. The karst erosion geological disasters, taken place or not, has become the main restrictive factors to the social stability and economic development. Thus the karst erosion process and mechanism of marlite of Badong formation are studied as the main content of this dissertation. The weakening characteristic of rock mass mechanics parameters are studied along with the rock mass structure deformation and failure processes in the course of the karst erosion. At first, the conditions and influencing factors of the karst erosion are analyzed in the investigative region, on the basis of different karst erosion phenomenon of the marlite and different failure modes of slope. Then via indoor the karst erosion tests, it is analyzed that the karst erosion will change the rock mass composition and its structure. Through test, the different karst erosion phenomena between micro and macro have been observed, and the karst erosion mechanism of the marlite has been summarized. Damage theory is introduced to explain the feature of dissolution pore and the law of crack propagation in the marlite. By microscope and the references data, it can be concluded that the karst erosion process can be divided into rock minerals damage and rock structural damage. And the percent of karst erosion volume is named damage factor, which can be used to describe the quantify karst erosion degree of marlite. Through test, the rock mechanical properties in the different period of karst erosion are studied. Based on the damage mechanics theory and the test result, the relation between the karst erosion degree of marlite and weakening degree of mechanical properties is summarized. By numerical simulations, the karst erosive rock mass mechanics is verified. The conclusion is drawn as below: to the rock mass of marlite, the karst erosion damage made mechanics parameters variation, the deformation modulus, cohesion, and inter friction angle reduce as the negative exponent with the increasing of the karst erosion volume, however, the Poisson ratio increases as the positive exponent with the karst erosion volume increasing. It should be noticed that the deduced formulations are limited to the test data and certain conditions. It is suitable to the rock mass parametric weakening process after the karst erosion of marlite in Three Gorges Reservoir area. Based on the failure types of marlite slope in the field, the karst erosion and weathering process of rock mass are analyzed. And the evolution law of deformation and failure of the marlite mass is studied. The main failure feature of the marlite slope is the karst erosive structure subsidence mode in Three Gorges Reservoir area. The karst erosive structure subsidence mode is explained as follows: the rock mass undergoes the synthetic influence, such as weathering, unloading, corrosion, and so on, many pores and cavities have been formed in the rock mass interior, the rock mass quality is worsen and the rock mass structure is changed, and then the inherent structure of rock mass is collapsed under its gravity, therefore, the failure mode of compaction and subsidence take place. Finally, two examples are used to verify the rock mass parameters in Three Gorges Reservoir area, and the relationship between the marlite slope stability and the time of karst erosion is proposed.

Measuring the state of consciousness in a free-living diving sea turtle

We report on results from two types of data-logger attached to hawksbill turtles (Eretmochelys coriacea) in the breeding season at the Seychelles, Indian Ocean. Conventional time-depth recorders (TDRs) showed prolonged bouts of long dives to the seabed, consistent with benthic resting. This behaviour has been widely reported in sea turtles and appears to be a common feature for energy conservation. An Inter-Mandibular Angle Sensor (IMASEN) recorded mouth opening and buccal pumping by one turtle for 2.5 days. Buccal pumping occurred widely while the turtle was submerged, consistent with a function of olfactory sensory perception of the turtle's environment. However, buccal pumping stopped during the middle of long benthic dives consistent with the turtle entering a phase of sleep. It therefore appears that by recording buccal oscillations, it is possible to assess the state of consciousness of turtles allowing the eco-physiology of diving to be more fully explored. (C) 2007 Elsevier B.V. All rights reserved.

Resonant States in Negative Ions

Resonant states are multiply excited states in atoms and ions that have enough energy to decay by emitting an electron. The ability to emit an electron and the strong electron correlation (which is extra strong in negative ions) makes these states both interesting and challenging from a theoretical point of view. The main contribution in this thesis is a method, which combines the use of B splines and complex rotation, to solve the three-electron Schrödinger equation treating all three electrons equally. It is used to calculate doubly excited and triply excited states of 4S symmetry with even parity in He-. For the doubly excited states there are experimental and theoretical data to compare with. For the triply excited states there is only theoretical data available and only for one of the resonances. The agreement is in general good. For the triply excited state there is a significant and interesting difference in the width between our calculation and another method. A cause for this deviation is suggested. The method is also used to find a resonant state of 4S symmetry with odd parity in H2-. This state, in this extremely negative system, has been predicted by two earlier calculations but is highly controversial. Several other studies presented here focus on two-electron systems. In one, the effect of the splitting of the degenerate H(n=2) thresholds in H-, on the resonant states converging to this threshold, is studied. If a completely degenerate threshold is assumed an infinite series of states is expected to converge to the threshold. Here states of 1P symmetry and odd parity are examined, and it is found that the relativistic and radiative splitting of the threshold causes the series to end after only three resonant states. Since the independent particle model completely fails for doubly excited states, several schemes of alternative quantum numbers have been suggested. We investigate the so called DESB (Doubly Excited Symmetry Basis) quantum numbers in several calculations. For the doubly excited states of He- mentioned above we investigate one resonance and find that it cannot be assigned DESB quantum numbers unambiguously. We also investigate these quantum numbers for states of 1S even parity in He. We find two types of mixing of DESB states in the doubly excited states calculated. We also show that the amount of mixing of DESB quantum numbers can be inferred from the value of the cosine of the inter-electronic angle. In a study on Li- the calculated cosine values are used to identify doubly excited states measured in a photodetachment experiment. In particular a resonant state that violates a propensity rule is found.

Dive track profile, intermandibular angel sensor profile and immobilisation data of adult female Weddell seals at Drescher Inlet from expedition DRE2003

Determination of when and where animals feed and how much they consume is fundamental to understand their ecology and role in ecosystems. However, the lack of reliable data on feeding habits of wild animals, and particularly in marine endotherms, attests to the difficulty in doing this. A promising recent development proposes using a Hall sensor-magnet System - the inter-mandibular angle sensor (IMASEN) attached to animals' jaws to elucidate feeding events. We conducted trials on captive pinnipeds by feeding IMASEN-equipped animals with prey to examine the utility of this system. Most feeding events were clearly distinguishable from other jaw movements; only small prey items might not be resolved adequately. Based on the results of this study we examined feeding events from free-ranging Weddell seals fitted with IMASENs and dead-reckoners during December 2003 at Drescher Inlet (Riiser Larsen Ice Shelf, eastern Weddell Sea coast), and present data on prey capture and ingestion in relation to the three-dimensionalmovement patterns of the seals. A total of 19 Weddell seals were immobilised by using a combination of ketamine, xylazine, and diazepam. Eight seals were drugged once, six two times, and two and three were drugged three and four times each, coming to a total of 38 immobilisation procedures. Narcoses were terminated with yohimbine (doi:10.1594/PANGAEA.438931).

Spectral sensitivity, spatial resolution and temporal resolution and their implications for conspecific signalling in cleaner shrimp.

Cleaner shrimp (Decapoda) regularly interact with conspecifics and client reef fish, both of which appear colourful and finely patterned to human observers. However, whether cleaner shrimp can perceive the colour patterns of conspecifics and clients is unknown, because cleaner shrimp visual capabilities are unstudied. We quantified spectral sensitivity and temporal resolution using electroretinography (ERG), and spatial resolution using both morphological (inter-ommatidial angle) and behavioural (optomotor) methods in three cleaner shrimp species: Lysmata amboinensis, Ancylomenes pedersoni and Urocaridella antonbruunii. In all three species, we found strong evidence for only a single spectral sensitivity peak of (mean ± s.e.m.) 518 ± 5, 518 ± 2 and 533 ± 3 nm, respectively. Temporal resolution in dark-adapted eyes was 39 ± 1.3, 36 ± 0.6 and 34 ± 1.3 Hz. Spatial resolution was 9.9 ± 0.3, 8.3 ± 0.1 and 11 ± 0.5 deg, respectively, which is low compared with other compound eyes of similar size. Assuming monochromacy, we present approximations of cleaner shrimp perception of both conspecifics and clients, and show that cleaner shrimp visual capabilities are sufficient to detect the outlines of large stimuli, but not to detect the colour patterns of conspecifics or clients, even over short distances. Thus, conspecific viewers have probably not played a role in the evolution of cleaner shrimp appearance; rather, further studies should investigate whether cleaner shrimp colour patterns have evolved to be viewed by client reef fish, many of which possess tri- and tetra-chromatic colour vision and relatively high spatial acuity.

Inter-observer agreement in evaluating the anterior chamber angle by ultrasound biomicroscopy

Purpose. To determine the agreement between observers in estimating the configuration of the human anterior chamber angle (ACA) using ultrasound biomicroscopy (UBM). Methods. Two masked clinicians used UBM images to estimate, in 41 eyes, the configuration of the ACA, especially (1) the position of contact between the peripheral iris and the inside of the eye wall, (2) the angularity of the approach to the ACA, and (3) the curvature of the peripheral iris. Agreement between observers was evaluated by the kappa statistic. Results. Inter-observer agreement in assessing the iris insertion (kappa = 0.79), angular width (Kappa = 0.95), and the peripheral iris curvature (kappa = 0.84) was high. Conclusions. The agreement between observers in evaluating the anterior chamber angle configuration by UBM was excellent.

Small-angle X-ray scattering and structural modeling of full-length: Cellobiohydrolase I from Trichoderma harzianum

Cellobiohydrolases hydrolyze cellulose releasing cellobiose units. They are very important for a number of biotechnological applications, such as, for example, production of cellulosic ethanol and cotton fiber processing. The Trichoderma cellobiohydrolase I (CBH1 or Cel7A) is an industrially important exocellulase. It exhibits a typical two domain architecture, with a small C-terminal cellulose-binding domain and a large N-terminal catalytic core domain, connected by an O-glycosylated linker peptide. The mechanism by which the linker mediates the concerted action of the two domains remains a conundrum. Here, we probe the protein shape and domain organization of the CBH1 of Trichoderma harzianum (ThCel7A) by small angle X-ray scattering (SAXS) and structural modeling. Our SAXS data shows that ThCel7A linker is partially-extended in solution. Structural modeling suggests that this linker conformation is stabilized by inter- and intra-molecular interactions involving the linker peptide and its O-glycosylations. © 2013 Springer Science+Business Media Dordrecht.

Flow around a hemisphere-cylinder at high angle of attack and low Reynolds number. Part II: POD and DMD applied to reduced domains

Three-dimensional direct numerical simulations (DNS) have been performed on a finite-size hemispherecylinder model at angle of attack AoA = 20◦ and Reynolds numbers Re = 350 and 1000. Under these conditions, massive separation exists on the nose and lee-side of the cylinder, and at both Reynolds numbers the flow is found to be unsteady. Proper orthogonal decomposition (POD) and dynamic mode decomposition (DMD) are employed in order to study the primary instability that triggers unsteadiness at Re = 350. The dominant coherent flow structures identified at the lower Reynolds number are also found to exist at Re = 1000; the question is then posed whether the flow oscillations and structures found at the two Reynolds numbers are related. POD and DMD computations are performed using different subdomains of the DNS computational domain. Besides reducing the computational cost of the analyses, this also permits to isolate spatially localized oscillatory structures from other, more energetic structures present in the flow. It is found that POD and DMD are in general sensitive to domain truncation and noneducated choices of the subdomain may lead to inconsistent results. Analyses at Re = 350 show that the primary instability is related to the counter rotating vortex pair conforming the three-dimensional afterbody wake, and characterized by the frequency St ≈ 0.11, in line with results in the literature. At Re = 1000, vortex-shedding is present in the wake with an associated broadband spectrum centered around the same frequency. The horn/leeward vortices at the cylinder lee-side, upstream of the cylinder base, also present finite amplitude oscillations at the higher Reynolds number. The spatial structure of these oscillations, described by the POD modes, is easily differentiated from that of the wake oscillations. Additionally, the frequency spectra associated with the lee-side vortices presents well defined peaks, corresponding to St ≈ 0.11 and its few harmonics, as opposed to the broadband spectrum found at the wake.

Use of the iPhone for Cobb angle measurement in scoliosis

Purpose: The Cobb technique is the universally accepted method for measuring the severity of spinal deformities. Traditionally, Cobb angles have been measured using protractor and pencil on hardcopy radiographic films. The new generation of mobile phones make accurate angle measurement possible using an integrated accelerometer, providing a potentially useful clinical tool for assessing Cobb angles. The purpose of this study was to compare Cobb angle measurements performed using an Apple iPhone and traditional protractor in a series of twenty Adolescent Idiopathic Scoliosis patients. Methods: Seven observers measured major Cobb angles on twenty pre-operative postero-anterior radiographs of Adolescent Idiopathic Scoliosis patients with both a standard protractor and using an Apple iPhone. Five of the observers repeated the measurements at least a week after the original measurements. Results: The mean absolute difference between pairs of iPhone/protractor measurements was 2.1°, with a small (1°) bias toward lower Cobb angles with the iPhone. 95% confidence intervals for intra-observer variability were ±3.3° for the protractor and ±3.9° for the iPhone. 95% confidence intervals for inter-observer variability were ±8.3° for the iPhone and ±7.1° for the protractor. Both of these confidence intervals were within the range of previously published Cobb measurement studies. Conclusions: We conclude that the iPhone is an equivalent Cobb measurement tool to the manual protractor, and measurement times are about 15% less. The widespread availability of inclinometer-equipped mobile phones and the ability to store measurements in later versions of the angle measurement software may make these new technologies attractive for clinical measurement applications.

The governance of inter-organisational decision-making : understanding the impacts of informal networks on formal decision-making regimes in the Brisbane Airport region

Airports and cities inevitably recognise the value that each brings the other; however, the separation in decision-making authority for what to build, where, when and how provides a conundrum for both parties. Airports often want a say in what is developed outside of the airport fence, and cities often want a say in what is developed inside the airport fence. Defining how much of a say airports and cities have in decisions beyond their jurisdictional control is likely to be a topic that continues so long as airports and cities maintain separate formal decision-making processes for what to build, where, when and how. However, the recent Green and White Papers for a new National Aviation Policy have made early inroads to formalising relationships between Australia’s major airports and their host cities. At present, no clear indication (within practice or literature) is evident to the appropriateness of different governance arrangements for decisions to develop in situations that bring together the opposing strategic interests of airports and cities; thus leaving decisions for infrastructure development as complex decision-making spaces that hold airport and city/regional interests at stake. The line of enquiry is motivated by a lack of empirical research on networked decision-making domains outside of the realm of institutional theorists (Agranoff & McGuire, 2001; Provan, Fish & Sydow, 2007). That is, governance literature has remained focused towards abstract conceptualisations of organisation, without focusing on the minutia of how organisation influences action in real-world applications. A recent study by Black (2008) has provided an initial foothold for governance researchers into networked decision-making domains. This study builds upon Black’s (2008) work by aiming to explore and understand the problem space of making decisions subjected to complex jurisdictional and relational interdependencies. That is, the research examines the formal and informal structures, relationships, and forums that operationalise debates and interactions between decision-making actors as they vie for influence over deciding what to build, where, when and how in airport-proximal development projects. The research mobilises a mixture of qualitative and quantitative methods to examine three embedded cases of airport-proximal development from a network governance perspective. Findings from the research provide a new understanding to the ways in which informal actor networks underpin and combine with formal decision-making networks to create new (or realigned) governance spaces that facilitate decision-making during complex phases of development planning. The research is timely, and responds well to Isett, Mergel, LeRoux, Mischen and Rethemeyer’s (2011) recent critique of limitations within current network governance literature, specifically to their noted absence of empirical studies that acknowledge and interrogate the simultaneity of formal and informal network structures within network governance arrangements (Isett et al., 2011, pp. 162-166). The combination of social network analysis (SNA) techniques and thematic enquiry has enabled findings to document and interpret the ways in which decision-making actors organise to overcome complex problems for planning infrastructure. An innovative approach to using association networks has been used to provide insights to the importance of the different ways actors interact with one another, thus providing a simple yet valuable addition to the increasingly popular discipline of SNA. The research also identifies when and how different types of networks (i.e. formal and informal) are able to overcome currently known limitations to network governance (see McGuire & Agranoff, 2011), thus adding depth to the emerging body of network governance literature surrounding limitations to network ways of working (i.e. Rhodes, 1997a; Keast & Brown, 2002; Rethemeyer & Hatmaker, 2008; McGuire & Agranoff, 2011). Contributions are made to practice via the provision of a timely understanding of how horizontal fora between airports and their regions are used, particularly in the context of how they reframe the governance of decision-making for airport-proximal infrastructure development. This new understanding will enable government and industry actors to better understand the structural impacts of governance arrangements before they design or adopt them, particularly for factors such as efficiency of information, oversight, and responsiveness to change.

Supine to standing Cobb Angle change in idiopathic scoliosis: The effect of endplate preselection

Introduction Standing radiographs are the ‘gold standard’ for clinical assessment of adolescent idiopathic scoliosis (AIS), with the Cobb Angle used to measure the severity and progression of the scoliotic curve. Supine imaging modalities can provide valuable 3D information on scoliotic anatomy, however, due to changes in gravitational loading direction, the geometry of the spine alters between the supine and standing position which in turn affects the Cobb Angle measurement. Previous studies have consistently reported a 7-10° [1-3] Cobb Angle increase from supine to standing, however, none have reported the effect of endplate pre-selection and which (if any) curve parameters affect the supine to standing Cobb Angle difference. Methods Female AIS patients with right-sided thoracic major curves were included in the retrospective study. Clinically measured Cobb Angles from existing standing coronal radiographs and fulcrum bending radiographs [4] were compared to existing low-dose supine CT scans taken within 3 months of the reference radiograph. Reformatted coronal CT images were used to measure Cobb Angle variability with and without endplate pre-selection (end-plates selected on the radiographs used on the CT images). Inter and intra-observer measurement variability was assessed. Multi-linear regression was used to investigate whether there was a relationship between supine to standing Cobb Angle change and patient characteristics (SPSS, v.21, IBM, USA). Results Fifty-two patients were included, with mean age of 14.6 (SD 1.8) years; all curves were Lenke Type 1 with mean Cobb Angle on supine CT of 42° (SD 6.4°) and 52° (SD 6.7°) on standing radiographs. The mean fulcrum bending Cobb Angle for the group was 22.6° (SD 7.5°). The 10° increase from supine to standing is consistent with existing literature. Pre-selecting vertebral endplates was found to increase the Cobb Angle difference by a mean 2° (range 0-9°). Multi-linear regression revealed a statistically significant relationship between supine to standing Cobb Angle change with: fulcrum flexibility (p=0.001), age (p=0.027) and standing Cobb Angle (p<0.001). In patients with high fulcrum flexibility scores, the supine to standing Cobb Angle change was as great as 20°.The 95% confidence intervals for intra-observer and inter-observer measurement variability were 3.1° and 3.6°, respectively. Conclusion There is a statistically significant relationship between supine to standing Cobb Angle change and fulcrum flexibility. Therefore, this difference can be considered a measure of spinal flexibility. Pre-selecting vertebral endplates causes only minor changes.

Supine to standing Cobb angle change in idiopathic scoliosis : the effect of endplate pre-selection

Background Supine imaging modalities provide valuable 3D information on scoliotic anatomy, but the altered spine geometry between the supine and standing positions affects the Cobb angle measurement. Previous studies report a mean 7°-10° Cobb angle increase from supine to standing, but none have reported the effect of endplate pre-selection or whether other parameters affect this Cobb angle difference. Methods Cobb angles from existing coronal radiographs were compared to those on existing low-dose CT scans taken within three months of the reference radiograph for a group of females with adolescent idiopathic scoliosis. Reformatted coronal CT images were used to measure supine Cobb angles with and without endplate pre-selection (end-plates selected from the radiographs) by two observers on three separate occasions. Inter and intra-observer measurement variability were assessed. Multi-linear regression was used to investigate whether there was a relationship between supine to standing Cobb angle change and eight variables: patient age, mass, standing Cobb angle, Risser sign, ligament laxity, Lenke type, fulcrum flexibility and time delay between radiograph and CT scan. Results Fifty-two patients with right thoracic Lenke Type 1 curves and mean age 14.6 years (SD 1.8) were included. The mean Cobb angle on standing radiographs was 51.9° (SD 6.7). The mean Cobb angle on supine CT images without pre-selection of endplates was 41.1° (SD 6.4). The mean Cobb angle on supine CT images with endplate pre-selection was 40.5° (SD 6.6). Pre-selecting vertebral endplates increased the mean Cobb change by 0.6° (SD 2.3, range −9° to 6°). When free to do so, observers chose different levels for the end vertebrae in 39% of cases. Multi-linear regression revealed a statistically significant relationship between supine to standing Cobb change and fulcrum flexibility (p = 0.001), age (p = 0.027) and standing Cobb angle (p < 0.001). The 95% confidence intervals for intra-observer and inter-observer measurement variability were 3.1° and 3.6°, respectively. Conclusions Pre-selecting vertebral endplates causes minor changes to the mean supine to standing Cobb change. There is a statistically significant relationship between supine to standing Cobb change and fulcrum flexibility such that this difference can be considered a potential alternative measure of spinal flexibility.

Solution NMR studies of acetohydroxy acid synthase I: Identification of the sites of inter-subunit interactions using multidimensional NMR methods

The novel multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been dissected to generate polypeptide fragments. These fragments when cloned, expressed and purified reassemble in the presence of cofactors to yield a catalytically competent enzyme. Structural characterization of AHAS has been impeded due to the fact that the holoenzyme is prone to dissociation leading to heterogeneity in samples. Our approach has enabled the structural characterization using high-resolution nuclear magnetic resonance methods. Near complete sequence specific NMR assignments for backbone H-N, N-15, C-13 alpha and C-13(beta) atoms of the FAD binding domain of ilvB have been obtained on samples isotopically enriched in H-2, C-13 and N-15. The secondary structure determined on the basis of observed C-13(alpha) secondary chemical shifts and sequential NOEs indicates that the secondary structure of the FAD binding domain of E. coli AHAS large Subunit (ilvB) is similar to the structure of this domain in the catalytic subunit of yeast AHAS. Protein-protein interactions involving the regulatory subunit (ilvN) and the domains of the catalytic subunit (ilvB) were studied using circular dichroic and isotope edited solution nuclear magnetic resonance spectroscopic methods. Observed changes in circular dichroic spectra indicate that the regulatory subunit (ilvN) interacts with ilvB alpha and ilvB beta domains of the catalytic subunit and not with the ilvB gamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvB beta and proximal to the intrasubunit ilvB alpha/ilvB beta domain interface. The implication of this interaction on the role of the regulatory subunit oil the activity of the holoenzyme is discussed. NMR studies of the regulatory domains show that these domains are structured in solution. Preliminary evidence for the interaction of ilvN with the metabolic end product of the pathway, viz., valine is also presented.

Inter-domain interactions influence the stability and catalytic activity of the bi-domain protein tyrosine phosphatase PTP99A

The two protein tyrosine phosphatase (PTP) domains in bi-domain PTPs share high sequence and structural similarity. However, only one of the two PIP domains is catalytically active. Here we describe biochemical studies on the two tandem PTP domains of the bi-domain PTP, PTP99A. Phosphatase activity, monitored using small molecule as well as peptide substrates, revealed that the inactive (D2) domain activates the catalytic (D1) domain. Thermodynamic measurements suggest that the inactive D2 domain stabilizes the bi-domain (D1-D2) protein. The mechanism by which the D2 domain activates and stabilizes the bi-domain protein is governed by few interactions at the inter-domain interface. In particular, mutating Lys990 at the interface attenuates inter-domain communication. This residue is located at a structurally equivalent location to the so-called allosteric site of the canonical single domain PIP, PTP1B. These observations suggest functional optimization in bi-domain PTPs whereby the inactive PTP domain modulates the catalytic activity of the bi-domain enzyme. (C) 2012 Elsevier B.V. All rights reserved.