Investigation of DMSO-Induced Conformational Transitions in Human Serum Albumin Using Two-Dimensional Raman Optical Activity Spectroscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Order-Disorder Transitions Govern Kinetic Cooperativity and Allostery of Monomeric Human Glucokinase

Glucokinase (GCK) catalyzes the rate-limiting step of glucose catabolism in the pancreas, where it functions as the body's principal glucose sensor. GCK dysfunction leads to several potentially fatal diseases including maturity-onset diabetes of the young type II (MODY-II) and persistent hypoglycemic hyperinsulinemia of infancy (PHHI). GCK maintains glucose homeostasis by displaying a sigmoidal kinetic response to increasing blood glucose levels. This positive cooperativity is unique because the enzyme functions exclusively as a monomer and possesses only a single glucose binding site. Despite nearly a half century of research, the mechanistic basis for GCK's homotropic allostery remains unresolved. Here we explain GCK cooperativity in terms of large-scale, glucose-mediated disorder-order transitions using 17 isotopically labeled isoleucine methyl groups and three tryptophan side chains as sensitive nuclear magnetic resonance (NMR) probes. We find that the small domain of unliganded GCK is intrinsically disordered and samples a broad conformational ensemble. We also demonstrate that small-molecule diabetes therapeutic agents and hyperinsulinemia-associated GCK mutations share a strikingly similar activation mechanism, characterized by a population shift toward a more narrow, well-ordered ensemble resembling the glucose-bound conformation. Our results support a model in which GCK generates its cooperative kinetic response at low glucose concentrations by using a millisecond disorder-order cycle of the small domain as a "time-delay loop," which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions.

The Role of Molecular Conformation and Polarizable Embedding for One- and Two-Photon Absorption of Disperse Orange 3 in Solution

Solvent effects on the one- and two-photon absorption (IPA and 2PA) of disperse orange 3 (DO3) in dimethyl sulfoxide (DMSO) are studied using a discrete polarizable embedding (PE) response theory. The scheme comprises a quantum region containing the chromophore and an atomically granulated classical region for the solvent accounting for full interactions within and between the two regions. Either classical molecular dynamics (MD) or hybrid Car-Parrinello (CP) quantum/classical (QM/MM) molecular dynamics simulations are employed to describe the solvation of DO3 in DMSO, allowing for an analysis of the effect of the intermolecular short-range repulsion, long-range attraction, and electrostatic interactions on the conformational changes of the chromophore and also the effect of the solute-solvent polarization. PE linear response calculations are performed to verify the character, solvatochromic shift, and overlap of the two lowest energy transitions responsible for the linear absorption spectrum of DO3 in DMSO in the visible spectral region. Results of the PE linear and quadratic response calculations, performed using uncorrelated solute-solvent configurations sampled from either the classical or hybrid CP QM/MM MD simulations, are used to estimate the width of the line shape function of the two electronic lowest energy excited states, which allow a prediction of the 2PA cross-sections without the use of empirical parameters. Appropriate exchange-correlation functionals have been employed in order to describe the charge-transfer process following the electronic transitions of the chromophore in solution.

P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity

The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp.

Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into β-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by β-strand exchange between the reactive loop of one molecule and β-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin α1-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into β-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma α1-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55–Pro mutation.

Managing infrastructure transitions : a complex adaptive systems perspective

Engineering assets such as roads, rail, bridges and other forms of public works are vital to the effective functioning of societies {Herder, 2006 #128}. Proficient provision of this physical infrastructure is therefore one of the key activities of government {Lædre, 2006 #123}. In order to ensure engineering assets are procured and maintained on behalf of citizens, government needs to devise the appropriate policy and institutional architecture for this purpose. The changing institutional arrangements around the procurement of engineering assets are the focus of this paper. The paper describes and analyses the transition to new, more collaborative forms of procurement arrangements which are becoming increasingly prevalent in Australia and other OECD countries. Such fundamental shifts from competitive to more collaborative approaches to project governance can be viewed as a major transition in procurement system arrangements. In many ways such changes mirror the shift from New Public Management, with its emphasis on the use of market mechanisms to achieve efficiencies {Hood, 1991 #166}, towards more collaborative approaches to service delivery, such as those under network governance arrangements {Keast, 2007 #925}. However, just as traditional forms of procurement in a market context resulted in unexpected outcomes for industry, such as a fragmented industry afflicted by chronic litigation {Dubois, 2002 #9}, the change to more collaborative forms of procurement is unlikely to be a panacea to the problems of procurement, and may well also have unintended consequences. This paper argues that perspectives from complex adaptive systems (CAS) theory can contribute to the theory and practice of managing system transitions. In particular the concept of emergence provides a key theoretical construct to understand the aggregate effect that individual project governance arrangements can have upon the structure of specific industries, which in turn impact individual projects. Emergence is understood here as the macro structure that emerges out of the interaction of agents in the system {Holland, 1998 #100; Tang, 2006 #51}.

Context-based chemistry : creating opportunities for fluid transitions between concepts and context

Nationally and internationally, context-based programs have been implemented in an attempt to engage students in chemistry through connecting the canonical science with the real-world. In Queensland, a context-based approach to chemistry was trialled in selected schools from 2002 but there is little research that investigates how students learn in a context-based setting. This paper presents one significant finding from an ethnographic study that explored the learning that occurred in an 11th grade context-based chemistry classroom in Queensland. The study found that by providing students with the opportunity to write, fluid transitions (or to-ing and fro-ing) between concepts and context were an outcome of context-based learning.

TAFE Governance Transitions Prophetical

Drama staff from QUT Creative Industries were commissioned by the Dept of Education, Training and the Arts (DETA) to devise a tailor made and highly targeted staff development program for TAFE Queensland Institute Directors and senior managers. The program was designed to assist these senior staff to address a range of change and governance issues in the context of TAFE Queensland’s transition to statutory authority status. In responding to this brief, the QUT team utilised an original applied performance technique known as a ‘Prophetical’ to research, devise and present an interactive case study to TAFE Institute Directors and senior managers. This particular Prophetical portrayed a senior staff team at the fictitious Massey Institute of TAFE, confronted by an escalating series of crises as they try to manage the transition to the fundamentally different business model of a statutory authority.

Generalised self-efficacy in relation to the life transitions of adult learners in a university setting : towards a narrative constructivist model of self-regulation

This paper demonstrates a model of self-regulation based on a qualitative research project with adult learners undertaking an undergraduate degree. The narrative about the participant’s life transitions, co-constructed with the researcher, yielded data about their generalised self-efficacy and resulted in a unique self-efficacy narrative for each participant. A model of self-regulation is proposed with potential applications for coaching, counselling and psychotherapy. A narrative method was employed to construct narratives about an individual’s self-efficacy in relation to their experience of learning and life transitions. The method involved a cyclical and iterative process using qualitative interviews to collect life history data from participants. In addition, research participants completed reflective homework tasks, and this data was included in the participant’s narratives. A highly collaborative method entailed narratives being co-constructed by researcher and research participants as the participants were guided in reflecting on their experience in relation to learning and life transitions; the reflection focused on behaviour, cognitions and emotions that constitute a sense of self-efficacy. The analytic process used was narrative analysis, in which life is viewed as constructed and experienced through the telling and retelling of stories and hence the analysis is the creation of a coherent and resonant story. The method of constructing self-efficacy narratives was applied to a sample of mature aged students starting an undergraduate degree. The research outcomes confirmed a three-factor model of self-efficacy, comprising three interrelated stages: initiating action, applying effort, and persistence in overcoming difficulties. Evaluation of the research process by participants suggested that they had gained an enhanced understanding of self-efficacy from their participation in the research process, and would be able to apply this understanding to their studies and other endeavours in the future. A model of self-regulation is proposed as a means for coaches, counsellors and psychotherapists working from a narrative constructivist perspective to assist clients facing life transitions by helping them generate selfefficacious cognitions, emotions and behaviour.

Transitions

Transitions represents a selection of works presented by 3rd year QUT design students undertaken as part of their coursework entitled "Environments in Transitions". The work focuses upon the migration of ideas and aesthetics from East to West, with particular consideration for the influence of Japanese woodblock prints, Wabi-Sabi themes and decorative motifs upon European design during the turn of the 19th century. The works exhibited included design artifacts of various themes, and associated print work which informed the design process. A small exhibition publication accompanied the exhibition and talk.

Education-to-work transitions of aspiring creatives

Despite some segments of the creative industries in Australia performing better than other segments in terms of earnings and employment growth, they all rely on highly skilled workers and face similar workforce challenges. Workers typically experience multiple entry attempts, spells of unemployment, short-term contracts, high degrees of mobility, casual/part-time employment within and outside the creative industries, and pressure to ensure their skills remain relevant. Skills shortages and gaps, an insufficient supply of high quality industry-ready graduates, difficulties in predicting demand for skills, weak linkages between industry and education providers, reliance on overseas talent in some segments, limited opportunities for workers to engage in skill development, and pressure on workers to keep abreast of technological developments are ongoing issues in the creative industries workforce. In response to these concerns, the Australian Research Council, three State Governments, industry, and a large vocational education and training (VET) provider funded Queensland University of Technology (QUT) to conduct the 60Sox project. This three-year project investigated the education, training, and work experiences of aspiring creatives defined as new entrants, recent graduates, and students enrolled in creative industries courses. It involved the largest survey of aspiring creatives ever undertaken in Australia, attracting 507 respondents, and a survey of 50 employers. Using the framework proposed by Hannan, Raffe, and Smyth (1996), this article presents findings from an analysis of the macro and micro labour market outcomes of aspiring creatives using data from the two 60Sox project surveys and publicly available sources. The analysis confirmed that many graduates of creative industries courses who participated in the 60Sox survey and the national surveys for the National Centre for Vocational Education Research and Graduate Careers Australia were struggling to make a successful transition from education to work. This article also discusses the causes of this key finding and possible solutions to address transition issues.

Kindergarten transitions and linkages to primary school-readiness reconceptualised

Young children’s transition into school has been constructed as a time-limited period around initial school entry, a set of teacher or school practices, a process of establishing continuity of experience, a multi-layered, multi-year set of experiences and a dynamic relationship-based process. Although preparedness issues continue to be addressed, there is a trend towards more complex understandings of transition emphasizing continuity, relationships amongst multiple stakeholders, system coherence across extended time periods and enhancement of resilience and transition capital. This article, in the early years of a new century, outlines some conceptualisations of readiness and transition as they relate to diverse children’s pathways through early childhood and early school settings.

Manifolds, patterns and transitions in a creative life

Using sculpture and drawing as my primary methods of investigation, this research explores ways of shifting the emphasis of my creative visual arts practice from object to process whilst still maintaining a primacy of material outcomes. My motivation was to locate ways of developing a sustained practice shaped as much by new works, as by a creative flow between works. I imagined a practice where a logic of structure within discrete forms and a logic of the broader practice might be developed as mutually informed processes. Using basic structural components of multiple wooden curves and linear modes of deployment – in both sculptures and drawings – I have identified both emergence theory and the image of rhizomic growth (Deleuze and Guattari, 1987) as theoretically integral to this imagining of a creative practice, both in terms of critiquing and developing works. Whilst I adopt a formalist approach for this exegesis, the emergence and rhizome models allow it to work as a critique of movement, of becoming and changing, rather than merely a formalism of static structure. In these models, therefore, I have identified a formal approach that can be applied not only to objects, but to practice over time. The thorough reading and application of these ontological models (emergence and rhizome) to visual arts practice, in terms of processes, objects and changes, is the primary contribution of this thesis. The works that form the major component of the research develop, reflect and embody these notions of movement and change.