18 research outputs found
Structural Heterogeneity and Quantitative FRET Efficiency Distributions of Polyprolines through a Hybrid Atomistic Simulation and Monte Carlo Approach
Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photon kinetics, provide a versatile tool to improve the accuracy of distance distributions that can be extracted from measured single molecule FRET efficiencies
Learning from fluorescence decay dynamics: A better read out from the spectroscopic ruler
The spectroscopic ruler is a noninvasive technique exploiting fluorescence resonance energy transfer (FRET) to determine intramolecular distances on the nm scale in proteins and other bio-molecules [1,2]. It is widely applied in the life sciences where it is used in vitro and in vivo to probe the separation between fluorescent markers attached to the molecules under investigation. Because FRET scales strongly with the separation distance and orientation between the optical dipole moments of the donor and acceptor molecules, the accuracy of the technique is limited when the molecules undergoes thermal fluctuations under physiological conditions and the orientation of the optical dipoles is not static. © 2011 IEEE
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Education and training futures in horticulture and horticultural science
Horticultural knowledge and skills training have been with humankind for some 10,000 to 20,000 years. With permanent settlement and rising wealth and trade, horticulture products and services became a source of fresh food for daily consumption, and a source of plant material in developing a quality environment and lifestyle. The knowledge of horticulture and the skills of its practitioners have been demonstrated through the advancing civilizations in both eastern and western countries. With the rise of the Agricultural Revolutions in Great Britain, and more widely across Continental Europe in the 17th and 18th centuries, as well as the move towards colonisation and early migration to the New Worlds, many westernised countries established the early institutions that would provide education and training in agriculture and horticulture. Today many of these colleges and universities provide undergraduate, postgraduate and vocational and technical training that specifically targets horticulture and/or horticultural science with some research and teaching institutions also providing extension and advisory services to industry. The objective of this chapter is to describe the wider pedagogic and educational context in which those concerned with horticulture operate, the institutional structures that target horticulture and horticultural science education and training internationally; examine changing educational formats, especially distance education; and consider strategies for attracting and retaining young people in the delivery of world-class horticultural education. In this chapter we set the context by investigating the horticultural education and training options available, the constraints that prevent young people entering horticulture, and suggest strategies that would attract and retain these students. We suggest that effective strategies and partnerships be put in place by the institution, the government and most importantly the industry to provide for undergraduate and postgraduate education in horticulture and horticultural science; that educational and vocational training institutions, government, and industry need to work more effectively together to improve communication about horticulture and horticultural science in order to attract enrolments of more and talented students; and that the horticulture curriculum be continuously evaluated and revised so that it remains relevant to future challenges facing the industries of horticulture in the production, environmental and social spheres. These strategies can be used as a means to develop successful programs and case studies that would provide better information to high school career counsellors, improve the image of horticulture and encourage greater involvement from alumni and the industries in recruitment, provide opportunities to improve career aspirations, ensure improved levels of remuneration, and promote the social features of the profession and greater awareness and recognition of the profession in the wider community. A successful career in horticulture demands intellectual capacities which are capable of drawing knowledge from a wide field of basic sciences, economics and the humanities and integrating this into academic scholarship and practical technologies