Structure of a low-population intermediate state in the release of an enzyme product.

Enzymes can increase the rate of biomolecular reactions by several orders of magnitude. Although the steps of substrate capture and product release are essential in the enzymatic process, complete atomic-level descriptions of these steps are difficult to obtain because of the transient nature of the intermediate conformations, which makes them largely inaccessible to standard structure determination methods. We describe here the determination of the structure of a low-population intermediate in the product release process by human lysozyme through a combination of NMR spectroscopy and molecular dynamics simulations. We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilise it, thus slowing down or speeding up, respectively, product release. These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and product

Nature and Regulation of Protein Folding on the Ribosome

Co-translational protein folding is an essential process by which cells ensure the safe and efficient production and assembly of new proteins in their functional native states following biosynthesis on the ribosome. In this review, we describe recent progress in probing the changes during protein synthesis of the free energy landscapes that underlie co-translational folding and discuss the critical coupling between these landscapes and the rate of translation that ultimately determines the success or otherwise of the folding process. Recent developments have revealed a variety of mechanisms by which both folding and translation can be modulated or regulated, and we discuss how these effects are utilised by the cell to optimise the outcome of protein biosynthesis

Particle-Based Monte-Carlo Simulations of Steady-State Mass Transport at Intermediate Péclet Numbers

Conventional approaches for simulating steady-state distributions of dilute particles under diffusive and advective transport involve solving the diffusion and advection equations in at least two dimensions. Here, we present an alternative computational strategy by combining a particle-based rather than a field-based approach with the initialisation of particles in proportion to their flux. This method allows accurate prediction of the steady state and is applicable even at intermediate and high Péclet numbers (Pe>1) swhere traditional particle-based Monte-Carlo methods starting from randomly initialised particle distributions fail. We demonstrate that generating a flux of particles according to a predetermined density and velocity distribution at a single fixed time and initial location allows for accurate simulation of mass transport under flow. Specifically, upon initialisation in proportion to their flux, these particles are propagated individually and detected by summing up their Monte-Carlo trajectories in predefined detection regions. We demonstrate quantitative agreement of the predicted concentration profiles with the results of experiments performed with fluorescent particles in microfluidic channels under continuous flow. This approach is computationally advantageous and readily allows non-trivial initial distributions to be considered. In particular, this method is highly suitable for simulating advective and diffusive transport in microfluidic devices, for instance in the context of diffusive sizing.Financial support from the Biotechnology and Biological Sciences Research Council (BBSRC), the European Research Council (ERC), the Frances and Augustus Newman Foundation as well as the Swiss National Science Foundation is gratefully acknowledged

An Environmentally Sensitive Fluorescent Dye as a Multidimensional Probe of Amyloid Formation

We have explored amyloid formation using poly(amino acid) model systems in which differences in peptide secondary structure and hydrophobicity can be introduced in a controlled manner. We show that an environmentally sensitive fluorescent dye, dapoxyl, is able to identify β-sheet structure and hydrophobic surfaces, structural features likely to be related to toxicity, as a result of changes in its excitation and emission profiles and its relative quantum yield. These results show that dapoxyl is a multidimensional probe of the time dependence of amyloid aggregation, which provides information about the presence and nature of metastable aggregation intermediates that is inaccessible to the conventional probes that rely on changes in quantum yield alone.The authors acknowledge the European Research Council (ERC), Biotechnology and Biological Sciences Research Council (BBSRC), Wellcome Trust, and Winston Churchill Foundation for financial support

Responsibility modelling for civil emergency planning

This paper presents a new approach to analysing and understanding civil emergency planning based on the notion of responsibility modelling combined with HAZOPS-style analysis of information requirements. Our goal is to represent complex contingency plans so that they can be more readily understood, so that inconsistencies can be highlighted and vulnerabilities discovered. In this paper, we outline the framework for contingency planning in the United Kingdom and introduce the notion of responsibility models as a means of representing the key features of contingency plans. Using a case study of a flooding emergency, we illustrate our approach to responsibility modelling and suggest how it adds value to current textual contingency plans

The extreme vulnerability of interdependent spatially embedded networks

Recent studies show that in interdependent networks a very small failure in one network may lead to catastrophic consequences. Above a critical fraction of interdependent nodes, even a single node failure can invoke cascading failures that may abruptly fragment the system, while below this "critical dependency" (CD) a failure of few nodes leads only to small damage to the system. So far, the research has been focused on interdependent random networks without space limitations. However, many real systems, such as power grids and the Internet, are not random but are spatially embedded. Here we analytically and numerically analyze the stability of systems consisting of interdependent spatially embedded networks modeled as lattice networks. Surprisingly, we find that in lattice systems, in contrast to non-embedded systems, there is no CD and \textit{any} small fraction of interdependent nodes leads to an abrupt collapse. We show that this extreme vulnerability of very weakly coupled lattices is a consequence of the critical exponent describing the percolation transition of a single lattice. Our results are important for understanding the vulnerabilities and for designing robust interdependent spatial embedded networks.Comment: 13 pages, 5 figure

Cross infection control measures and the treatment of patients at risk of Creutzfeldt Jakob disease in UK general dental practice

AIMS: To determine the suitability of key infection control measures currently employed in UK dental practice for delivery of dental care to patients at risk of prion diseases. MATERIALS AND METHODS: Subjects: Five hundred dental surgeons currently registered with the General Dental Council of the UK. Data collection: Structured postal questionnaire. Analysis: Frequencies, cross-tabulations and chi-squared analysis. RESULTS: The valid response rate to the questionnaire was 69%. 33% of practices had no policy on general disinfection and sterilisation procedures. Only 10 of the 327 responding practices (3%) possessed a vacuum autoclave. 49% of dentists reported using the BDA medical history form but less than 25% asked the specific questions recommended by the BDA to identify patients at risk of iatrogenic or familial CJD. However, 63% of practitioners would refer such patients, if identified, to a secondary care facility. Of the 107 practitioners who were prepared to provide dental treatment, 75 (70%) would do so using routine infection control procedures. CONCLUSIONS: Most of the dental practices surveyed were not actively seeking to identify patients at risk of prion diseases. In many cases, recommended procedures for providing safe dental care for such patients were not in place

The Significance of the Location of Mutations for the Native-State Dynamics of Human Lysozyme

The conversion of human lysozyme into amyloid fibrils is associated with a rare but fatal hereditary form of nonneuropathic systemic amyloidosis. The accumulation of large amounts of aggregated protein is thought to be initiated by the formation of transient intermediate species of disease-related lysozyme variants, essentially due to the loss of global cooperativity under physiologically relevant conditions. Interestingly, all five naturally occurring, amyloidogenic, single-point mutations are located in the β-domain of lysozyme, the region that is predominantly unfolded during the formation of the transient intermediate species. Given the lack of known naturally occurring, amyloidogenic, single-point mutations in the α-domain, we chose three specific mutations to address the effects that location may have on native-state dynamics, as studied by hydrogen-deuterium (HD) exchange experiments analyzed by NMR spectroscopy, and mass spectrometry. We compared the effect of a destabilizing α-domain mutation (I23A) with that of the well-characterized I59T β-domain variant. We also investigated the effect of a mutation that has minor effects on native-state stability at the domain interface (I56V) and compared it with that of a variant with similar stability within the C-helix (I89V). We show that when variants have similar reduced native-state stabilities, the location of the mutation (I23A versus I59T) is crucial to the native-state dynamics, with the α-domain mutation having a significantly lower ability to populate transient intermediate species under physiologically relevant conditions. Interestingly, the mutation at the interface (I56V) has a greater effect in facilitating the formation of transient intermediate species at elevated temperatures compared with the variants containing α-domain mutations, even though this mutation results in only minor changes to the native-state stability of lysozyme. These findings reveal that the location of specific mutations is an important factor in determining the native-state dynamical properties of human lysozyme in the context of its propensity to populate the aggregation-prone transient intermediate species associated with pathogenic amyloid formation.This research was supported by the Biotechnology and Biological Sciences Research Council (BB/E019927/1 to C.M.D., C.V.R., and J.R.K.), the Medical Research Council (E.D.G. and C.M.D.), the Belgian Program of Interuniversity Attraction Poles administered by the Federal Office for Scientific Technical and Cultural Affairs (PAI numbers P6/19 and P7144 to C.M.D. and M.D.), the European Union’s Sixth Framework Program (LSHM-CT-2006-037525 to C.M.D. and M.D.), and Programme grants from the Wellcome Trust and the Leverhulme Trust (C.M.D.). It was also supported by a Korean Government Scholarship for Overseas Studies (M.A.), the Winston Churchill Foundation (C.L.H.), and Boerhinger Ingleheim funds (A.D.). The NMR facility at the Department of Chemistry, University of Cambridge, is supported in part by an EPSRC Core Capability grant (EP/K039520/1)