Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state

Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles

The stability, activity, and solubility of a protein sequence are determined by a delicate balance of molecular interactions in a variety of conformational states. Even so, most computational protein design methods model sequences in the context of a single native conformation. Simulations that model the native state as an ensemble have been mostly neglected due to the lack of sufficiently powerful optimization algorithms for multistate design. Here, we have applied our multistate design algorithm to study the potential utility of various forms of input structural data for design. To facilitate a more thorough analysis, we developed new methods for the design and high-throughput stability determination of combinatorial mutation libraries based on protein design calculations. The application of these methods to the core design of a small model system produced many variants with improved thermodynamic stability and showed that multistate design methods can be readily applied to large structural ensembles. We found that exhaustive screening of our designed libraries helped to clarify several sources of simulation error that would have otherwise been difficult to ascertain. Interestingly, the lack of correlation between our simulated and experimentally measured stability values shows clearly that a design procedure need not reproduce experimental data exactly to achieve success. This surprising result suggests potentially fruitful directions for the improvement of computational protein design technology

Ab initio molecular dynamics using density based energy functionals: application to ground state geometries of some small clusters

The ground state geometries of some small clusters have been obtained via ab initio molecular dynamical simulations by employing density based energy functionals. The approximate kinetic energy functionals that have been employed are the standard Thomas-Fermi $(T_{TF})$ along with the Weizsacker correction $T_W$ and a combination $F(N_e)T_{TF} + T_W$. It is shown that the functional involving $F(N_e)$ gives superior charge densities and bondlengths over the standard functional. Apart from dimers and trimers of Na, Mg, Al, Li, Si, equilibrium geometries for $Li_nAl, n=1,8$ and $Al_{13}$ clusters have also been reported. For all the clusters investigated, the method yields the ground state geometries with the correct symmetries with bondlengths within 5\% when compared with the corresponding results obtained via full orbital based Kohn-Sham method. The method is fast and a promising one to study the ground state geometries of large clusters.Comment: 15 pages, 3 PS figure

Forecasting the Impacts of Silver and Bighead Carp on the Lake Erie Food Web

Nonindigenous bigheaded carps (Bighead Carp Hypophthalmichthys nobilis and Silver Carp H. molitrix; hereafter, “Asian carps” [AC]) threaten to invade and disrupt food webs and fisheries in the Laurentian Great Lakes through their high consumption of plankton. To quantify the potential effects of AC on the food web in Lake Erie, we developed an Ecopath with Ecosim (EwE) food web model and simulated four AC diet composition scenarios (high, low, and no detritus and low detritus with Walleye Sander vitreus and Yellow Perch Perca flavescens larvae) and two nutrient load scenarios (the 1999 baseline load and 2× the baseline [HP]). We quantified the uncertainty of the potential AC effects by coupling the EwE model with estimates of parameter uncertainty in AC production, consumption, and predator diets obtained using structured expert judgment. Our model projected mean ± SD AC equilibrium biomass ranging from 52 ± 34 to 104 ± 75 kg/ha under the different scenarios. Relative to baseline simulations without AC, AC invasion under all detrital diet scenarios decreased the biomass of most fish and zooplankton groups. The effects of AC in the HP scenario were similar to those in the detrital diet scenarios except that the biomasses of most Walleye and Yellow Perch groups were greater under HP because these fishes were buffered from competition with AC by increased productivity at lower trophic levels. Asian carp predation on Walleye and Yellow Perch larvae caused biomass declines among all Walleye and Yellow Perch groups. Large food web impacts of AC occurred in only 2% of the simulations, where AC biomass exceeded 200 kg/ha, resulting in biomass declines of zooplankton and planktivorous fish near the levels observed in the Illinois River. Our findings suggest that AC would affect Lake Erie's food web by competing with other planktivorous fishes and by providing additional prey for piscivores. Our methods provide a novel approach for including uncertainty into forecasts of invasive species' impacts on aquatic food webs. Received December 6, 2014; accepted July 15, 201

Four small puzzles that Rosetta doesn't solve

A complete macromolecule modeling package must be able to solve the simplest structure prediction problems. Despite recent successes in high resolution structure modeling and design, the Rosetta software suite fares poorly on deceptively small protein and RNA puzzles, some as small as four residues. To illustrate these problems, this manuscript presents extensive Rosetta results for four well-defined test cases: the 20-residue mini-protein Trp cage, an even smaller disulfide-stabilized conotoxin, the reactive loop of a serine protease inhibitor, and a UUCG RNA tetraloop. In contrast to previous Rosetta studies, several lines of evidence indicate that conformational sampling is not the major bottleneck in modeling these small systems. Instead, approximations and omissions in the Rosetta all-atom energy function currently preclude discriminating experimentally observed conformations from de novo models at atomic resolution. These molecular "puzzles" should serve as useful model systems for developers wishing to make foundational improvements to this powerful modeling suite.Comment: Published in PLoS One as a manuscript for the RosettaCon 2010 Special Collectio

Forecasting the Impacts of Silver and Bighead Carp on the Lake Erie Food Web

Nonindigenous bigheaded carps (Bighead Carp Hypophthalmichthys nobilis and Silver Carp H. molitrix; hereafter, “Asian carps” [AC]) threaten to invade and disrupt food webs and fisheries in the Laurentian Great Lakes through their high consumption of plankton. To quantify the potential effects of AC on the food web in Lake Erie, we developed an Ecopath with Ecosim (EwE) food web model and simulated four AC diet composition scenarios (high, low, and no detritus and low detritus with Walleye Sander vitreus and Yellow Perch Perca flavescens larvae) and two nutrient load scenarios (the 1999 baseline load and 2× the baseline [HP]). We quantified the uncertainty of the potential AC effects by coupling the EwE model with estimates of parameter uncertainty in AC production, consumption, and predator diets obtained using structured expert judgment. Our model projected mean ± SD AC equilibrium biomass ranging from 52 ± 34 to 104 ± 75 kg/ha under the different scenarios. Relative to baseline simulations without AC, AC invasion under all detrital diet scenarios decreased the biomass of most fish and zooplankton groups. The effects of AC in the HP scenario were similar to those in the detrital diet scenarios except that the biomasses of most Walleye and Yellow Perch groups were greater under HP because these fishes were buffered from competition with AC by increased productivity at lower trophic levels. Asian carp predation on Walleye and Yellow Perch larvae caused biomass declines among all Walleye and Yellow Perch groups. Large food web impacts of AC occurred in only 2% of the simulations, where AC biomass exceeded 200 kg/ha, resulting in biomass declines of zooplankton and planktivorous fish near the levels observed in the Illinois River. Our findings suggest that AC would affect Lake Erie's food web by competing with other planktivorous fishes and by providing additional prey for piscivores. Our methods provide a novel approach for including uncertainty into forecasts of invasive species' impacts on aquatic food webs. Received December 6, 2014; accepted July 15, 201

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

The promiscuous activity of the enzyme o-succinylbenzoate synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both density functional theory and semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylamino acids, with N-succinyl-R-phenylglycine being the best substrate. We investigated the molecular mechanisms for both reactions exploring the potential energy surface. Then, molecular dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues with the reacting system. Our results confirm the plausibility of the reaction mechanisms proposed in the literature, with a good agreement between theoretical and experimentally derived activation free energies. Our simulations unravel the role played by the different residues in each of the two possible reactions. The presence of flexible loops in the active site and the selection of structural modifications in the substrate seem to be key elements to promote the promiscuity of this enzyme.This work was supported by the Spanish Ministerio de Economia y Competitividad project CTQ2012-36253-C03-03 ́ and FEDER funds. K.S. thanks the Polish National Science Center (NCN) for Grant 2011/02/A/ST4/00246. The authors acknowledge computational facilities of the Servei d’Informatica ̀ de la Universitat de Valencia in the ̀ “Tirant” supercomputer, which is part of the Spanish Supercomputing Network

Synthetic biology: Understanding biological design from synthetic circuits

An important aim of synthetic biology is to uncover the design principles of natural biological systems through the rational design of gene and protein circuits. Here, we highlight how the process of engineering biological systems — from synthetic promoters to the control of cell–cell interactions — has contributed to our understanding of how endogenous systems are put together and function. Synthetic biological devices allow us to grasp intuitively the ranges of behaviour generated by simple biological circuits, such as linear cascades and interlocking feedback loops, as well as to exert control over natural processes, such as gene expression and population dynamics

Shells and humans: molluscs and other coastal resources from the earliest human occupations at the Mesolithic shell midden of El Mazo (Asturias, Northern Spain)

Human populations exploited coastal areas with intensity during the Mesolithic in Atlantic Europe, resulting in the accumulation of large shell middens. Northern Spain is one of the most prolific regions, and especially the so-called Asturian area. Large accumulations of shellfish led some scholars to propose the existence of intensification in the exploitation of coastal resources in the region during the Mesolithic. In this paper, shell remains (molluscs, crustaceans and echinoderms) from stratigraphic units 114 and 115 (dated to the early Mesolithic c. 9 kys cal BP) at El Mazo cave (Asturias, northern Spain) were studied in order to establish resource exploitation patterns and environmental conditions. Species representation showed that limpets, top shells and sea urchins were preferentially exploited. One-millimetre mesh screens were crucial in establishing an accurate minimum number of individuals for sea urchins and to determine their importance in exploitation patterns. Environmental conditions deduced from shell assemblages indicated that temperate conditions prevailed at the time of the occupation and the morphology of the coastline was similar to today (rocky exposed shores). Information recovered relating to species representation, collection areas and shell biometry reflected some evidence of intensification (reduced shell size, collection in lower areas of exposed shores, no size selection in some units and species) in the exploitation of coastal resources through time. However, the results suggested the existence of changes in collection strategies and resource management, and periods of intense shell collection may have alternated with times of shell stock recovery throughout the Mesolithic.This research was performed as part of the project “The human response to the global climatic change in a littoral zone: the case of the transition to the Holocene in the Cantabrian coast (10,000–5000 cal BC) (HAR2010-22115-C02-01)” funded by the Spanish Ministry of Economy and Competitiveness. AGE was funded by the University of Cantabria through a predoctoral grant and IGZ was funded by the Spanish Ministry of Economy and Competitiveness through a Juan de la Cierva grant. We also would like to thank the University of Cantabria and the IIIPC for providing support, David Cuenca-Solana, Alejandro García Moreno and Lucia Agudo Pérez for their help. We also thank Jennifer Jones for correcting the English. Comments from two anonymous reviewers helped to improve the paper

The Role of Tourism and Recreation in the Spread of Non-Native Species: A Systematic Review and Meta-Analysis

Managing the pathways by which non-native species are introduced and spread is considered the most effective way of preventing species invasions. Tourism and outdoor recreation involve the frequent congregation of people, vehicles and vessels from geographically diverse areas. They are therefore perceived to be major pathways for the movement of non-native species, and ones that will become increasingly important with the continued growth of these sectors. However, a global assessment of the relationship between tourism activities and the introduction of non-native species–particularly in freshwater and marine environments–is lacking. We conducted a systematic review and meta-analysis to determine the impact of tourism and outdoor recreation on non-native species in terrestrial, marine and freshwater environments. Our results provide quantitative evidence that the abundance and richness of non-native species are significantly higher in sites where tourist activities take place than in control sites. The pattern was consistent across terrestrial, freshwater and marine environments; across a variety of vectors (e.g. horses, hikers, yachts); and across a range of taxonomic groups. These results highlight the need for widespread biosecurity interventions to prevent the inadvertent introduction of invasive non-native species (INNS) as the tourism and outdoor recreation sectors grow