Analytical ultracentrifugation as a tool for studying protein interactions

The last two decades have led to significant progress in the field of analytical ultracentrifugation driven by instrumental, theoretical, and computational methods. This review will highlight key developments in sedimentation equilibrium (SE) and sedimentation velocity (SV) analysis. For SE, this includes the analysis of tracer sedimentation equilibrium at high concentrations with strong thermodynamic non-ideality, and for ideally interacting systems, the development of strategies for the analysis of heterogeneous interactions towards global multi-signal and multi-speed SE analysis with implicit mass conservation. For SV, this includes the development and applications of numerical solutions of the Lamm equation, noise decomposition techniques enabling direct boundary fitting, diffusion deconvoluted sedimentation coefficient distributions, and multi-signal sedimentation coefficient distributions. Recently, effective particle theory has uncovered simple physical rules for the co-migration of rapidly exchanging systems of interacting components in SV. This has opened new possibilities for the robust interpretation of the boundary patterns of heterogeneous interacting systems. Together, these SE and SV techniques have led to new approaches to study macromolecular interactions across the entire spectrum of affinities, including both attractive and repulsive interactions, in both dilute and highly concentrated solutions, which can be applied to single-component solutions of self-associating proteins as well as the study of multi-protein complex formation in multi-component solutions.     

Understanding investment in biodiversity conservation in Mexico

Documenting financial resources in biodiversity conservation is a key aspect worldwide in order to set priorities and use effectively the limited resources available. In Mexico, a megadiverse country, studies on financial resources invested in biodiversity conservation are scarce and do not address funding for conservation comprehensively. Using recent data from several sources and applying criteria based on the national priorities for conservation, we compiled, systematized and analyzed data at a national scale on financing sources, financial resources and conservation organizations and their projects. The information obtained is presented in various ways and part of it (case study) is already an information system that can be continuously up-dated. Some of the results show the following: a steady diversification of mechanisms and methods for raising funds for conservation; an increase in governmental budgets; the acknowledgment by the private sector of the importance of biodiversity conservation; a greater technical capacity in people and organizations working in conservation; a greater accessibility of financial resources to support and maintain conservation projects; yet a short term vision in conservation projects; among other. Although the results obtained through this study are a first approach, they can now be used as a baseline to continue gathering and analyzing information on conservation financing in Mexico.     

Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition

Sedimentation equilibrium is a powerful tool for the characterization of protein self-association and heterogeneous protein interactions. Frequently, it is applied in a configuration with relatively long solution columns and with equilibrium profiles being acquired sequentially at several rotor speeds. The present study proposes computational tools, implemented in the software SEDPHAT, for the global analysis of equilibrium data at multiple rotor speeds with multiple concentrations and multiple optical detection methods. The detailed global modeling of such equilibrium data can be a nontrivial computational problem. It was shown previously that mass conservation constraints can significantly improve and extend the analysis of heterogeneous protein interactions. Here, a method for using conservation of mass constraints for the macromolecular redistribution is proposed in which the effective loading concentrations are calculated from the sedimentation equilibrium profiles. The approach is similar to that described by Roark (Biophys. Chem. 5 (1976) 185-196), but its utility is extended by determining the bottom position of the solution columns from the macromolecular redistribution. For analyzing heterogeneous associations at multiple protein concentrations, additional constraints that relate the effective loading concentrations of the different components or their molar ratio in the global analysis are introduced. Equilibrium profiles at multiple rotor speeds also permit the algebraic determination of radial-dependent baseline profiles, which can govern interference optical ultracentrifugation data, but usually also occur, to a smaller extent, in absorbance optical data. Finally, the global analysis of equilibrium profiles at multiple rotor speeds with implicit mass conservation and computation of the bottom of the solution column provides an unbiased scale for determining molar mass distributions of noninteracting species. The properties of these tools are studied with theoretical and experimental data sets.     

Analytical ultracentrifugation for the study of protein association and assembly

Analytical ultracentrifugation remains pre-eminent among the methods used to study the interactions of macromolecules under physiological conditions. Recent developments in analytical procedures allow the high resolving power of sedimentation velocity methods to be coupled to sedimentation equilibrium approaches and applied to both static and dynamic associations. Improvements in global modeling based on numerical solutions of the Lamm equation have generated new sedimentation velocity applications with an emphasis on data interpretation using sedimentation coefficient or molar mass distributions. Procedures based on the use of multiple optical signals from absorption and interference optics for the analysis of the sedimentation velocity and equilibrium behavior of more complex interactions have now been developed. New applications of tracer sedimentation equilibrium experiments and the development of a fluorescence optical system for the analytical ultracentrifuge extend the accessible concentration range over several orders of magnitude and, coupled with the new analytical procedures, provide powerful new tools for studies of both weak and strong macromolecular interactions in solution.     

Versatile vectors to study recoding: conservation of rules between yeast and mammalian cells.

In many viruses and transposons, expression of some genes requires alternative reading of the genetic code, also called recoding. Such events depend on specific mRNA sequences and can lead to read through of an in-frame stop codon or to +1 or -1 frameshifting. Here, we addressed the issue of conservation of recoding rules between the yeast Saccharomyces cerevisiae and mammalian cells by establishing a versatile vector that can be used to study recoding in both species. We first assessed this vector by analysing the site of +1 frameshift of the Ty1 transposon. Two sequences from higher organisms were then tested in both yeast and mammalian cells: the gag-pol junction of human immunodeficiency virus type 1 (HIV-1) (a site of -1 frameshift), and the stop codon region of the replicase cistron from the tobacco mosaic virus (a site of UAG read through). We show that both sequences direct a high level of recoding in yeast. Furthermore, different mutations of the target sequences have similar effects on recoding in yeast and in mouse cells. Most notably, a strong decrease of frameshifting was observed in the absence of the HIV-1 stem-loop stimulatory signal. Taken together, these data suggest that mechanisms of some recoding events are conserved between lower and higher eukaryotes, thus allowing the use of S. cerevisiae as a model system to study recoding on target sequences from higher organisms.     

Is time on our side? Strengthening the link between field efforts and conservation needs

The collection and organization of distributional data is the first crucial stage of any conservation planning action: therefore the decline in field research has implications in both the systematic, floristic and conservation fields. The aim of this paper is to analyze the effects of data updating on conservation planning and priorities. Focusing on the short time frame ranging from 2006 to 2011, we present a case study showing the rate of increase of collected data (taxa and records) and the consequential effects on the definition of areas of priority interest for plant conservation (Important Plant Areas—IPAs). We gathered data on a total of 193 taxa and 849 records with a mean rate of increase of +97 % for taxa and +166 % for records (2006/2011). This increase caused a positive rate of change in high ranking cells (+78 %) defining IPAs, while the number of low ranking cells and no data cells slightly decreased (?12 and ?8 %, respectively). Our results suggest that specific investment to complete the knowledge on the distribution of selected taxa (e.g. 193 taxa represent the 7.5 % of the total vascular flora of Sardinia) would dramatically reduce both the Linnean and Wallacean shortfalls and would allow robust conservation programs to preserve the diversity of the island. Updating the IPAs on a regular basis is a good example of a process that has a low impact as well as a big potential gain especially when field research can only be performed with low intensity and small monetary investments.     

Smallholder perceptions and communication gaps shape East African riparian ecosystems

Human livelihood needs and nature conservation often contradict. Yet, healthy ecosystems are crucial for human livelihood quality. The semi-arid regions of East Africa suffer under demographic pressure and soil depletion. Ecosystem degradation becomes particularly visible along rivers in semiarid regions of south-east Kenya, where former pristine riparian forests have been transformed into agricultural fields and settlements with negative effects on ecosystem services. In this study, we aim to understand how local smallholders perceive the challenges for the riparian ecosystems and what factors affect their engagement in environmental conservation. We surveyed about 200 farmers and performed expert interviews with representatives from governmental institutions from the field of land- and resource management along Nzeeu River in south-east Kenya. We assessed the level of education, land use practices, environmental knowledge, attitudes and the willingness to contribute to nature conservation. We tested for spatial bias to understand smallholders’ perceptions on environmental challenges. Our data show that land division due to inheritance is not perceived as a problem by the farmers. However, owners holding?<?1 ha of land property are less willing to spare some of their land for conservation, as opposed to those holding land plots above this size. Despite a high level of general willingness to conserve ecosystems, our data underline that local people hardly actively engage in conservation action. Furthermore, our data indicate a communication gap between local smallholders and regional governmental officers as well as overconfidence in mass media through the radio which can contradict successful adoption of pro-environment behavior. Sustainable land management in our study area is not a matter of education, but depends from the size of land property. There is an urgent need to bridge this communication gap, as a prerequisite to improve sustainable land management.     

MultiSig: a new high-precision approach to the analysis of complex biomolecular systems

MultiSig is a newly developed mode of analysis of sedimentation equilibrium (SE) experiments in the analytical ultracentrifuge, having the capability of taking advantage of the remarkable precision (~0.1 % of signal) of the principal optical (fringe) system employed, thus supplanting existing methods of analysis through reducing the ‘noise’ level of certain important parameter estimates by up to orders of magnitude. Long-known limitations of the SE method, arising from lack of knowledge of the true fringe number in fringe optics and from the use of unstable numerical algorithms such as numerical differentiation, have been transcended. An approach to data analysis, akin to ‘spatial filtering’, has been developed, and shown by both simulation and practical application to be a powerful aid to the precision with which near-monodisperse systems can be analysed, potentially yielding information on protein-solvent interaction. For oligo- and poly-disperse systems the information returned includes precise average mass distributions over both cell radial and concentration ranges and mass-frequency histograms at fixed radial positions. The application of MultiSig analysis to various complex heterogenous systems and potentially multiply-interacting carbohydrate oligomers is described.     

Boundary analysis of sedimentation-velocity experiments with monodisperse and paucidisperse solutes

A method is presented which allows one to calculate a distribution of sedimentation coefficients from the boundries of sedimentation-velocity experiments with mono- or pauci-disperse solutes. With two solutes differences in S value as small as 20% can be resolved. In absence of heterogeneity and concentration-dependent effects, the analysis also provides values for the diffusion coefficient within an accuracy of ?10 to +5%. Tests with both simulated data and ultracentrifugation experiments on short DNA fragments show the value and the limitations of the method.     

Automatic collection and processing of data from the ultracentrifuge using a programmable desk calculator.

Previous investigators [Trautman, R., Spragg, S. P., and Halsall, H. B. (1969) Anal. Biochem.28, 396–415] have published a detailed protocol for the analysis of sedimentation velocity measurements which is adaptable to data generated by an ultraviolet scanning system. The advent of programmable desk calculators capable of sampling the output of digital measuring devices has made it possible to develop inexpensive and highly convenient systems for collecting and processing scanner data. Basing our approach on the referenced protocol, we have developed algorithms for dealing with real data, that is, data characterized by a relatively high level of noise. The techniques are applieable to both sedimentation equilibrium and sedimentation velocity measurements using the seanning system and multicell rotors. With known concentration dependence, valid estimates of weight-average sedimentation coefficients, diffusion coefficients, and heterogeneity parameters have been obtained for both simulated and actual sedimenting anddiffusing macromolecular solutes. We find, however, that concentration dependence derived internally from a single sedimentation velocity measurement is unreliable.     

Unprotecting the rare species: a niche‐based gap analysis for odonates in a core Cerrado area

Aim We evaluated Odonata distribution data and predicted the compositional resemblance based on niche‐based species distribution models to analyse the following questions: (1) How is estimated species richness distributed, and how can it be preserved under the actual network of conservation units (a gap analysis approach)? (2) How is the estimated odonate beta diversity distributed, and is there a better distribution of conservation units (a priority setting approach)? (3) Is the probability of being under protection a function of the potential species range size? and (4) Will the current conservation network proposals protect odonate taxa? Location Central Brazil in a core Cerrado area. Methods We generated odonate species distribution predictions based on MaxEnt and maps derived from estimated species richness, beta diversity and gap analysis for all species predicted to occur in the study area. Then, we compared these maps with current conservation units, land‐use patterns and proposals for the establishment of conservation units. Results Raw odonate species records provided limited utility for setting conservation priorities without the use of niche‐based models. However, area under the receiver operating curve (AUC) values were characterized by substantial variation that was related to the number of records. No current conservation units overlapped the areas with higher predicted richness and beta diversity, and in general, conservation units were not preserving restricted/rare species. There was a direct linear correlation between species range size and the proportion of its range protected in the current network of conservation units. Finally, we identified three areas with high odonate beta diversity where conservationist actions should be implemented. Main conclusions Current conservation units and future suggested areas do not overlap regions with high conservation values for odonates. Conservation units protect species at random, and the level of protection has a direct relationship with species range size; thus, wide‐range species are expected to be more protected than restricted or threatened species.     

Using prior knowledge in the determination of macromolecular size-distributions by analytical ultracentrifugation

Analytical ultracentrifugation has reemerged as a widely used tool for the study of ensembles of biological macromolecules to understand, for example, their size-distribution and interactions in free solution. Such information can be obtained from the mathematical analysis of the concentration and signal gradients across the solution column and their evolution in time generated as a result of the gravitational force. In sedimentation velocity analytical ultracentrifugation, this analysis is frequently conducted using high resolution, diffusion-deconvoluted sedimentation coefficient distributions. They are based on Fredholm integral equations, which are ill-posed unless stabilized by regularization. In many fields, maximum entropy and Tikhonov-Phillips regularization are well-established and powerful approaches that calculate the most parsimonious distribution consistent with the data and prior knowledge, in accordance with Occam's razor. In the implementations available in analytical ultracentrifugation, to date, the basic assumption implied is that all sedimentation coefficients are equally likely and that the information retrieved should be condensed to the least amount possible. Frequently, however, more detailed distributions would be warranted by specific detailed prior knowledge on the macromolecular ensemble under study, such as the expectation of the sample to be monodisperse or paucidisperse or the expectation for the migration to establish a bimodal sedimentation pattern based on Gilbert-Jenkins' theory for the migration of chemically reacting systems. So far, such prior knowledge has remained largely unused in the calculation of the sedimentation coefficient or molecular weight distributions or was only applied as constraints. In the present paper, we examine how prior expectations can be built directly into the computational data analysis, conservatively in a way that honors the complete information of the experimental data, whether or not consistent with the prior expectation. Consistent with analogous results in other fields, we find that the use of available prior knowledge can have a dramatic effect on the resulting molecular weight, sedimentation coefficient, and size-and-shape distributions and can significantly increase both their sensitivity and their resolution. Further, the use of multiple alternative prior information allows us to probe the range of possible interpretations consistent with the data.     

Sedimentation equilibrium analysis of interference optical data by systematic noise decomposition.

An analytical method is described for removal of systematic signal offsets from interference optical data of sedimentation equilibrium gradients. It is demonstrated that the time-invariant signal contributions can be extracted from hydrodynamic modeling of interference profiles acquired during the approach to sedimentation equilibrium. This method is based on a technique for the explicit algebraic calculation of time-invariant noise components from sedimentation data, recently described for the direct modeling of sedimentation velocity experiments (P. Schuck and B. Demeler, Biophys. J. 76, 2288-2296, 1999). The calculated systematic signal offset is very well defined by the experimental data, stable over time, and its calculation is robust and to a large extent independent of the hydrodynamic model. The calculated time-invariant signal can be used to reduce the systematic errors in the measured sedimentation equilibrium profiles by more than an order of magnitude. It is shown that the resulting net equilibrium fringe profiles after subtraction of the time-invariant noise component allow equilibrium analyses consistent with those obtained from absorbance profiles. However, due to a higher dynamic range and the higher number of data points, the parameters derived from the net interference analysis can exhibit significantly improved precision. The presented study demonstrates the feasibility and potential of this analytical method for full exploitation of the remarkable precision of the interference optical data acquisition system, allowing sedimentation equilibrium experiments at loading concentrations below 0.05 mg/ml.     

A collaborative GIS method for integrating local and technical knowledge in establishing biodiversity conservation priorities

Methods for establishing biodiversity conservation priorities are urgently required, as the number of species and habitats that are threatened increases relative to the material resources available for their conservation. The identification of priority areas demands the integration of biophysical data on ecosystems together with social data on human pressures and planning opportunities. But comprehensive and reliable data are rarely available to demarcate where the need for action is most urgent and where the benefits of conservation strategies can be maximized. Strategic conservation initiatives cannot wait for the creation of comprehensive databases. In order to fill the missing data gaps, the combined knowledge of local and technical experts can be used. This study presents a collaborative geographic information system (GIS) method for integrating the knowledge of local and technical experts with existing spatial environmental data to establish priority areas for biodiversity conservation. Procedures for structuring and framing the discussions, establishing assessment criteria, integrating knowledge with data, and building consensus are incorporated into the method. The method provides a novel cooperative mechanism to aid spatial knowledge management and inclusive biodiversity planning.     

Characterization of macromolecular heterogeneity by equilibrium sedimentation techniques

New graphical procedures have been developed to investigate the heterogeneity of protein preparations using sedimentation equilibrium. The heterogeneous systems that can be studied include self-associating systems contaminated by incompetent monomer, self-associating systems contaminated by non-dissociating oligomer and simple non-interacting monomer-oligmer disperse systems. The new procedures are based on the concentration dependence of the apparent association constants estimated by a non-linear least square fitting program (NONLIN), on the assumption of conservation of mass during sedimentation and on the applications of several standard techniques for statistical inferences of NONLIN estimations. The procedures outlined here can detect various types of heterogeneity, discriminate amongst different types of heterogeneity, estimate the amount of contaminant causing heterogeneity and determine the true equilibrium constant of the self-associating components. The procedures appear to be sensitive, accurate and easily applicable when tested using both protein samples and computer simulated data.     

Metabolic control analysis of developing oilseed rape (Brassica napus cv Westar) embryos shows that lipid assembly exerts significant control over oil accumulation

? Metabolic control analysis allows the study of metabolic regulation. We applied both single- and double-manipulation top-down control analysis to examine the control of lipid accumulation in developing oilseed rape (Brassica napus) embryos. ? The biosynthetic pathway was conceptually divided into two blocks of reactions (fatty acid biosynthesis (Block A), lipid assembly (Block B)) connected by a single system intermediate, the acyl-coenzyme A (acyl-CoA) pool. Single manipulation used exogenous oleate. Triclosan was used to inhibit specifically Block A, whereas diazepam selectively manipulated flux through Block B. ? Exogenous oleate inhibited the radiolabelling of fatty acids from [1-(14) C]acetate, but stimulated that from [U-(14) C]glycerol into acyl lipids. The calculation of group flux control coefficients showed that c. 70% of the metabolic control was in the lipid assembly block of reactions. Monte Carlo simulations gave an estimation of the error of the resulting group flux control coefficients as 0.27?±?0.06 for Block A and 0.73?±?0.06 for Block B. ? The two methods of control analysis gave very similar results and showed that Block B reactions were more important under our conditions. This contrasts notably with data from oil palm or olive fruit cultures and is important for efforts to increase oilseed rape lipid yields.     

Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents

We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D(o) (1 + k(D)c), and the reciprocal sedimentation coefficient s = s(o)/(1 + k(s)c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k(s) and s degrees and also for D degrees and the buoyant molar mass were obtained. It was also possible to obtain good estimates for k(D) and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.     

Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation

A method is presented for preparing very large DNA from Bacillus subtilis protoplasts. When the DNA is characterized by sedimentation in neutral sucrose gradients, a fast-sedimenting component is found whose sedimentation coefficient varies with centrifuge speed. By use of [³H]thymine label for the DNA and a ¹⁴C-labeled amino acid, it is shown that less than 5% cellular material other than DNA is associated with this component. Irradiation of this DNA in solution with gamma rays forms a slower component, called the “main peak”, whose sedimentation coefficient also depends on centrifuge speed. More irradiation breaks down this main peak into even slower-sedimenting DNA; it is shown that for low doses, double-strand breaks are formed in both the B. subtilis DNA and in bacteriophage T2 DNA at the same rate linear in dose, 0.018 double-strand breaks per kilorad per mass equal to that of T2 DNA.The speed dependence of the DNA sedimenting at the main peak is compared with an approximate theory of the speed dependence of the sedimentation coefficient of linear DNA by B. H. Zimm (unpublished calculations). The comparison suggests that for sufficiently high centripedal acceleration, DNA molecules larger than a critical mass will sediment at much the same velocity. The theory, and data on the break-up of the DNA with gamma rays, are used to estimate that the DNA extracted is at least 13 times the mass of T2 DNA, and possibly larger.In the Appendix, data from the literature are put together with data taken during this work to make plausible the assumption that the usual theory for the sedimentation of DNA molecules, experimentally tested in salt solutions, may also be applied to sucrose solutions. If, in neutral sucrose gradients, the distance sedimented is proportional to a power α of the mass, the best value of α = 0.38.     

A web-based GIS tool for exploring the world's biodiversity: The Global Biodiversity Information Facility Mapping and Analysis Portal Application (GBIF-MAPA)

Legacy biodiversity data from natural history and survey collections are rapidly becoming available in a common format over the Internet. Over 110 million records are already being served from the Global Biodiversity Information Facility (GBIF). However, our ability to use this information effectively for ecological research, management and conservation lags behind. A solution is a web-based Geographic Information System for enabling visualization and analysis of this rapidly expanding data resource. In this paper we detail a case study system, GBIF Mapping and Analysis Portal Application (MAPA), developed for deployment at distributed database portals. Building such a system requires overcoming a series of technical and research challenges. These challenges include: assuring fast speed of access to the vast amounts of data available through these distributed biodiversity databases; developing open standards based access to suitable environmental data layers for analyzing biodiversity distribution; building suitably flexible and intuitive map interfaces for refining the scope and criteria of an analysis; and building appropriate web-services based analysis tools that are of primary importance to the ecological community and make manifest the value of online biodiversity GBIF data. After discussing how we overcome these challenges, we provide case studies showing two examples of the use of GBIF-MAPA analysis tools.     

石器研究中“操作链”的概念、内涵及应用

作为旧石器研究中十分重要的研究概念之一, 本文对"操作链"的发展史、理论内涵、实践方法等方面进行讨论, 认为"操作链"概念是一种动态的、综合的理论视角和研究体系, 强调了石器技术系统的两个行为过程(技术表现与思维运作)和一个互动关系(操作序列)。实践应用和术语对比, 为更好地运用"操作链"研究石器并复原史前技术体系提供了参考。文章还提出了"操作链"概念本身存在的问题, 希望能在今后的工作中得到完善。