How far in-silico computing meets real experiments. A study on the structure and dynamics of spin labeled vinculin tail protein by molecular dynamics simulations and EPR spectroscopy.

BACKGROUND: Investigation of conformational changes in a protein is a prerequisite to understand its biological function. To explore these conformational changes in proteins we developed a strategy with the combination of molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy. The major goal of this work is to investigate how far computer simulations can meet the experiments. METHODS: Vinculin tail protein is chosen as a model system as conformational changes within the vinculin protein are believed to be important for its biological function at the sites of cell adhesion. MD simulations were performed on vinculin tail protein both in water and in vacuo environments. EPR experimental data is compared with those of the simulated data for corresponding spin label positions. RESULTS: The calculated EPR spectra from MD simulations trajectories of selected spin labelled positions are comparable to experimental EPR spectra. The results show that the information contained in the spin label mobility provides a powerful means of mapping protein folds and their conformational changes. CONCLUSIONS: The results suggest the localization of dynamic and flexible regions of the vinculin tail protein. This study shows MD simulations can be used as a complementary tool to interpret experimental EPR data.

Net energy of cellulosic ethanol from switchgrass.

Perennial herbaceous plants such as switchgrass (Panicum virgatum L.) are being evaluated as cellulosic bioenergy crops. Two major concerns have been the net energy efficiency and economic feasibility of switchgrass and similar crops. All previous energy analyses have been based on data from research plots (<5 m2) and estimated inputs. We managed switchgrass as a biomass energy crop in field trials of 3-9 ha (1 ha = 10,000 m2) on marginal cropland on 10 farms across a wide precipitation and temperature gradient in the midcontinental U.S. to determine net energy and economic costs based on known farm inputs and harvested yields. In this report, we summarize the agricultural energy input costs, biomass yield, estimated ethanol output, greenhouse gas emissions, and net energy results. Annual biomass yields of established fields averaged 5.2-11.1 Mg x ha(-1) with a resulting average estimated net energy yield (NEY) of 60 GJ x ha(-1) x y(-1). Switchgrass produced 540% more renewable than nonrenewable energy consumed. Switchgrass monocultures managed for high yield produced 93% more biomass yield and an equivalent estimated NEY than previous estimates from human-made prairies that received low agricultural inputs. Estimated average greenhouse gas (GHG) emissions from cellulosic ethanol derived from switchgrass were 94% lower than estimated GHG from gasoline. This is a baseline study that represents the genetic material and agronomic technology available for switchgrass production in 2000 and 2001, when the fields were planted. Improved genetics and agronomics may further enhance energy sustainability and biofuel yield of switchgrass.

Genetic modification of lignin concentration affects fitness of perennial herbaceous plants.

Populations of four perennial herbaceous species that were genetically modified for altered lignin content (or associated forage digestibility) by conventional plant breeding were evaluated for two agricultural fitness traits, plant survival and plant biomass, in three Northcentral USA environments for more than 4 years. Reduced lignin concentration or increased digestibility resulted in increased winter mortality in two of four species and reduced biomass in one species. Results from other experiment indicate that these apparent genetic correlations may be ephemeral, suggesting that selection for fitness can be successful within high-digestibility or low-lignin germplasm. Results indicate that perennial plants genetically engineered with altered lignin concentration or composition for use in livestock, pulp and paper, or bioenergy production should be evaluated for fitness in field environments prior to use in agriculture.

Influence of lignin on digestibility of forage cell wall material.

One-hundred-ninety-four grass samples, representing eight species, were used to determine the relationships of in vitro forage dry matter, cell wall, hemicellulose and cellulose digestibilities with lignin concentration. Linear regressions indicated that dry matter digestion was inhibited to a lesser degree (P less than .05) by lignin concentration than was cell wall digestion. Results for linear regressions of hemicellulose and cellulose digestibilities as functions of lignin concentration showed a greater (P less than .05) effect of lignin on cellulose digestion. Smooth bromegrass and switchgrass were collected at both Clay Center and Mead, NE; for all digestibility measures, the Clay Center samples gave stronger (P less than .05) negative correlations with lignin. A comparison of linear and curvilinear models indicated that, for all digestion measurements, the curvilinear model was a better (P less than .05) description of relationships with lignin concentration. For all digestibility measures, lignin's inhibiting effect declined at higher lignin concentrations. The curvilinear models did not show significant differences among the digestibility measures for effect of lignin. The demonstration that the forage digestibility response to lignin's inhibitory effect is curvilinear in nature suggests that the mechanism of lignin's inhibition is complex.

Grazing selectivity and in vivo digestibility of switchgrass strains selected for differing digestibility.

Animal selectivity and digestibility differences among switchgrass strains selected for different in vitro dry matter digestibilities (IVDMD) were measured in a grazing trial with esophageally fistulated steers and a sheep digestion trial. Extrusa selected by esophageally fistulated steers grazing high-IVDMD (Trailblazer), Pathfinder and low-IVDMD strains of switchgrass were compared, as were top and whole plant hand-clipped samples from each strain. Trailblazer extrusa had higher (P less than .1) in vitro organic matter disappearance (IVOMD) and lower (P less than .1) NDF and ADF than Pathfinder extrusa. Extrusa from all three strains appeared to be of higher quality than top or whole plant hand-clipped samples. In vitro organic matter disappearance tended to be highest for Trailblazer top hand-clipped samples. Composition of hand-clipped samples among strains was not significantly different. Mature crossbred wethers were used to compare Trailblazer and Pathfinder switchgrass hay in a digestion trial. No differences (P greater than .1) were detected between strains for DMI or apparent digestibility of DM, NDF, ADF and CP. Extrusa from Trailblazer switchgrass that had been selected for whole plant IVDMD had higher IVOMD; however, there was no indication that steers selected a differentially higher IVOMD for one strain than another.

Using plant breeding and genetics to overcome the incidence of grass tetany.

Plant breeders developing cultivars to minimize the hazards of grass tetany are concentrating largely on increasing herbage Mg concentrations in cool-season (C3) grasses. Significant genetic variation has been found for Mg, Ca and K concentrations within C3 grass species studied to date. For most C3 forage grass species, heritability estimates are highest for Mg, slightly lower for Ca and lowest for K concentrations. The largest genotype x environmental interactions are found for K values, whereas small environmental effects have been observed for Mg and Ca values. No C3 forage grass cultivar has been developed to date that would eliminate hypomagnesemia. Grass breeders need to develop more experimental C3 plant populations that have high Mg and Ca concentrations. These experimental synthetics with genetically altered mineral concentrations need to be fed to ruminants susceptible to grass tetany to determine whether grass tetany can be eliminated or reduced. Limited feeding trials using ruminants show that improved animal performance can be expected when feeding forage grasses bred for higher Mg concentrations.

Genotypic variability in mineral composition of switchgrass.

Switchgrass (Panicum virgatum L.) is a warm season perennial grass with great potential as an energy crop in the USA. It is widely adapted to many regions of the country, produces large amounts of biomass, serves as a useful forage grass, and provides ecosystem services that benefit soil and water quality and wildlife. Biological and thermochemical technologies are being developed to convert herbaceous biomass, including switchgrass, to energy. The objective of this research was to determine the effect of genotype and production environment on the concentration of minerals that affect the suitability of switchgrass for thermochemical conversion and to quantify the amount of potassium (K) and phosphorus (P) removed from the production system by harvest of the aboveground biomass, a measure of the sustainability of the practice. Straw dry biomass contained from 1.3 to 6.4 kg Mg(-1) and from 6.2 to 15.8 kg Mg(-1) of P and K, respectively. Variability in aluminum (Al), calcium (Ca), chloride (Cl), K, P, silicon (Si), and sulfur (S) concentrations across locations was relatively high, ranging from twofold (Al) to eightfold (Cl). Location had a strong impact on mineral concentrations among switchgrass genotypes evaluated in this study. Latitude of origin impacted the Cl and Si concentrations measured in plant tissues, but none of the other minerals analyzed in this study. Upland and lowland cytotypes explained some of the observed differences, but populationxlocation interactions were the primary source of variability in the concentration of these minerals.

In vitro gas production as a surrogate measure of the fermentability of cellulosic biomass to ethanol.

Current methods for measuring ethanol yields from lignocellulosic biomass are relatively slow and are not well geared for analyzing large numbers of samples generated by feedstock management and breeding research. The objective of this study was to determine if an in vitro ruminal fermentation assay used in forage quality research was predictive of results obtained using a conventional biomass-to-ethanol conversion assay. In the conventional assay, herbaceous biomass samples were converted to ethanol by Saccharomyces cerevisiae cultures in the presence of cellulase enzymes. Cultures were grown in sealed serum bottles and gas production monitored by measuring increasing head space pressure. Gas accumulation as calculated from the pressure measurements was highly correlated (r(2)>0.9) with ethanol production measured by gas chromatography at 24 h or 7 days. The same feedstocks were also analyzed by in vitro ruminal digestion, as also measured by gas accumulation. Good correlations (r(2) approximately 0.63-0.82) were observed between ethanol production during simultaneous saccharification and fermentation and gas accumulation in parallel in vitro ruminal fermentations. Because the in vitro ruminal fermentation assay can be performed without sterilization of the medium and does not require aseptic conditions, this assay may be useful for biomass feedstock agronomic and breeding research.

The isolation and identification of steroidal sapogenins in switchgrass.

Switchgrass (Panicum virgatum L.) has been reported to be hepatotoxic, causing photosensitization in lambs and horses. In this study we show the presence of steroidal saponins in two samples of switchgrass that has been implicated in the poisonings of sheep and horses. After hydrolysis of the saponins, diosgenin was determined to be the major sapogenin in both switchgrass samples. We also confirmed the presence of diosgenin in kleingrass after hydrolysis of saponins extracted from it.