Structural transformations in protein crystals caused by controlled dehydration

Recent experiments in this laboratory on structural transformations caused by controlled dehydration of protein crystals have been reviewed. X-ray diffraction patterns of the following crystals have been examined under varying conditions of environmental humidity in the relative humidity range of 100-75%: a new crystal form of bovine pancreatic ribonuclease A grown from acetone solution in tris buffer (I), the well-known monoclinic form of the protein grown from aqueous ethanol (II), the same form grown from a solution of 2-methyl pentan-2,4-diol in phosphate buffer (III), tetragonal (IV), orthorhombic (V), monoclinic (VI) and triclinic (VII) hen egg white lysozyme, porcine 2 Zn insulin (VIII), porcine 4 Zn insulin (IX) and the crystals of concanavalin A(X). I, II, IV, V and VI undergo one or more transformations as evidenced by discontinuous changes in the unit cell dimensions, the diffraction pattern and the solvent content. Such water-mediated transformations do not appear to occur in the remaining crystals in the relative humidity range explored. The relative humidity at which the transformation occurs is reduced when 2-methyl pentan-2,4-diol is present in the mother liquor. The transformations are affected by the crystal structure but not by the amount of solvent in the crystals. The X-ray investigations reviewed here and other related investigations emphasize the probable importance of water-mediated transformations in exploring hydration of proteins and conformational transitions in them

Effects of nutrient addition and soil drainage on germination of N-fixing and non-N-fixing tropical dry forest tree species

To develop generalised predictions regarding the effects of atmospheric nitrogen (N) and phosphorus (P) deposition on vegetation communities, it is necessary to account for the impacts of increased nutrient availability on the early life history stages of plants. Additionally, it is important to determine if these responses (a) differ between plant functional groups and (b) are modulated by soil drainage, which may affect the persistence of added nutrients. We experimentally assessed seed germination responses (germination proportion and germination energy, i.e. time to germination) of commonly occurring N-fixing and non-N-fixing tropical dry forest tree species found in India to simulated N and P deposition in well-drained soils, as well as soils with impeded drainage. When soils were not allowed to drain, germination proportion declined with nutrient addition, while germination energy remained unchanged. Stronger declines in germination proportion were observed for N-fixing species. In free-draining soils, nutrient addition did not affect germination proportion in either functional group. However, we detected a trend of delayed germination with nutrient addition, especially in N-fixers. Our results suggest that nutrient deposition can lead to potential shifts in functional dominance and tree community composition of tropical dry forests in the long term through its effects on early life stages of trees, although the mechanisms underlying the observed germination responses remain unclear. Further, such effects are likely to be spatially variable across the geographic range in which tropical dry forests occur depending on soil drainage properties

Structural Modeling and DNA Binding Autoinhibition Analysis of Ergp55, a Critical Transcription Factor in Prostate Cancer

BACKGROUND: The Ergp55 protein belongs to Ets family of transcription factor. The Ets proteins are highly conserved in their DNA binding domain and involved in various development processes and regulation of cancer metabolism. To study the structure and DNA binding autoinhibition mechanism of Ergp55 protein, we have produced full length and smaller polypeptides of Ergp55 protein in E. coli and characterized using various biophysical techniques. RESULTS: The Ergp55 polypeptides contain large amount of α-helix and random coil structures as measured by circular dichorism spectroscopy. The full length Ergp55 forms a flexible and elongated molecule as revealed by molecular modeling, dynamics simulation and structural prediction algorithms. The binding analyses of Ergp55 polypeptides with target DNA sequences of E74 and cfos promoters indicate that longer fragments of Ergp55 (beyond the Ets domain) showed the evidence of auto-inhibition. This study also revealed the parts of Ergp55 protein that mediate auto-inhibition. SIGNIFICANCE: The current study will aid in designing the compounds that stabilize the inhibited form of Ergp55 and inhibit its binding to promoter DNA. It will contribute in the development of drugs targeting Ergp55 for the prostate cancer treatment

Nitrogen fixation ability explains leaf chemistry and arbuscular mycorrhizal responses to fertilization

Atmospheric nitrogen (N) and phosphorus (P) deposition rates are predicted to drastically increase in the coming decades. The ecosystem level consequences of these increases will depend on how plant tissue nutrient concentrations, stoichiometry and investment in nutrient uptake mechanisms such as arbuscular mycorrhizal fungi (AMF) change in response to increased nutrient availability, and how responses differ between plant functional types. Using a factorial nutrient addition experiment with seedlings of multiple N-fixing and non-N-fixing tree species, we examined whether leaf chemistry and AMF responses differ between these dominant woody plant functional groups of tropical savanna and dry forest ecosystems. We found that N-fixers have remarkably stable foliar chemistry that stays constant with external input of nutrients. Non-N-fixers responded to N and N + P addition by increasing both concentrations and total amounts of foliar N, but showed a corresponding decrease in P concentrations while total amounts of foliar P stayed constant, suggesting a ‘dilution’ of tissue P with increased N availability. Non-N-fixers also showed an increase in N:P ratios with N and N + P addition, probably driven by both an increase in N and a decrease in P concentrations. AMF colonization decreased with N + P addition in non-N-fixers and increased with N and N + P addition in N-fixers, suggesting differences in their nutrient acquisition roles in the two plant functional groups. Our results suggest that N-fixers and non-N-fixers can differ significantly in their responses to N and P deposition, with potential consequences for future nutrient and carbon cycling in savanna and dry forest ecosystems

Protein hydration and water structure: X-ray analysis of a closely packed protein crystal with very low solvent content

Low-humidity monoclinic lysozyme, resulting from a water-mediated transformation, has one of the lowest solvent contents (22% by volume) observed in a protein crystal. Its structure has been solved by the molecular replacement method and refined to an R value of 0.175 for 7684 observed reflections in the 10-1.75 Å resolution shell. 90% of the solvent in the well ordered crystals could be located. Favourable sites of hydration on the protein surface include side chains with multiple hydrogen-bonding centres, and regions between short hydrophilic side chains and the main-chain CO or NH groups of the same or nearby residues. Major secondary structural features are not disrupted by hydration. However, the free CO groups at the C terminii and, to a lesser extent, the NH groups at the N terminii of helices provide favourable sites for water interactions, as do reverse turns and regions which connect β-structure and helices. The hydration shell consists of discontinuous networks of water molecules, the maximum number of molecules in a network being ten. The substrate-binding cleft is heavily hydrated, as is the main loop region which is stabilized by water interactions. The protein molecules are close packed in the crystals with a molecular coordination number of 14. Arginyl residues are extensively involved in intermolecular hydrogen bonds and water bridges. The water molecules in the crystal are organized into discrete clusters. A distinctive feature of the clusters is the frequent occurrence of three-membered rings. The protein molecules undergo substantial rearrangement during the transformation from the native to the low-humidity form. The main-chain conformations in the two forms are nearly the same, but differences exist in the side-chain conformation. The differences are particularly pronounced in relation to Trp 62 and Trp 63. The shift in Trp 62 is especially interesting as it is also known to move during inhibitor binding

Protein Hydration and Water Structure: X-ray Analysis of a Closely Packed Protein Crystal with Very Low Solvent Content

Low-humidity monoclinic lysozyme, resulting from a water-mediated transformation, has one of the lowest solvent contents (22% by volume) observed in a protein crystal. Its structure has been solved by the molecular replacement method and refined to an R value of 0.175 for 7684 observed reflections in the 10–1.75 Å resolution shell. 90% of the solvent in the well ordered crystals could be located. Favourable sites of hydration on the protein surface include side chains with multiple hydrogen-bonding centres, and regions between short hydrophilic side chains and the main-chain CO or NH groups of the same or nearby residues. Major secondary structural features are not disrupted by hydration. However, the free CO groups at the C terminii and, to a lesser extent, the NH groups at the N terminii of helices provide favourable sites for water interactions, as do reverse turns and regions which connect β-structure and helices. The hydration shell consists of discontinuous networks of water molecules, the maximum number of molecules in a network being ten. The substrate-binding cleft is heavily hydrated, as is the main loop region which is stabilized by water interactions. The protein molecules are close packed in the crystals with a molecular coordination number of 14. Arginyl residues are extensively involved in intermolecular hydrogen bonds and water bridges. The water molecules in the crystal are organized into discrete clusters. A distinctive feature of the clusters is the frequent occurrence of three-membered rings. The protein molecules undergo substantial rearrangement during the transformation from the native to the low-humidity form. The main-chain conformations in the two forms are nearly the same, but differences exist in the side-chain conformation. The differences are particularly pronounced in relation to Trp 62 and Trp 63. The shift in Trp 62 is especially interesting as it is also known to move during inhibitor binding

Conservation Threat of Increasing Fire Frequencies in the Western Ghats, India

The acceleration of processes such as forest fragmentation and forest fires in landscapes under intense human pressures makes it imperative to quantify and understand the effects of these processes oil the conservation of biodiversity in these landscapes. We combined information from remote-sensing, imagery and ground maps of all fires in the Mudumalai Wildlife Sanctuary (MWLS) in the Western Ghats of India over 14 years (1989-2002). These spatial data on fire occurrence were integrated with maps of vegetation types found in the MWLS to examine fire conditions in each. We calculated the average fire-return interval for. each of the vegetation types individually, and for the MWLS as a whole. Using vegetation data from the larger Nilgiri Biosphere Reserve and the entire Western Ghats region, we conservatively estimated fire frequency information for these larger regions. Because the MWLS does not contain tropical evergreen or montane forests, we were unable to estimate fire conditions in these fire conditions in these forest types, which represent 31% of all Western Ghats vegetation cover. For the MWLS, all vegetation types had average fire-return intervals of < 7 years, and the sanctuary as a whole had a fire-return interval of 3.3 years. Compared with a 13-year MWLS fire data set from 1909-1921, this represents a threefold increase in fire frequency over the last 80 years. We estimated average fire-return intervals of roughly 5 years for both the larger Nilgiri Biosphere Reserve and the entire Western Ghats region. Given other recent reports, the estimated fire frequencies for the Western Ghats forests outside protected reserves are conservative. We conclude that the current fire regime of the Western Ghats poses a severe and persistent conservation threat to forests both within and outside protected reserves

Conservation threat of increasing fire frequencies in the Western Ghats, India

The acceleration of processes such as forest fragmentation and forest fires in landscapes under intense human pressures makes it imperative to quantify and understand the effects of these processes on the conservation of biodiversity in these landscapes. We combined information from remote-sensing imagery and ground maps of all fires in the Mudumalai Wildlife Sanctuary (MWLS) in the Western Ghats of India over 14 years (1989-2002). These spatial data on fire occurrence were integrated with maps of vegetation types found in the MWLS to examine fire conditions in each. We calculated the average fire-return interval for each of the vegetation types individually and for the MWLS as a whole. Using vegetation data from the larger Nilgiri Biosphere Reserve and the entire Western Ghats region, we conservatively estimated fire-frequency information for these larger regions. Because the MWLS does not contain tropical evergreen or montane forests, we were unable to estimate fire conditions in these forest types, which represent 31% of all Western Ghats vegetation cover. For the MWLS, all vegetation types had average fire-return intervals of &#x003C; 7 years, and the sanctuary as a whole had a fire-return interval of 3.3 years. Compared with a 13-year MWLS fire data set from 1909-1921, this represents a threefold increase in fire frequency over the last 80 years. We estimated average fire-return intervals of roughly 5 years for both the larger Nilgiri Biosphere Reserve and the entire Western Ghats region. Given other recent reports, the estimated fire frequencies for the Western Ghats forests outside protected reserves are conservative. We conclude that the current fire regime of the Western Ghats poses a severe and persistent conservation threat to forests both within and outside protected reserves

A comparative analysis of spatial, temporal, and ecological characteristics of forest fires in seasonally dry tropical ecosystems in the Western Ghats, India

The Western Ghats in India is one of the 25 global hotspots of biodiversity, and it is the hotspot with the highest human density. This study considers variations in the regional fire regime that are related to vegetation type and past human disturbances in a landscape. Using a combination of remote sensing data and GIS techniques, burnt areas were delineated in three different vegetation types and various metrics of fire size were estimated. Belt transects were enumerated to assess the vegetation characteristics and fire effects in the landscape. Temporal trends suggest increasingly short fire-return intervals in the landscape. In the tropical dry deciduous forest, the mean fire-return interval is 6 years, in the tropical dry thorn forest mean fire-return interval is 10 years, and in the tropical moist deciduous forest mean fire-return interval is 20 years. Tropical dry deciduous forests burned more frequently and had the largest number of fires in any given year as well as the single largest fire (9900 ha). Seventy percent, 56%, and 30% of the tropical moist deciduous forests, tropical dry thorn forests, and tropical dry deciduous forests, respectively have not burned during the 7-year period of study. The model of fire-return interval as a function of distance from park boundary explained 63% of the spatial variation of fire-return interval in the landscape. Forest fires had significant impacts on species diversity and regeneration in the tropical dry deciduous forests. Species diversity declined by 50% and 60% in the moderate and high frequency classes, respectively compared to the low fire frequency class. Sapling density declined by ca. 30% in both moderate and high frequency classes compared to low frequency class. In tropical moist deciduous ecosystems, there were substantial declines in species diversity, tree density, seedling and sapling densities in burned forests compared to the unburned forests. In contrast forest fires in tropical dry thorn forests had a marginal positive effect on ecosystem diversity, structure, and regeneration

A comparative analysis of spatial, temporal, and ecological characteristics of forest fires in seasonally dry tropical ecosystems in the Western Ghats, India

The Western Ghats in India is one of the 25 global hotspots of biodiversity, and it is the hotspot with the highest human density. This study considers variations in the regional fire regime that are related to vegetation type and past human disturbances in a landscape. Using a combination of remote sensing data and GIS techniques, burnt areas were delineated in three different vegetation types and various metrics of fire size were estimated. Belt transects were enumerated to assess the vegetation characteristics and fire effects in the landscape. Temporal trends suggest increasingly short fire-return intervals in the landscape. In the tropical dry deciduous forest, the mean fire-return interval is 6 years, in the tropical dry thorn forest mean fire-return interval is 10 years, and in the tropical moist deciduous forest mean fire-return interval is 20 years. Tropical dry deciduous forests burned more frequently and had the largest number of fires in any given year as well as the single largest fire (9900 ha). Seventy percent, 56%, and 30% of the tropical moist deciduous forests, tropical dry thorn forests, and tropical dry deciduous forests, respectively have not burned during the 7-year period of study. The model of fire-return interval as a function of distance from park boundary explained 63% of the spatial variation of fire-return interval in the landscape. Forest fires had significant impacts on species diversity and regeneration in the tropical dry deciduous forests. Species diversity declined by 50% and 60% in the moderate and high frequency classes, respectively compared to the low fire frequency class. Sapling density declined by ca. 30% in both moderate and high frequency classes compared to low frequency class. In tropical moist deciduous ecosystems, there were substantial declines in species diversity, tree density, seedling and sapling densities in burned forests compared to the unburned forests. In contrast forest fires in tropical dry thorn forests had a marginal positive effect on ecosystem diversity, structure, and regeneration