Molecular dynamics analysis of the structures of ras-guanine nucleotide exchange protein (SOS) bound to wild-type and oncogenic ras-p21. Identification of effector domains of SOS

The X-ray crystal structure of the ras oncogene-encoded p21 protein bound to SOS, the guanine nucleotide exchange-promoting protein, has been determined. We have undertaken to determine if there are differences between the three-dimensional structures of SOS bound to normal and oncogenic (Val 12-p21) proteins. Using molecular dynamics, we have computed the average structures for both complexes and superimposed them. We find four domains of SOS that differ markedly in structure: 631–641, 676–691, 718–729, and 994–1004. Peptides corresponding to these sequences have been synthesized and found to be powerful modulators of oncogenic p21 in cells as described in an accompanying paper. We find that the SOS segment from 809–815 makes contacts with multiple domains of ras-p21 and can facilitate correlated conformational changes in these domains.     

Influence of the completeness of chemical shift assignments on NMR structures obtained with automated NOE assignment

Reliable automated NOE assignment and structure calculation on the basis of a largely complete, assigned input chemical shift list and a list of unassigned NOESY cross peaks has recently become feasible for routine NMR protein structure calculation and has been shown to yield results that are equivalent to those of the conventional, manual approach. However, these algorithms rely on the availability of a virtually complete list of the chemical shifts. This paper investigates the influence of incomplete chemical shift assignments on the reliability of NMR structures obtained with automated NOESY cross peak assignment. The program CYANA was used for combined automated NOESY assignment with the CANDID algorithm and structure calculations with torsion angle dynamics at various degrees of completeness of the chemical shift assignment which was simulated by random omission of entries in the experimental ¹H chemical shift lists that had been used for the earlier, conventional structure determinations of two proteins. Sets of structure calculations were performed choosing the omitted chemical shifts randomly among all assigned hydrogen atoms, or among aromatic hydrogen atoms. For comparison, automated NOESY assignment and structure calculations were performed with the complete experimental chemical shift but under random omission of NOESY cross peaks. When heteronuclear-resolved three-dimensional NOESY spectra are available the current CANDID algorithm yields in the absence of up to about 10% of the experimental ¹H chemical shifts reliable NOE assignments and three-dimensional structures that deviate by less than 2 Å from the reference structure obtained using all experimental chemical shift assignments. In contrast, the algorithm can accommodate the omission of up to 50% of the cross peaks in heteronuclear- resolved NOESY spectra without producing structures with a RMSD of more than 2 Å to the reference structure. When only homonuclear NOESY spectra are available, the algorithm is slightly more susceptible to missing data and can tolerate the absence of up to about 7% of the experimental ¹H chemical shifts or of up to 30% of the NOESY peaks.Abbreviations: BmPBP^A – Bombyx mori pheromone binding protein form A; CYANA – combined assignment and dynamics algorithm for NMR applications; NMR – nuclear magnetic resonance; NOE – nuclear Overhauser effect; NOESY – NOE spectroscopy; RMSD – root-mean-square deviation; WmKT – Williopsis mrakii killer toxin     

A comparison of structure,development and function in cluster roots of Lupinus albus L. under phosphate and iron stress

Cluster roots are adaptations for nutrient acquisition, found throughout the world in many different plant families and habitats. They arise from changes in root initiation, meristem maintenance and physiology. In Lupinus albus cluster roots form under low internal plant phosphate and low internal plant iron levels. In this study, we compare morphology, structure and physiology of cluster roots formed under –P and –Fe conditions. –Fe cluster roots had a lower density of shorter rootlets than –P roots, and were yellow in colour, probably because of increased phenolics due to down-regulation of peroxidase. Rootlet length and width was reduced in –Fe conditions. The change in exudation of citrate, over time, of –P and –Fe cluster roots shared identical temporal dynamics, with an exudative burst occurring in day 3. However, the –Fe cluster roots displayed much higher rates of exudation than the –P cluster roots. Results are discussed within the context of structural and functional control.     

Some characteristics of the community of autotrophs of Lake Sevan in connection with its eutrophication

Eutrophication of Lake Sevan caused by the artificial lowering of water level was accompanied by changes in the structure and dynamics of the planktonic communities. A dominance of diatoms up to 1983 was changed to that of green algae in the last years. Primary production of plankton rose and then decreased in the process of eutrophication. The annual average primary production in 1982–1986 — 250 g C m^–2 yr^–1 — is evidently close to the steady state production under the present morphometry of the lake. The activity coefficient of phytoplanktonic photosynthesis changed within relatively narrow limits, in spite of significant changes in the concentrations of major nutrients and in the structure and productivity of the phytoplankton.     

Molecular dynamics as a tool to help design mutants

A 200 ps MD trajectory of wild type PCI and a 120 ps one for the Pro36Gly putative mutant are studied and compared with the structure of PCI in its complex with carboxypeptidase A (CPA). It is first established that the structures of PCI from X-ray and from MD simulation are essentially equal. Thereafter, data from the PCI-CPA and average MD structures together with available biochemical information are used to identify possible structural factors that may determine the inhibitory power of PCI. These structural determinants are used to analyze the mutant structure. The fold of the mutant protein shows a large degree of stability. The N-terminal tail in PCIm differs from the X-ray structure as it does in PCIw, while the mutant's C-terminal tail (which is the primary binding site with CPA) and residues 13–17 present deviations. Differences in fluctuation patterns exist between PCIm and PCIw in residues 2–4 (the N-terminal tail), 13–17, 22–23, 28–81 (the secondary contact site with CPA), and 37–38 (the C-terminal tail); the latter region is rigidified in PCIm. Results show that the MD method is able to sense long-range as well as local perturbative effects produced by amino-acid substitutions in flexible regions of this protein. The simulations suggest that the conformation of the C-terminal tail is less favorable for interaction with the target protein in the mutant than it is in the wild type protein. The Pro-36-Gly mutant is predicted to be a less potent inhibitor.Abbreviations CPA carboxypeptidase A - MD molecular dynamics - NIS non-inertial solvent - PCI potato carboxypeptidase A inhibitor - PCIm mutated inhibitor - PCIw wild inhibitor     

Interannual Dynamics of the Trophic Structure of Zooplankton of the Southern Kuril Region

Based on the data on feeding ecology of common species of zooplankton, the main trophic groups of plankton of the southern Kuril region are identified. These are phytophages, zoophages, and omnivores. The interannual dynamics of trophic characteristics of the plankton community are examined. Predatory plankton significantly dominated in 1992–1994; however, in 1995–1996 its biomass decreased markedly. The biomass dynamics of predatory and nonpredatory zooplankton, as well as changes in the structure of nonpredatory zoolankton, suggest structural changes in the planktonic community.     

Comparison of the computed three-dimensional structures of oncogenic forms (bound to GDP) of theras-Gene-Encoded p21 protein with the structure of the normal (non-transforming) wild-type protein

Theras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10–16, 32–47, 55–74, 85–89, 100–110, and 119–134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32–47 and 55–74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55–74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.     

Inhibition of Oncogenic and Activated Wild-Type ras-p21 Protein-Induced Oocyte Maturation by Peptides from the ras-Binding Domain of the raf-p74 Protein, Identified from Molecular Dynamics Calculations

In the preceding paper we found from molecular dynamics calculations that the structure of the ras-binding domain (RBD) of raf changes predominantly in three regions depending upon whether it binds to ras-p21 protein or to its inhibitor protein, rap-1A. These three regions of the RBD involve residues from the protein–protein interaction interface, e.g., between residues 60 and 72, residues 97–110, and 111–121. Since the rap-1A–RBD complex is inactive, these three regions are implicated in ras-p21-induced activation of raf. We have therefore co-microinjected peptides corresponding to these three regions, 62–76, 97–110, and 111–121, into oocytes with oncogenic p21 and microinjected them into oocytes incubated in in insulin, which activates normal p2l. All three peptides, but not a control peptide, strongly inhibit both oncogenic p21- and insulin-induced oocyte maturation. These findings corroborate our conclusions from the theoretical results that these three regions constitute raf effector domains. Since the 97–110 peptide is the strongest inhibitor of oncogenic p21, while the 111–121 peptide is the strongest inhibitor of insulin-induced oocyte maturation, the possibility exists that oncogenic and activated normal p21 proteins interact differently with the RBD of raf.     

The latitude-elevation relationship for spruce-fir forest and treeline along the Appalachian mountain chain

Spruce-fir forests extend along the Appalachian Mountains of eastern North America from 35° to 49° N. This montane vegetation differs from boreal spruce-fir forest in that it is dominated by Picea rubens, has a higher vascular species richness, has wind, rather than fire, dominated dynamics, and has a mean annual temperature above 2 °C. Using field reconnaissance, remote sensing, and literature review we described and modeled the latitude-elevation relationship for Appalachian spruce-fir. The elevation of the sprucefir/deciduous forest ecotone decreases from 1,680 m at 35° N to 150 m at 49° N, while the elevation of treeline (spruce-fir/tundra ecotone) decreases from 1,480 m at 44° N to 550 m at 55° N. Linear regressions gave latitude-elevation relationships of –100 m/l^o Latitude for the spruce-fir/deciduous forest ecotone and –83 m/l^o Latitude for treeline. These values compare to literature reports of –54 to –230 m/l^o Latitude and are most similar to values reported from eastern Asia. The latitude-elevation relationship for eman July temperature ( –94 to –121 m/l^o Latitude) was more similar to the slopes of these ecotones than is the slope for mean annual temperature ( –170 to –220 m/l^o Latitude). The spruce-fir/deciduous forest ecotone was correlated with a mean July temperature of approximately 17 °C. Treeline was correlated with a mean July temperature of approximately 13 °C.     

Inhibition of oncogenic and activated wild-type ras-p21 protein-induced oocyte maturation by peptides from the guanine-nucleotide exchange protein, SOS, identified from molecular dynamics calculations. Selective inhibition of oncogenic ras-p21

In the preceding paper we performed molecular dynamics calculations of the average structures of the SOS protein bound to wild-type and oncogenic ras–p21. Based on these calculations, we have identified four major domains of the SOS protein, consisting of residues 631–641, 676–691, 718–729, and 994–1004, which differ in structure between the two complexes. We have now microinjected synthetic peptides corresponding to each of these domains into Xenopus laevis oocytes either together with oncogenic (Val 12)-p21 or into oocytes subsequently incubated with insulin. We find that the first three peptides inhibit both oncogenic and wild-type p21-induced oocyte maturation, while the last peptide much more strongly inhibits oncogenic p21 protein-induced oocyte maturation. These results suggest that each identified SOS region is involved in ras–stimulated signal transduction and that the 994–1004 domain is involved uniquely with oncogenic ras–p21 signaling.     

Temperature Broadening of LH2 Absorption in Glycerol Solution

In order to determine the relationship between the pigment–protein and the pigment–pigment interactions, the measurements of absorption spectra of the peripheral light-harvesting complex LH2 from the purple bacteria Rhodobacter sphaeroides solvated in glycerol/buffer solution were carried out in a wide temperature range, from 4 to 250 K. The SDFs used for simulating the temperature dependence of B800 and B850 bands were determined in a parametric form. To fit experimental spectra the overall exciton–phonon coupling had to be assumed to be weak for B850 (λ/2V ≈ 0.3, where λ is the reorganization energy and V is the nearest-neighbor dipole–dipole coupling for bacteriochlorophylls). At physiological temperatures the intermediate nuclear bath dynamics compares with the magnitude of energy gap fluctuations. Slower dynamics with κ ≈ 0.39, where κ is the ratio of the nuclear relaxation rate and the line width parameter, determines the spectral shape of B850 whilst faster modulations characterize B800 (κ ≈2.39). The static disorder for the B800 band is relatively high with the characteristic value of the inhomogeneous bandwidth Γ^inh ≈120 cm⁻¹, while for the B850 band this value is almost equal to the dipole–dipole coupling strength (Γ^inh ≈360 cm⁻¹). It has been found that the LH2 absorption spectrum is likely to be influenced by the temperature dependence of the dielectric constant of the solution in the high temperature range, when the glycerol/buffer solution is in the liquid state.     

Identification, using molecular dynamics, of an effector domain of the ras-binding domain of the raf-p74 protein that is uniquely involved in oncogenic ras-p21 signaling

By comparing the average structures, computed using molecular dynamics, of the ras-binding domain of raf (RBD) bound to activated wild-type ras-p21 and its homologous inhibitory protein, rap-1A, we formerly identified three domains of the RBD that changed conformation between the two complexes, residues 62–76, 97–110, and 111–121. We found that one synthetic peptide, corresponding to RBD residues 97–110, selectively inhibited oncogenic ras-p21-induced oocyte maturation. In this study, we performed molecular dynamics on the Val 12-ras-p21-RBD complex and compared its average structure with that for the wild-type protein. We find that there is a large displacement of a loop involving these residues when the structures of the two complexes are compared. This result corroborates our former finding that the RBD 97–110 peptide inhibits only signal transduction by oncogenic ras-p21 and suggests that oncogenic p21 uses this loop to interact with raf in a unique manner.     

Macrozoobenthic community structure in a poikilohaline Mediterranean lagoon (Laki Lagoon, northern Aegean)

The community structure of the macrobenthic fauna was studied in the overall area of Laki Lagoon in September 1997 (salinity 32–35 psu) and monthly from February 1998 to February 1999 in the innermost part of the lagoon (salinity 0.1–6.8 psu). Community structure was analyzed by means of uni- and multivariate methods. In September 1997, the macrofauna in the outer part of the lagoon was characterized by a higher diversity and the occurrence of both lagoonal and marine species, and in the innermost part by a higher total abundance and the occurrence of a few lagoonal species. The combination of distance from the sea, depth, salinity and sediment organic matter correlated best with the spatial distribution pattern of the macrobenthic fauna. Community structure in the innermost part of the lagoon showed a seasonal periodicity. Species composition during spring 1998, at 0.1–2.0 psu, was similar to that in September 1997. During summer the macrobenthic fauna became impoverished, but recovered from late summer onwards. The salinity increase during summer (up to 5–7 psu) was followed by the appearance of marine species in the innermost part of the lagoon. Total abundance displayed a peak in late spring and a lower one in mid-autumn. The seasonal dynamics of the faunal assemblage was mainly governed by water temperature. Predation pressure by Atherina boyeri may have contributed to quantitative community changes during autumn.     

Dynamics of the Variables of Neonates' Physical Development and Geomagnetic Variables over a Many-Year Period

The study of the dynamics of the variables of physical development of neonates in the first months of life and geomagnetic field fluctuations in the period from 1990 to 2001 demonstrated their complex rhythmic structure. In the studied series of birth weight and the parameters of the 84-h rhythms of daily body weight gain, as well as the parameters of the 84-h rhythms of the K-index, there are ranges of increased spectral density coinciding in frequency. In these intervals (1.1–1.3 and 5–10 years), the temporal behavior of the series of physiological and geomagnetic variables is correlated.     

Power law dispersion in animal horn

The dielectric response of sheep horn has been measured in the frequency range from 10^–3–10⁵ Hz and over temperatures in the range 304–500 K. The dynamic behaviour of the conductance and capacitance in sheep horn has been observed to follow fractional power law dependences on frequency. It is shown that the over all dielectric response of these dead cells correspond to a dispersive imperfect bulk in series with a dispersive barrier region. It is further shown that the increase in temperature influences the reponse by eliminating the room temperature dc conductance and affecting the magnitude of the dispersion in capacitance. The magnitudes of activation energies are found as 0.33±0.02 eV for conductance, 0.40±0.02 eV for relaxation and 0.33±0.02 eV for the frequency shift.     

Root dynamics in bottomland hardwood forests of the Southeastern United States Coastal Plain

Effects of flooding on root dynamics appear nonlinear and therefore difficult to predict, leading to disparate and often contradictory reports of flooding impacts on production in bottomland hardwood forests. We explored root dynamics in two adjacent wetland habitats by comparing results obtained from several methods of estimating root processes. Also, we tested the influence of flooding on root dynamics of cherrybark, overcup, water and swamp chestnut oaks. Fine root biomass in the laurel oak habitat was greater (< 0.05) than in the swamp tupelo habitat (5.7 vs. 2.4 Mg ha^–1), as was fine root necromass (2.4 vs. 1.3 Mg ha^–1), productivity (2.3 vs. 0.3 Mg ha^–1 yr^–1 when the sum of significant increments method was used, 5.6 vs. 2.5 Mg ha ^–1 yr^–1 when the maximum minus minimum method was used, and 1.2 vs. 1.0 Mg ha^–1 yr ^–1, when the root screen method was used), and turnover (40% and 12% per year). Mortality estimates were lower in the laurel oak habitat (1.3 and 1.2 Mg ha^–1 yr^–1) than in the swamp tupelo community (2.8 and 2.1 Mg ha^–1 yr^–1) when significant increment and maximum minus minimum methods were used, respectively. This apparent contradiction between estimates of production and mortality may be due to more rapid decomposition rates in the more aerated soil of the laurel oak than in the swamp tupelo forest type. Roots in the swamp tupelo habitat appeared to be longer-lived than in the laurel oak habitat. We concluded that there was greater investment in roots in the laurel oak habitat, where a shallow rooting zone and episodes of flooding and drought required drastic changes in root structure and physiology. In contrast, the swamp tupelo habitat had a deeper rooting zone and more consistently moist to flooded hydroperiod, allowing flood adapted roots to persist. The four oak species varied in their phenology of root production and response to flooding, from no difference among treatments for overcup oak to dramatic reductions in root growth during and after flooding for cherrybark oak. Flooding enhanced or at least did not negatively influence root growth in overcup oak, but seriously impacted root growth and survival of cherrybark oak and swamp chestnut oak. Different responses were attributed to the timing of root production: root growth began early for cherrybark oak so spring flooding severely affected this species. Growth in overcup oak began later and ended earlier than the other species tested, allowing the species a means of avoiding flood stress.     

The X-ray crystal structure of pyrrolidone–carboxylate peptidase from hyperthermophilic archaea Pyrococcus horikoshii

The crystal structure of pyrrolidone–carboxylate peptidase (PCP) from hyperthermophilic archaea Pyrococcus horikoshii (PhoPCP) has been determined at 1.6-A resolution by X-ray crystallography. PCP belongs to the C15 family of cysteine protease, and specifically removes the amino terminal pyroglutamate residue from a wide range of N-terminal-blocking peptides. The crystal structure is very similar to that of other hyperthermophiles, Pyrococcus friosus and Thermococcus litoralis, and even that from the mesophile, Bacillus amyloliquefacience. The inter-subunit disulfide bonds, which have been proposed as one of the thermostabilizing factors of the PCP from such hyperthermophiles, was not present in PhoPCP. The result suggests that the thermostability of PhoPCP may be obtained by the accumulation of many weak factors. Abbreviations: NCS – non-crystallographic symmetry; PCP – pyrrolidone-carboxylate peptidase; rmsd – root mean square deviation     

Palaeoecology and plant population dynamics

The pollen record of the past 10–20 thousand years is a source of data both on long-term climatic change and on the dynamics of plant populations in response to climatic change. Time sequences of pollen accumulation rates record invasions of tree taxa over 10¹–10³ years. Palaeoecologists have fifted such data with simple population dynamic models that assume a constant climate. Population doubling times estimated from the pollen record are consistent with species' life-history characteristics and with estimates based on the population structure of modern forests. This palaeo-ecological approach complements palaeoclimatological studies of longeer-term (10³–10⁵-year) population shifts, in which population response is assumed instantaneous. Both approaches depend on population responses being fast compared to the climatic changes that cause them. Pollen data also record the more complex interactions between climate and vegetation that occur during periods of rapid climatic change, and could be used to test more realistic models of vegetation dynamics in a changing environment.     

Solution structure of a PAN module from the apicomplexan parasite Eimeria tenella

Micronemes, specialised organelles found in all apicomplexan parasites, secrete molecules that are essential for parasite attachment and invasion of host cells. EtMIC5 is one such microneme protein that contains eleven tandemly repeating modules. These modules have homology with the PAN module superfamily. Members of this family are found in blood clotting proteins, some growth factors and some nematode proteins. This paper presents the structure of the 9^th PAN module in EtMIC5, determined using high resolution NMR. The structure shows similarities to and some differences from the N-terminal module of hepatocyte growth factor (HGF), the only previous member of the PAN family with known structure. AbbreviationsNMR – nuclear magnetic resonance; NOE – nuclear Overhauser enhancement; NOESY – NOE spectroscopy; COSY – correlated spectroscopy; TOCSY – total correlated spectroscopy; HSQC – hetero nuclear single quantum coherence; HMQC-J – hetero nuclear multiple quantum coherence-J coupling; MICs – microneme proteins; EtMIC5 – a microneme protein from Eimeria tenella; Apple9 – the ninth Apple repeat of EtMIC5; FXI – blood coagulation factor XI; PK – plasma prekallikrein; HGF – hepatocyte growth factor.     

Formation of Chromoneme-like Fibrils Induced in Infusorian Somatic Nuclei by Magnesium Ions

A study was made of the effect of Mg²⁺ on higher-order chromatin structure in macronuclei (Ma) of infusoria Paramecium aurelia and Bursaria truncatella. In infusorian Ma, inactive chromatin is commonly packed in chromatin bodies sized 60–200 nm. When isolated chromatin or Ma were treated with Mg²⁺ (about 3 mM), chromatin bodies arranged into fibrils 100–300 nm in diameter, which resembled higher eukaryotic chromonemes. The dynamics of chromoneme-like fibril formation was described. The results testified to the similarity of chromatin bodies to chromomeres of higher eukaryotes. Structurally intact central chromomere cores proved to be essential for the formation of chromoneme-like fibrils. Chromatin organization in infusoria was shown to follow the discrete-level model (nucleosomes–nucleomeres–chromomeres–chromonemes) assumed for higher eukaryotes.