Identification of HN-methyl NOEs in large proteins using simultaneous amide-methyl TROSY-based detection

A pair of HN-methyl NOESY experiments that are based on simultaneous TROSY-type detection of amide and methyl groups is described. The preservation of cross-peak symmetry in the simultaneous ¹H–¹⁵N/¹³CH₃ NOE spectra enables straightforward assignments of HN-methyl NOE cross-peaks in large and complex protein structures. The pulse schemes are designed to preserve the slowly decaying components of both ¹H–¹⁵N and methyl ¹³CH₃ spin-systems in the course of indirect evolution (t ₂) and acquisition period (t ₃) of 3D NOESY experiments. The methodology has been tested on {U-[¹⁵N,²H]; Ileδ1-[¹³CH₃]; Leu,Val-[¹³CH₃,¹²CD₃]}-labeled 82-kDa enzyme Malate Synthase G (MSG). A straightforward procedure that utilizes the symmetry of NOE cross-peaks in the time-shared 3D NOE data sets allows unambiguous assignments of more than 300 HN-methyl interactions in MSG from a single 3D data set providing important structural restraints for derivation of the backbone global fold.     

Reducing uncertainty in projections of extinction risk from climate change

Aim Concern over the implications of climate change for biodiversity has led to the use of species–climate ‘envelope’ models to forecast risks of species extinctions under climate change scenarios. Recent studies have demonstrated significant variability in model projections and there remains a need to test the accuracy of models and to reduce uncertainties. Testing of models has been limited by a lack of data against which projections of future ranges can be tested. Here we provide a first test of the predictive accuracy of such models using observed species’ range shifts and climate change in two periods of the recent past. Location Britain. Methods Observed range shifts for 116 breeding bird species in Britain between 1967 and 1972 (t₁) and 1987–91 (t₂) are used. We project range shifts between t₁ and t₂ for each species based on observed climate using 16 alternative models (4 methods × 2 data parameterizations × 2 rules to transform probabilities of occurrence into presence and absence records). Results Modelling results were extremely variable, with projected range shifts varying both in magnitude and in direction from observed changes and from each other. However, using approaches that explore the central tendency (consensus) of model projections, we were able to improve agreement between projected and observed shifts significantly. Conclusions Our results provide the first empirical evidence of the value of species–climate ‘envelope’ models under climate change and demonstrate reduction in uncertainty and improvement in accuracy through selection of the most consensual projections.     

Biomolecule Association Rates Do Not Provide a Complete Description of Bond Formation

The efficiency of many cell-surface receptors is dependent on the rate of binding soluble or surface-attached ligands. Much effort was exerted to measure association rates between soluble molecules (three-dimensional k_on) and, more recently, between surface-attached molecules (two-dimensional [2D] k_on). According to a generally accepted assumption, the probability of bond formation between receptors and ligands is proportional to the first power of encounter duration. Here we provide new experimental evidence and review published data demonstrating that this simple assumption is not always warranted. Using as a model system the (2D) interaction between ICAM-1-coated surfaces and flowing microspheres coated with specific anti-ICAM-1 antibodies, we show that the probability of bond formation may scale as a power of encounter duration that is significantly higher than 1. Further, we show that experimental data may be accounted for by modeling ligand-receptor interaction as a displacement along a single path of a rough energy landscape. Under a wide range of conditions, the probability that an encounter of duration t resulted in bond formation varied as erfc[(t₀/t)^1/2], where t₀ was on the order of 10 ms. We conclude that the minimum contact time for bond formation may be a useful parameter to describe a ligand-receptor interaction, in addition to conventional association rates.     

Characterization of a putative thioredoxin peroxidase prx1 of <Emphasis Type="Italic">Candida albicans</Emphasis>

In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H₂O₂ scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H₂O₂, menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H₂O₂. These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H₂O₂, but its cellular function is specified to t-BOOH.     

The Estimation of a Multivariate Fitness Function from Several Samples Taken from a Population

The situation is considered where the multivariate distribution of certain variables X₁, X₂, …, X_p is changing with time in a population because natural selection related to the X's is taking place. It is assumed that random samples taken from the population at times t₁, t₂, …, t_s are available and it is desirable to estimate the fitness function w_t(x₁, x₂,…,x_p) which shows how the number of individuals with X_i = x_i, i = 1, 2, …, p at time t is related to the number of individuals with the same X values at time zero. Tests for population changes are discussed and indices of the selection on the population dispersion and the population mean are proposed. The situation with a multivariate normal distribution is considered as a special case. A maximum likelihood method that can be applied with any form of population distribution is proposed for estimating w_t. The methods discussed in the paper are illustrated with data on four dimensions of male Egyptian skulls covering a time span from about 4500 B.C. to about 300 A.D. In this case there seems to have been very little selection on the population dispersion but considerable selection on means.     

Modeling the dynamics of a mutated stem-loop in the SL1 domain of HIV-1Lai genomic RNA by 1H-NOESY spectra

The cross-peaks of ¹H-NOESY spectra at different time delays are compared to a mode-coupling diffusion (MCD) calculation, including the evaluation of the full ¹H relaxation matrix, in the case of a 23 nucleotide fragment of the stem-loop SL1 domain of HIV-1_Lai genomic RNA mutated in a single position. The MCD theory gives significant agreement with ¹H relaxation experiments enabling a thorough understanding of the differential local dynamics along the sequence and particularly of the dynamics of nucleotides in the stem and in the loop. The differential dynamics of this hairpin structure is important in directing the dimerization of the retroviral genome, a fundamental step in the infectious process. The demonstration of a reliable use of time dependent NOE cross-peaks, largely available from NMR solution structure determination, coupled to MCD analysis, to probe the local dynamics of biological macromolecules, is a result of general interest of this paper.     

Note on a Test for Trend in Binomial Proportions Using Ranks

For a 2xk contingency table when all the k groups fall into a natural order and for which a sequence of k values × may be assigned, ARMITAGE (1955) has suggested a X²₍₁₎ test statistic for the hypothesis H_t of a trend in the proportions; This note considers a statistic for H_t based on midrank values for the binary observations. It is shown that the proposed rank-order test for H_t coincides with that of ARMITAGE (1955).     

Rhodopsin-transducin heteropentamer: three-dimensional structure and biochemical characterization

The process of vision is initiated when the G protein-coupled receptor, rhodopsin (Rho), absorbs a photon and transitions to its activated Rho^∗ form. Rho^∗ binds the heterotrimeric G protein, transducin (G_t) inducing GDP to GTP exchange and G_t dissociation. Using nucleotide depletion and affinity chromatography, we trapped and purified the resulting nucleotide-free Rho^∗·G_t complex. Quantitative SDS–PAGE suggested a 2:1 molar ratio of Rho^∗ to G_t in the complex and its mass determined by scanning transmission electron microscopy was 221 ± 12 kDa. A 21.6 Å structure was calculated from projections of negatively stained Rho^∗·G_t complexes. The molecular envelope thus determined accommodated two Rho molecules together with one G_t heterotrimer, corroborating the heteropentameric structure of the Rho^∗·G_t complex.     

Thick-to-Thin Filament Surface Distance Modulates Cross-Bridge Kinetics in Drosophila Flight Muscle

The demembranated (skinned) muscle fiber preparation is widely used to investigate muscle contraction because the intracellular ionic conditions can be precisely controlled. However, plasma membrane removal results in a loss of osmotic regulation, causing abnormal hydration of the myofilament lattice and its proteins. We investigated the structural and functional consequences of varied myofilament lattice spacing and protein hydration on cross-bridge rates of force development and detachment in Drosophila melanogaster indirect flight muscle, using x-ray diffraction to compare the lattice spacing of dissected, osmotically compressed skinned fibers to native muscle fibers in living flies. Osmolytes of different sizes and exclusion properties (Dextran T-500 and T-10) were used to differentially alter lattice spacing and protein hydration. At in vivo lattice spacing, cross-bridge attachment time (t_on) increased with higher osmotic pressures, consistent with a reduced cross-bridge detachment rate as myofilament protein hydration decreased. In contrast, in the swollen lattice, t_on decreased with higher osmotic pressures. These divergent responses were reconciled using a structural model that predicts t_on varies inversely with thick-to-thin filament surface distance, suggesting that cross-bridge rates of force development and detachment are modulated more by myofilament lattice geometry than protein hydration. Generalizing these findings, our results suggest that cross-bridge cycling rates slow as thick-to-thin filament surface distance decreases with sarcomere lengthening, and likewise, cross-bridge cycling rates increase during sarcomere shortening. Together, these structural changes may provide a mechanism for altering cross-bridge performance throughout a contraction-relaxation cycle.     

Position of residues in transmembrane peptides with respect to the lipid bilayer: A combined lipid NOEs and water chemical exchange approach in phospholipid bicelles

The model transmembrane peptide P16 (Ac-KKGLLLALLLLALLLALLLKKA-NH₂) was incorporated into small unaligned phospholipid bicelles, which provide a `native-like' lipid bilayer compatible with high-resolution solution NMR techniques. Using amide-water chemical exchange and amide-lipid cross-relaxation measurements, the interactions between P16 and bicelles were investigated. Distinctive intermolecular NOE patterns observed in band-selective 2D-NOESY spectra of bicellar solutions with several lipid deuteration schemes indicated that P16 is preferentially interacting with the `bilayered' region of the bicelle rather than with the rim. Furthermore, when amide-lipid NOEs were combined with amide-water chemical exchange cross-peaks of selectively ¹⁵N-labeled P16 peptides, valuable information was obtained about the position of selected residues relative to the membrane-water interface. Specifically, three main classes were identified. Class I residues lie outside the bilayer and show amide-water exchange cross-peaks but no amide-lipid NOEs. Class II residues reside in the bilayer-water interface and show both amide-water exchange cross-peaks and amide-lipid NOEs. Class III residues are embedded within the hydrophobic core of the membrane and show no amide-water exchange cross-peaks but strong amide-lipid NOEs.     

The Matched Pairs Problem with Exponential Variates

Two classes of tests for the hypothesis of bivariate symmetry are studied. For paired exponential survival times (t_1j, t_2j), the classes of tests are those based on t_1j-t_2j and those based on log t_1j–log t_2j. For each class the sign, signed ranks, t and likelihood ratio tests are compared via Pitman's criterion of asymptotic relative efficiency (ARE). For tests based on t_1j — t_2j, it is found that: (1) the efficacy of the paired t depends on the coefficient of variation (CV) of the pair means, (2) the signed rank test has the same ARE to the sign test as for the usual location problem. For tests based on log t_1j — log t_2j, the ARE comparisons reduce to the well-known results for the one-sample location problem for samples from a logistic density. Hence, the signed rank test is asymptotically efficient. Furthermore, analyses based on log t_1j — log t_2j are not complicated by the underlying pairing mechanism.     

Symmetry of a Vertebrate Muscle Basketweave Z-Band

The Z-band in vertebrate striated muscle links actin filaments of opposite polarity in adjacent sarcomeres to form a regular structure based on a tetragonal lattice. In transverse sections there are two commonly observed appearances of the Z-band seen in different muscles, namely, the small-square lattice and the basketweave forms. A clear example of the latter occurs in the fin muscle of the flatfish plaice and its symmetry is described here. Improved methods over previous work include fast freezing/freeze-substitution and lattice straightening of the scanned images. It is demonstrated here that when a longitudinal section is tilted in the electron microscope about the myofibril axis, the 10 and 01 projections are mirror images of each other about the centre of the Z-hand. By examining the symmetry relationships between these views and a longitudinal 11 projection and a transverse view, it is concluded that the symmetry is best described by the two-sided plane group c12. The twofold axis lies in the central plane of the Z-band along the diagonal of the primitive lattice and runs normal to the actin filaments. In contrast, the symmetry of the simple Z-band in fish myotomal white muscle, which in longitudinal sections has the appearance of a single zigzag structure, is p12₁ (Luther, P. K. (1991), J. Cell Biol. 113, 1043-1055).     

Total Relaxivities of Material Content in Various Cysts and Ameloblastoma: Implications for Discriminating Different Fluids

Nuclear magnetic resonance T ₁ and T ₂ relaxivities (r ₁ and r ₂) exhibit efficiency of a material to alter the relaxation rates (1/T ₁ and 1/T ₂), and they are being used for diagnostic purposes. The determination of total relaxivities (r _1t and r _2t) of cystic fluid content and ameloblastoma may therefore be useful for discriminative purposes. In order to determine what makes total relaxivities of hemorrhagic cysts, four sets of tubes containing pooled cyst were doped with increasing concentrations of iron, copper, albumin, and γ-globulins. These sets were replaced in a phantom together with six individual cysts and one ameloblastoma. The relaxation times were measured by magnetic resonance imaging operating at 1.5 T. The relaxivities of individual ions and proteins were determined from the slope of the relation between relaxation rates and concentration, while total relaxivities were determined by using the increases in relaxation rates and material content of cystic fluid (MC). Iron, copper, albumin, and γ-globulins were found to be the sources of r _1t and r _2t. Each of r _1t, r _2t, r _1tMC, r _2tMC, and r _2t/r _1t are distinctive parameters for each cystic category and ameloblastoma. Except for MC, the parameters measured for ameloblastoma are significantly smaller than those of cysts. The similarity of the present results to those used in clinical applications suggests that each of r _1t, r _2t, r _1tMC, r _2tMC, and r _2t/r _1t has an ability to discriminate various fluids and masses. The present work also suggests that r _1tMC, r _2tMC, and r _2t/r _1t can be determined in vivo.     

Fast reconstruction of four-dimensional NMR spectra from plane projections

Four-dimensional NMR spectroscopy can be speeded up by a large factor by a projection-reconstruction technique related to that used in X-ray tomography. The limited amount of information recorded in a few suitably-tilted projection planes suffices to recreate the entire four-dimensional spectrum. The method is demonstrated by reference to the 500 MHz spectrum of 1 millimolar aqueous solution of ubiquitin by dispersing the responses of the three-dimensional HNCO spectrum into the C(alpha) dimension of a four-dimensional HNCOCA spectrum obtained by projection-reconstruction.     

Optimizing resolution in multidimensional NMR by three-way decomposition

Resolution depends on the number of points sampled in a FID; in indirectly detected dimensions it is an important determinant of the total experiment time. Based on the high redundancy present in NMR data, we propose the following timesaving scheme for three-dimensional spectra. An extensive grid of discrete t1- and t2-values is used, which increases resolution while preserving the spectral width. Total experiment time is reduced by avoiding the recording of t3-FIDs for selected pairs of t1 and t2; typically the recording is omitted for about 75% of the (t1,t2) combinations. These data sets are referred to as sparse, and post-experimental processing making optimal use of spectral redundancy provides the missing, non-recorded data. We have previously shown that three-way decomposition (TWD) within the MUNIN approach provides a practical way to process dense NMR data sets. Here, a novel TWD algorithm [Ibraghimov, (2002) Numer. Linear Algebra Appl. 9, 551–565] is used to complement a sparselyrecorded time-domain data set by providing the missing FIDs for all (t1,t2) combinations omitted in the experiment. A necessary condition is that for each t1-value at least a few FIDs are recorded, and similar for each t2-value. The method is demonstrated on non-uniformly sampled ¹⁵N-NOESY-HSQC data sets recorded for the 14 kD protein azurin. The spectra obtained by TWD, reconstruction and ordinary transform to frequency-domain are, in spite of the large number of signals and the high dynamic range typical for NOESYs, highly similar to a corresponding reference spectrum, for which all (t1,t2) combinations were recorded.     

Crystallization and Analyses of Crystals of Various Chemotypes of R-Form Lipopolysaccharides from Salmonella Spp.

Various chemotypes (Re, Rd₂, Rd₁P^–, Rd₁, RcP^–, Rc, Rb₃, Rb₂, Rb₁, and Ra) of R-form lipopolysaccharides (LPSs) of Salmonella spp. were crystallized by treatment with 70% ethanol containing 250 mM MgCl₂, and crystals of the LPSs were observed electron microscopically and analyzed by electron diffraction and synchrotron X-ray diffraction. All the LPSs tested formed three-dimensional crystals showing very similar shapes; hexagonal plate, solid column, discoid, square or rectangular plate, lozenge plate and truncated hexangular or rectangular pyramid forms. Electron diffraction patterns from the hexagonal plate crystals of all these LPSs obtained by electron irradiation from the direction perpendicular to the basal plane showed that they consist of hexagonal lattices with the lattice constant of 4.62 Å. The crystals of all the LPSs thus formed gave ring-like X-ray diffraction patterns because of their small sizes. The long-axis values were calculated from the X-ray diffraction patterns from crystals of all the LPSs in the low-angle region and they corresponded roughly to the length of the proposed primary chemical structures of the R cores of the LPSs. The volume occupied by a single molecule of all the LPSs were calculated from the molecular weights based on the proposed structures and the crystallographic data obtained by electron diffraction, X-ray diffraction, and density determination.     

Galerkin-Ritz procedures for approximate solutions to systems of reaction-diffusion equations

For any essentially nonlinear system of reaction-diffusion equations of the generic form ∂c_i/∂t=D_i∇²c_i+Q_i(c,x,t) supplemented with Robin type boundary conditions over the surface of a closed bounded three-dimensional region, it is demonstrated that all solutions for the concentration distributionn-tuple function c=(c ₁(x,t),...,c _n (x,t)) satisfy a differential variational condition. Approximate solutions to the reaction-diffusion intial-value boundary-value problem are obtainable by employing this variational condition in conjunction with a Galerkin-Ritz procedure. It is shown that the dynamical evolution from a prescribed initial concentrationn-tuple function to a final steady-state solution can be determined to desired accuracy by such an approximation method. The variational condition also admits a systematic Galerkin-Ritz procedure for obtaining approximate solutions to the multi-equation elliptic boundary-value problem for steady-state distributions c=−c(x). Other systems of phenomenological (non-Lagrangian) field equations can be treated by Galerkin-Ritz procedures based on analogues of the differential variational condition presented here. The method is applied to derive approximate nonconstant steady-state solutions for ann-species symbiosis model.     

A model approach to project the start of egg laying of Great Tit (Parus major L.) in response to climate change

The aim of this study was to select a phenological model that is able to calculate the beginning of egg laying of Great Tit (Parus major) for both current and future climate conditions. Four models (M1–M4) were optimised on long-term phenological observations from the Ecological Research Centre Schlüchtern (Hessen/Germany). Model M1 was a common thermal time model that accumulates growing degree days (GDD) on an optimised starting date t ₁. Since egg laying of Great Tit is influenced not only by air temperature but also by photoperiod, model M1 was extended by a daylength term to give M2. The other two models, M3 and M4, correspond to M1 and M2, but t ₁ was intentionally set to 1 January, in order to consider already rising temperatures at the beginning of the year. A comparison of the four models led to following results: model M1 had a relatively high root mean square error at verification (RMSE_ver) of more than 4 days and can be used only to calculate the start of egg laying for current climate conditions because of the relatively late starting date for GDD calculation. The model failed completely if the starting date was set to 1 January (M3). Consideration of a daylength term in models M2 and M4 improved the performance of both models strongly (RMSE_ver of only 3 days or less), increased the credibility of parameter estimation, and was a precondition to calculate reliable projections in the timing of egg laying in birds for the future. These results confirm that the start of egg laying of Great Tit is influenced not only by air temperature, but also by photoperiod. Although models M2 and M4 both provide comparably good results for current climate conditions, we recommend model M4–with a starting date of temperature accumulation on 1 January–for calculating possible future shifts in the commencement of egg laying. Our regional projections in the start of egg laying, based on five regional climate models (RCMs: REMO-UBA, ECHAM5-CLM, HadCM3-CLM, WETTREG-0, WETTREG-1, GHG emission scenario A1B), indicate that in the near future (2011–2040) no significant change will take place. However, in the mid- (2041–2070) and long-term (2071–2100) range the beginning of egg laying could be advanced significantly by up to 11 days on average of all five RCMs. This result corresponds to the already observed shift in the timing of egg laying by about 1 week, due mainly to an abrupt increase in air temperature at the end of the 1980s by 1.2 K between April and May. The use of five regional climate scenarios additionally allowed to estimate uncertainties among the RCMs.