Objectivity for the research worker.

In the last decade, many problematic cases of scientific conduct have been diagnosed; some of which involve outright fraud (e.g., Stapel, 2012) others are more subtle (e.g., supposed evidence of extrasensory perception; Bem, 2011). These and similar problems can be interpreted as caused by lack of scientific objectivity. The current philosophical theories of objectivity do not provide scientists with conceptualizations that can be effectively put into practice in remedying these issues. We propose a novel way of thinking about objectivity for individual scientists; a negative and dynamic approach.We provide a philosophical conceptualization of objectivity that is informed by empirical research. In particular, it is our intention to take the first steps in providing an empirically and methodologically informed inventory of factors that impair the scientific practice. The inventory will be compiled into a negative conceptualization (i.e., what is not objective), which could in principle be used by individual scientists to assess (deviations from) objectivity of scientific practice. We propose a preliminary outline of a usable and testable instrument for indicating the objectivity of scientific practice.

Sensitized photo-oxidation of plant cytokinin-specific binding protein - Does the environment of the thioether group influence the oxidation reaction? From primary intermediates to stable products.

The reactions of protein oxidation play a significant role in many biological processes, especially in diseases development. Therefore, it is important to understand, how the protein molecule behaves in the presence of oxidants. In the present work, photo-oxidation of phytohormone-binding plant protein (VrPhBP) was investigated using light and 3-carboxybenzophenone (3CB) as a sensitizer (one electron oxidant). The protein interacts with the sensitizer in the ground state forming a weak binding complex leading to the presence of bound and free 3CB in solution. The early events and transient species (such as radicals and radical ions) formed during irradiation were characterised by transient spectroscopy showing the formation of the sulphur radical cation Met>S●+ (stabilized by (S∴N)+)and the tyrosyl radical TyrO● on VrPhBP. Thus the 3CB excited triplet state was quenched by the Met and Tyr residues and mostly by Met (based on the deconvoluted transient absorption spectra).The presence of a Tyr side chain in the vicinity of a Met residue results in intramolecular electron transfer from Tyr to the Met>S●+ radical cation, leading to regeneration of the thioether side chain and formation of TyrO●. The presence of other side chains close to Met, such as Arg or Lys can induce the stabilization of Met>S●+ via the formation of two-centered three-electron bonded species (S∴N)+. The transient species were additionally confirmed by stable product analysis. Based on SDS-PAGE, chromatography and mass spectrometry, the formation of methionine sulphoxide and Met-3CB adduct was identified together with di-Tyr cross links. On the basis of the experimental results the overall mechanism of VrPhBP photo-oxidation, from its early events to the formation of stable products, is described. In addition, a good correlation between the mechanisms of photooxidation of model compounds such as Met derivatives and peptides and those for real biological systems is emphasized.

In-vitro anti-fungal assay and association analysis reveal a role for the Pinus monticola PR10 gene (PmPR10-3.1) in quantitative disease resistance to white pine blister rust.

Pathogenesis-related (PR) proteins play important roles in plant defense response. However, functional investigation of PR10 genes is still limited and their physiological roles have not been conclusively characterized in biological processes of conifer trees. Here, we identified multiple novel members in the western white pine (Pinus monticola) PmPR10 family by bioinformatic mining available transcriptomic data. Phylogenetic analysis of protein sequences revealed four PR10 and two PR10-like clusters with a high synteny across different species of five-needle pines. Of 10 PmPR10 genes, PmPR10-3.1 was selected and expressed in Escherichia coli. The purified recombinant protein exhibited inhibitory effects on spore hyphal growth of fungal pathogens Cronartium ribicola, Phoma exigua, and Phoma argillacea by in-vitro anti-fungal analysis. Genetic variation analysis detected a total of 21 single nucleotide polymorphisms (SNPs) within PmPR10-3.1 in a collection of P. monticola seed families. A nonsynonymous SNP (t178g) showed significant association with relative levels of quantitative disease resistance (QDR), explaining about 8.7% of phenotypic variation as the peak value across all SNPs. Our results provide valuable insight into the genetic architecture underlying P. monticola QDR and imply that PmPR10-3.1 may function as an important component in conifer basal immunity for non-specific resistance to a wide spectrum of pathogens.

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity.

Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31-Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

PR-10 proteins as potential mediators of melatonin-cytokinin cross-talk in plants: crystallographic studies of LlPR-10.2B isoform from yellow lupine.

LlPR-10.2B, a Pathogenesis-related class 10 (PR-10) protein from yellow lupine (Lupinus luteus) was crystallized in complex with melatonin, an emerging important plant regulator and antioxidant. The structure reveals two molecules of melatonin bound in the internal cavity of the protein, plus a very well-defined electron density near the cavity entrance, corresponding to an unknown ligand molecule comprised of two flat rings, which is most likely a product of melatonin transformation. In a separate LlPR-10.2B co-crystallization experiment with an equimolar mixture of melatonin and trans-zeatin, which is a cytokinin phytohormone well recognized as a PR-10-binding partner, a quaternary 1 : 1 : 1 : 1 complex was formed, in which one of the melatonin-binding sites has been substituted with trans-zeatin, whereas the binding of melatonin at the second binding site and binding of the unknown ligand are undisturbed. This unusual complex, when compared with the previously described PR-10/trans-zeatin complexes and with the emerging structural information about melatonin binding by PR-10 proteins, provides intriguing insights into the role of PR-10 proteins in phytohormone regulation in plants, especially with the involvement of melatonin, and implicates the PR-10 proteins as low-affinity melatonin binders under the conditions of elevated melatonin concentration. DATABASES: Atomic coordinates and processed structure factors corresponding to the final models of the LlPR-10.2B/melatonin and LlPR-10.2B/melatonin + trans-zeatin complexes have been deposited with the Protein Data Bank (PDB) under the accession codes 5MXB and 5MXW. The corresponding raw X-ray diffraction images have been deposited in the RepOD Repository at the Interdisciplinary Centre for Mathematical and Computational Modelling (ICM) of the University of Warsaw, Poland, and are available for download with the following Digital Object Identifiers (DOI): https://doi.org/10.18150/repod.9923638 and https://doi.org/10.18150/repod.6621013.

Crystallographic and CD probing of ligand-induced conformational changes in a plant PR-10 protein.

Plant pathogenesis-related class 10 (PR-10) proteins are a family of abundant proteins initially identified as elements of the plant defense system. The key structural feature suggesting PR-10 functionality is a huge hydrophobic cavity created in the protein interior by a scaffold composed of an extended ß-sheet wrapped around a long and flexible C-terminal α-helix. Several crystallographic and NMR studies have shown that the cavity can accommodate a variety of small molecule ligands, including phytohormones. The article describes ∼1.3 Å resolution crystal structures of a Lupinus luteus PR-10 isoform LlPR-10.1A, in its free form and in complex with trans-zeatin, a naturally occurring plant hormone belonging to the cytokinin group. Moreover we present the structure of the same protein where the saturation with zeatin is not complete. This set of three crystal structures allows us to track the structural adaptation of the protein upon trans-zeatin docking, as well as the sequence of the ligand-binding events, step-by-step. In addition, titration of LlPR-10.1A with trans-zeatin monitored in solution by CD spectra, confirmed the pattern of structural adaptations deduced from the crystallographic studies. The ligand-biding mode shows no similarity to other zeatin complexes of PR-10 proteins. The present work, which describes the first atomic models of the same PR-10 protein with and without a physiological ligand, reveals that the conformation of LlPR-10.1A undergoes a significant structural rearrangement upon trans-zeatin binding.

Specific binding of gibberellic acid by cytokinin-specific binding proteins: a new aspect of plant hormone-binding proteins with the PR-10 fold.

Pathogenesis-related proteins of class 10 (PR-10) are a family of plant proteins with the same fold characterized by a large hydrophobic cavity that allows them to bind various ligands, such as phytohormones. A subfamily with only ~20% sequence identity but with a conserved canonical PR-10 fold have previously been recognized as Cytokinin-Specific Binding Proteins (CSBPs), although structurally the binding mode of trans-zeatin (a cytokinin phytohormone) was found to be quite diversified. Here, it is shown that two CSBP orthologues from Medicago truncatula and Vigna radiata bind gibberellic acid (GA3), which is an entirely different phytohormone, in a conserved and highly specific manner. In both cases a single GA3 molecule is found in the internal cavity of the protein. The structural data derived from high-resolution crystal structures are corroborated by isothermal titration calorimetry (ITC), which reveals a much stronger interaction with GA3 than with trans-zeatin and pH dependence of the binding profile. As a conclusion, it is postulated that the CSBP subfamily of plant PR-10 proteins should be more properly linked with general phytohormone-binding properties and termed phytohormone-binding proteins (PhBP).

Analysis of boutique arrays: a universal method for the selection of the optimal data normalization procedure.

DNA microarrays, which are among the most popular genomic tools, are widely applied in biology and medicine. Boutique arrays, which are small, spotted, dedicated microarrays, constitute an inexpensive alternative to whole-genome screening methods. The data extracted from each microarray-based experiment must be transformed and processed prior to further analysis to eliminate any technical bias. The normalization of the data is the most crucial step of microarray data pre-processing and this process must be carefully considered as it has a profound effect on the results of the analysis. Several normalization algorithms have been developed and implemented in data analysis software packages. However, most of these methods were designed for whole-genome analysis. In this study, we tested 13 normalization strategies (ten for double-channel data and three for single-channel data) available on R Bioconductor and compared their effectiveness in the normalization of four boutique array datasets. The results revealed that boutique arrays can be successfully normalized using standard methods, but not every method is suitable for each dataset. We also suggest a universal seven-step workflow that can be applied for the selection of the optimal normalization procedure for any boutique array dataset. The described workflow enables the evaluation of the investigated normalization methods based on the bias and variance values for the control probes, a differential expression analysis and a receiver operating characteristic curve analysis. The analysis of each component results in a separate ranking of the normalization methods. A combination of the ranks obtained from all the normalization procedures facilitates the selection of the most appropriate normalization method for the studied dataset and determines which methods can be used interchangeably.

Medicago truncatula histidine-containing phosphotransfer protein: structural and biochemical insights into the cytokinin transduction pathway in plants.

Histidine-containing phosphotransfer proteins (HPts) take part in hormone signal transduction in higher plants. The overall pathway of this process is reminiscent of the two-component system initially identified in prokaryotes. HPts function in histidine-aspartate phosphorelays in which they mediate the signal from sensory kinases (usually membrane proteins) to RRs in the nucleus. Here, we report the crystal structure of an HPt protein from Medicago truncatula (MtHPt1) determined at 1.45 Å resolution and refined to an R-factor of 16.7% using low-temperature synchrotron-radiation X-ray diffraction data. There is one MtHPt1 molecule in the asymmetric unit of the crystal lattice with P2(1)2(1)2(1) symmetry. The protein fold consists of six α helices, four of which form a C-terminal helix bundle. The coiled-coil structure of the bundle is stabilized by a network of S-aromatic interactions involving highly conserved sulfur-containing residues. The structure reveals a solvent-exposed side chain of His79, which is the phosphorylation site, as demonstrated by autoradiography combined with site-directed mutation. It is surrounded by highly conserved residues present in all plant HPts. These residues form a putative docking interface for either the receiver domain of the sensory kinase, or for the RR. The biological activity of MtHPt1 was tested by autoradiography. It demonstrated phosphorylation by the intracellular kinase domain of the cytokinin receptor MtCRE1. Complex formation between MtHPt1 and the intracellular fragment of MtCRE1 was confirmed by thermophoresis, with a dissociation constant K(d) of 14 µM.

Structural and functional aspects of PR-10 proteins.

Physical, chemical and biological stress factors, such as microbial infection, upregulate the transcription levels of a number of plant genes, coding for the so-called pathogenesis-related (PR) proteins. For PR proteins of class-10 (PR-10), the biological function remains unclear, despite two decades of scientific research. PR-10 proteins have a wide distribution throughout the plant kingdom and the class members share size and secondary structure organization. Throughout the years, we and other groups have determined the structures of a number of PR-10 proteins, both in the crystalline state by X-ray diffraction and in solution by NMR spectroscopy. Despite the accumulating structural information, our understanding of PR-10 function is still limited. PR-10 proteins are rather small (~ 160 amino acids) with a fold consisting of three α helices and seven antiparallel ß strands. These structural elements enclose a large hydrophobic cavity that is most probably the key to their functional relevance. Also, the outer surface of these proteins is of extreme interest, as epitopes from a PR-10 subclass cause allergic reactions in humans.

Crystal structures of NodS N-methyltransferase from Bradyrhizobium japonicum in ligand-free form and as SAH complex.

NodS is an S-adenosyl-L-methionine (SAM)-dependent N-methyltransferase that is involved in the biosynthesis of Nod factor (NF) in rhizobia, which are bacterial symbionts of legume plants. NF is a modified chitooligosaccharide (COS) signal molecule that is recognized by the legume host, where it initiates symbiotic processes leading to atmospheric nitrogen fixation. We report the crystal structure of recombinant NodS protein from Bradyrhizobium japonicum, which infects lupine and serradella legumes. Two crystal forms--ligand-free NodS and NodS in complex with S-adenosyl-L-homocysteine, which is a by-product of the methylation reaction--were obtained, and their structures were refined to resolutions of 2.43 Å and 1.85 Å, respectively. Although the overall fold (consisting of a seven-stranded ß-sheet flanked by layers of helices) is similar to those of other SAM-dependent methyltransferases, NodS has specific features reflecting the unique character of its oligosaccharide substrate. In particular, the N-terminal helix and its connecting loop get ordered upon SAM binding, thereby closing the methyl donor cavity and shaping a long surface canyon that is clearly the binding site for the acceptor molecule. Comparison of the two structural forms of NodS suggests that there are also other conformational changes taking place upon the binding of the donor substrate. As an enzyme that methylates a COS substrate, NodS is the first example among all SAM-dependent methyltransferases to have its three-dimensional structure elucidated. Gaining insight about how NodS binds its donor and acceptor substrates helps to better understand the mechanism of NodS activity and the basis of its functional difference in various rhizobia.

Crystal structure of Hyp-1, a St. John's wort protein implicated in the biosynthesis of hypericin.

Hypericin, a red-colored naphtodianthrone, is a natural product synthesized in the medicinal plant Hypericum perforatum, widely known as St. John's wort. Hypericin has been attracting a growing attention of the pharmaceutical industry because of its potential application in various therapies, including the treatment of depression. In vivo, hypericin is synthesized by dimerization of emodin in a complicated multistep reaction that is reportedly catalyzed by a small (17.8kDa) protein, Hyp-1. Based on relatively low sequence similarity ( approximately 50%), Hyp-1 has been tentatively classified as a plant PR-10 (pathogenesis-related class 10) protein. Members of the PR-10 family are ubiquitous plant proteins associated with stress control and tissue differentiation but with no clearly understood molecular mechanism. They have, however, a well-defined folding canon, consisting of an extended antiparallel beta-sheet wrapped around a C-terminal alpha-helix, enclosing in the protein interior a huge cavity, in which various hydrophobic ligands can be bound. Apart from Hyp-1, only two other PR-10 members have been found to possess enzymatic activity (S-norcoclaurine synthase and TcmN aromatase/cyclase). In this paper, we report a high-resolution crystal structure of Hyp-1, confirming that it indeed has a PR-10 fold. The protein binds multiple polyethylene glycol molecules, some of which occupy the hydrophobic cavity. The crystallographic model illustrates a high degree of conformational adaptability of both interacting partners for efficient binding. We have been unable, however, to dimerize emodin to hypericin using Hyp-1 as biocatalyst. This puzzling result does not have a clear explanation at this time.

Cytokinin-induced structural adaptability of a Lupinus luteus PR-10 protein.

Plant pathogenesis-related (PR) proteins of class 10 are the only group among the 17 PR protein families that are intracellular and cytosolic. Sequence conservation and the wide distribution of PR-10 proteins throughout the plant kingdom are an indication of an indispensable function in plants, but their true biological role remains obscure. Crystal and solution structures for several homologues have shown a similar overall fold with a vast internal cavity which, together with structural similarities to the steroidogenic acute regulatory protein-related lipid transfer domain and cytokinin-specific binding proteins, strongly indicate a ligand-binding role for the PR-10 proteins. This article describes the structure of a complex between a classic PR-10 protein [Lupinus luteus (yellow lupine) PR-10 protein of subclass 2, LlPR-10.2B] and N,N'-diphenylurea, a synthetic cytokinin. Synthetic cytokinins have been shown in various bioassays to exhibit activity similar to that of natural cytokinins. The present 1.95 A resolution crystallographic model reveals four N,N'-diphenylurea molecules in the hydrophobic cavity of the protein and a degree of conformational changes accompanying ligand binding. The structural adaptability of LlPR-10.2B and its ability to bind different cytokinins suggest that this protein, and perhaps other PR-10 proteins as well, can act as a reservoir of cytokinin molecules in the aqueous environment of a plant cell.

Cloning, expression, purification, crystallization and preliminary X-ray analysis of NodS N-methyltransferase from Bradyrhizobium japonicum WM9.

The Nod factor (NF) is a rhizobial signal molecule that is involved in recognition of a legume host and the formation of root and stem nodules. Some unique enzymes are involved in the biosynthesis of NF, which is a variously but specifically substituted lipochitooligosaccharide. One of these enzymes is NodS, an N-methyltransferase that methylates end-deacetylated chitooligosaccharide substrates. In the methylation reaction, NodS uses S-adenosyl-L-methionine (SAM) as a methyl donor. To date, no structural information is available about NodS from any rhizobium. X-ray crystallographic studies of the NodS protein from Bradyrhizobium japonicum WM9, which infects the legumes lupin and serradella, have been undertaken. The nodS gene was cloned and the recombinant protein was expressed in Escherichia coli cells using natural amino acids and as an SeMet derivative. NodS without ligands was crystallized in the presence of PEG 3350 and MgCl(2). The protein was also crystallized in complex with S-adenosyl-L-homocysteine (SAH) in the presence of PEG 8000 and MgCl(2). SAH is produced from SAM as a byproduct of the methylation reaction. The crystals of apo NodS are tetragonal and diffracted X-rays to 2.42 A resolution. The NodS-SAH complex crystallizes in an orthorhombic space group and the crystals diffracted X-rays to 1.85 A resolution.

MAD phasing using the (Ta6Br12)2+ cluster: a retrospective study.

The crystal structure of cytokinin-specific binding protein (CSBP) containing four independent molecules with 4 x 155 = 620 residues in the asymmetric unit of the P6(4) unit cell has been solved by three-wavelength MAD using 1.8 angstroms resolution data recorded from a crystal derivatized with the dodecabromohexatantalum cation (Ta6Br12)2+. The diffraction data contained a very strong anomalous signal (allowing successful phasing even using peak SAD data alone) despite the fact that the five (Ta6Br12)2+ clusters found in the asymmetric unit have low occupancy (about 0.3). The derivative structure has been successfully refined to R = 0.158, providing interesting details on the geometry of the (Ta6Br12)2+ cluster, its interactions with the protein and on the backsoaking of a cytokinin ligand that was originally part of a CSBP-cytokinin complex in the native crystals used for (Ta6Br12)2+ derivatization. A simulation analysis of the phasing power of the (Ta6Br12)2+ ions at artificially imposed resolution limits shows that it is not possible to resolve the individual Ta atoms if the dmin limit of the data is higher than 2.9 angstroms. Additionally, for successful Ta identification the (Ta6Br12)2+ complex should be specifically bound and ordered. Good binding at the protein surface is facilitated by the presence of acidic groups, indicating higher pH buffer conditions to be preferable. In addition, the water channels in the crystal should be sufficiently wide (at least 11 angstroms) to allow free diffusion of the (Ta6Br12)2+ ions on soaking. A retrospective look at the initial molecular-replacement calculations provides interesting insights into how the peculiar packing mode and strong bias of the molecular-replacement-phased electron-density maps had hindered successful solution of the structure by this method.

Crystallization and preliminary crystallographic studies of Hyp-1, a St John's wort protein implicated in the biosynthesis of hypericin.

According to a debated hypothesis, the biosynthesis from emodin of the medicinally important natural compound hypericin is catalyzed in St John's wort (Hypericum perforatum) by the phenolic oxidative-coupling protein Hyp-1. Recombinant St John's wort Hyp-1 has been overexpressed in Escherichia coli and obtained in single-crystal form. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell parameters a = 37.5, b = 76.7, c = 119.8 A, contain two protein molecules in the asymmetric unit and diffract X-rays to 1.73 A resolution.

Lupinus luteus pathogenesis-related protein as a reservoir for cytokinin.

Plant pathogenesis-related (PR) proteins of class 10 (PR-10) are small and cytosolic. The main feature of their three-dimensional structure is a large cavity between a seven-stranded antiparallel beta-sheet and a long C-terminal alpha-helix. Although PR-10 proteins are abundant in plants, their physiological role remains unknown. Recent data have indicated ligand binding as their possible biological function. The article describes the structure of a complex between a classic PR-10 protein (yellow lupine LlPR-10.2B) and the plant hormone, trans-zeatin. Previously, trans-zeatin binding has been reported in a structurally related cytokinin-specific binding protein, which has a distant sequence relation with classic PR-10 proteins. In the present 1.35 A resolution crystallographic model, three perfectly ordered zeatin molecules are found in the binding cavity of the protein. The fact that three zeatin molecules are bound by the protein when only a fourfold molar excess of the ligand was used indicates an unusual type of affinity for this ligand and suggests that LlPR-10.2B, and perhaps other PR-10 proteins as well, acts as a reservoir of cytokinin molecules in the aqueous environment of the cell.

Structural and functional characteristics of two novel members of pathogensis-related multigene family of class 10 from yellow lupine+.

PR-10 proteins (pathogensis-related), ubiquitous within the plant kingdom, are usually encoded by multigene families. To date we have identified 10 homologous pr-10 genes in a yellow lupine cDNA library. Here, the structure and expression of two newly identified yellow lupine pr-10 genes (LlYpr10-2b and LlYpr10-2f) are presented. Many potential regulatory sites were found in both gene promoters including common ones as well as those unique for each gene. However, promoter deletion analysis in transgenic tobacco plants revealed similar patterns of reporter gene (gus) expression. Shortened fragments of both gene promoters studied caused high GUS activity in leaves (along vascular bundles), stamen stigma, anthers and pollen grains. When conjugated with longer LlYpr-10.2 promoter fragments, GUS was additionally present in petal edges. Only a long fragment of the LlYpr10-2b gene promoter caused GUS expression in the stem. In yellow lupine the pr-10.2 genes are present in all studied organs, but their level of expression depends on the stage of development and is affected by wounding, oxidative stress and salicylic acid treatment. Silencing of the Llpr-10.2b gene in 4-week-old yellow lupine plants did not lead to any visible symptoms, which suggests that the function of the silenced gene is supplemented by its close homologues, still present in the studied plants.

Selection in a cycling population: differential response among skeletal traits.

Population density cycles influence phenotypic evolution through both density-dependent selection during periods of high density and through enhanced genetic drift during periods of low density. We investigated the response of different phenotypic traits to the same density cycles in a population of the yellow-necked mouse, Apodemus flavicollis, from Bialowieza National Park in Poland. We examined nonmetric skull traits, skull and mandible size, skull and mandible shape, and transferrin allele frequencies. We found that all of the traits changed significantly over the seven-year study period. The greatest changes in nonmetric traits and mandible size occurred during periods of increasing density, and the magnitude of changes in skull and mandible shape was correlated with the magnitude of density changes. Frequencies of transferrin alleles changed the most when population density was in decline. Changes among the five phenotypic traits were generally uncorrelated with one another, except for skull and mandible shape. Nonmetric traits were selectively neutral when assessed with Q(ST)/F(ST) analysis, whereas mandible size, mandible shape, and skull shape showed evidence of fairly strong selection. Selection on skull size was weak or nonexistent. We discuss how different assumptions about the genetic components of variance affect Q(ST) estimates when phenotypic variances are substituted for genetic ones. We also found that change in mandible size, mandible shape, skull size, and skull shape were greater than expected under a neutral model given reasonable assumptions about heritability and effective population size.

Crystal structure of Vigna radiata cytokinin-specific binding protein in complex with zeatin.

The cytosolic fraction of Vigna radiata contains a 17-kD protein that binds plant hormones from the cytokinin group, such as zeatin. Using recombinant protein and isothermal titration calorimetry as well as fluorescence measurements coupled with ligand displacement, we have reexamined the K(d) values and show them to range from approximately 10(-6) M (for 4PU30) to 10(-4) M (for zeatin) for 1:1 stoichiometry complexes. In addition, we have crystallized this cytokinin-specific binding protein (Vr CSBP) in complex with zeatin and refined the structure to 1.2 A resolution. Structurally, Vr CSBP is similar to plant pathogenesis-related class 10 (PR-10) proteins, despite low sequence identity (<20%). This unusual fold conservation reinforces the notion that classic PR-10 proteins have evolved to bind small-molecule ligands. The fold consists of an antiparallel beta-sheet wrapped around a C-terminal alpha-helix, with two short alpha-helices closing a cavity formed within the protein core. In each of the four independent CSBP molecules, there is a zeatin ligand located deep in the cavity with conserved conformation and protein-ligand interactions. In three cases, an additional zeatin molecule is found in variable orientation but with excellent definition in electron density, which plugs the entrance to the binding pocket, sealing the inner molecule from contact with bulk solvent.