An investigation into effects of long-distance seed dispersal on organelle population genetic structure and colonization rate: a model analysis

A simulation-based modelling approach is used to examine the effects of stratified seed dispersal (representing the distribution of the majority of dispersal around the maternal parent and also rare long-distance dispersal) on the genetic structure of maternally inherited genomes and the colonization rate of expanding plant populations. The model is parameterized to approximate postglacial oak colonization in the UK, but is relevant to plant populations that exhibit stratified seed dispersal. The modelling approach considers the colonization of individual plants over a large area (three 500 km x 10 km rolled transects are used to approximate a 500 km x 300 km area). Our approach shows how the interaction of plant population dynamics with stratified dispersal can result in a spatially patchy haplotype structure. We show that while both colonization speeds and the resulting genetic structure are influenced by the characteristics of the dispersal kernel, they are robust to changes in the periodicity of long-distance events, provided the average number of long-distance dispersal events remains constant. We also consider the effects of additional physical and environmental mechanisms on plant colonization. Results show significant changes in genetic structure when the initial colonization of different haplotypes is staggered over time and when a barrier to colonization is introduced. Environmental influences on survivorship and fecundity affect both the genetic structure and the speed of colonization. The importance of these mechanisms in relation to the postglacial spread and genetic structure of oak in the UK is discussed.

Structural analysis and molecular model of a self-incompatibility RNase from wild tomato

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S-3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S-3-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S-3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S-3-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.

SVMtm: Support vector machines to predict transmembrane segments

A new method has been developed for prediction of transmembrane helices using support vector machines. Different coding schemes of protein sequences were explored, and their performances were assessed by crossvalidation tests. The best performance method can predict the transmembrane helices with sensitivity of 93.4% and precision of 92.0%. For each predicted transmembrane segment, a score is given to show the strength of transmembrane signal and the prediction reliability. In particular, this method can distinguish transmembrane proteins from soluble proteins with an accuracy of similar to99%. This method can be used to complement current transmembrane helix prediction methods and can be Used for consensus analysis of entire proteomes . The predictor is located at http://genet.imb.uq.edu.au/predictors/ SVMtm. (C) 2004 Wiley Periodicals, Inc.

SCORPION2: A database for structure-function analysis of scorpion toxins

Scorpion toxins are important experimental tools for characterization of vast array of ion channels and serve as scaffolds for drug design. General public database entries contain limited annotation whereby rich structure-function information from mutation studies is typically not available. SCORPION2 contains more than 800 records of native and mutant toxin sequences enriched with binding affinity and toxicity information, 624 three-dimensional structures and some 500 references. SCORPION2 has a set of search and prediction tools that allow users to extract and perform specific queries: text searches of scorpion toxin records, sequence similarity search, extraction of sequences, visualization of scorpion toxin structures, analysis of toxic activity, and functional annotation of previously uncharacterized scorpion toxins. The SCORPION2 database is available at http://sdmc.i2r.a-star.edu.sg/scorpion/. (c) 2006 Elsevier Ltd. All rights reserved.

3D protein structure matching by patch signatures

For determining functionality dependencies between two proteins, both represented as 3D structures, it is an essential condition that they have one or more matching structural regions called patches. As 3D structures for proteins are large, complex and constantly evolving, it is computationally expensive and very time-consuming to identify possible locations and sizes of patches for a given protein against a large protein database. In this paper, we address a vector space based representation for protein structures, where a patch is formed by the vectors within the region. Based on our previews work, a compact representation of the patch named patch signature is applied here. A similarity measure of two patches is then derived based on their signatures. To achieve fast patch matching in large protein databases, a match-and-expand strategy is proposed. Given a query patch, a set of small k-sized matching patches, called candidate patches, is generated in match stage. The candidate patches are further filtered by enlarging k in expand stage. Our extensive experimental results demonstrate encouraging performances with respect to this biologically critical but previously computationally prohibitive problem.

Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor

Two synthetic analogues of murine epidermal. growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its H-1 chemical shifts suggested that its structure was also very similar to native.

The Three-dimensional Solution Structure of NaD1, a New Floral Defensin from Nicotiana alata and its Application to a Homology Model of the Crop Defense Protein alfAFP

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first example of a plant defensin from flowers to be characterized structurally. Its three-dimensional structure consists of an a-helix and a triple-stranded anti-parallel beta-sheet that are stabilized by four intramolecular disulfide bonds. NaD1 features all the characteristics of the cysteine-stabilized up motif that has been described for a variety of proteins of differing functions ranging from antibacterial insect defensins and ion channel-perturbing scorpion toxins to an elicitor of the sweet taste response. The protein is biologically active against insect pests, which makes it a potential candidate for use in crop protection. NaD1 shares 31% sequence identity with alfAFP, an antifungal protein from alfalfa that confers resistance to a fungal pathogen in transgenic potatoes. The structure of NaD1 was used to obtain a homology model of alfAFP, since NaD1 has the highest level of sequence identity with alfAFP of any structurally characterized antifungal defensin. The structures of NaD1 and alfAFP were used in conjunction with structure - activity data for the radish defensin Rs-AFP2 to provide an insight into structure-function relationships. In particular, a putative effector site was identified in the structure of NaD1 and in the corresponding homology model of alfAFP. (C) 2002 Elsevier Science Ltd. All rights reserved.

Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization

Background: The redox proteins that incorporate a thioredoxin fold have diverse properties and functions. The bacterial protein-folding factor DsbA is the most oxidizing of the thioredoxin family. DsbA catalyzes disulfide-bond formation during the folding of secreted proteins, The extremely oxidizing nature of DsbA has been proposed to result from either domain motion or stabilizing active-site interactions in the reduced form. In the domain motion model, hinge bending between the two domains of DsbA occurs as a result of redox-related conformational changes. Results: We have determined the crystal structures of reduced and oxidized DsbA in the same crystal form and at the same pH (5.6). The crystal structure of a lower pH form of oxidized DsbA has also been determined (pH 5.0). These new crystal structures of DsbA, and the previously determined structure of oxidized DsbA at pH 6.5, provide the foundation for analysis of structural changes that occur upon reduction of the active-site disulfide bond. Conclusions: The structures of reduced and oxidized DsbA reveal that hinge bending motions do occur between the two domains. These motions are independent of redox state, however, and therefore do not contribute to the energetic differences between the two redox states, instead, the observed domain motion is proposed to be a consequence of substrate binding. Furthermore, DsbA's highly oxidizing nature is a result of hydrogen bond, electrostatic and helix-dipole interactions that favour the thiolate over the disulfide at the active site.

Probing the S100 protein family through genomic and functional analysis

The EF-hand superfamily of calcium binding proteins includes the S100, calcium binding protein, and troponin subfamilies. This study represents a genome, structure, and expression analysis of the S100 protein family, in mouse, human, and rat. We confirm the high level of conservation between mammalian sequences but show that four members, including S100A12, are present only in the human genome. We describe three new members of the S100 family in the three species and their locations within the S100 genomic clusters and propose a revised nomenclature and phylogenetic relationship between members of the EF-hand superfamily. Two of the three new genes were induced in bone-marrow-derived macrophages activated with bacterial lipopolysaccharide, suggesting a role in inflammation. Normal human and murine tissue distribution profiles indicate that some members of the family are expressed in a specific manner, whereas others are more ubiquitous. Structure-function analysis of the chemotactic properties of murine S100A8 and human S100A12, particularly within the active hinge domain, suggests that the human protein is the functional homolog of the murine protein. Strong similarities between the promoter regions of human S100A12 and murine S100A8 support this possibility. This study provides insights into the possible processes of evolution of the EF-hand protein superfamily. Evolution of the S100 proteins appears to have occurred in a modular fashion, also seen in other protein families such as the C2H2-type zinc-finger family. (C) 2004 Elsevier Inc. All rights reserved.

The 2.1 angstrom crystal structure of the far-red fluorescent protein HcRed: Inherent conformational flexibility of the chromophore

We have determined the crystal structure of HcRed, a far-red fluorescent protein isolated from Heteractis crispa, to 2.1 resolution. HcRed was observed to form a dimer, in contrast to the monomeric form of green fluorescent protein (GFP) or the tetrameric forms of the GFP-like proteins (eqFP611, Rtms5 and DsRed). Unlike the well-defined chromophore conformation observed in GFP and the GFP-like proteins, the HcRed chromophore was observed to be considerably mobile. Within the HcRed structure, the cyclic tripeptide chromophore, Glu64-Tyr65-Gly66, was observed to adopt both a cis coplanar and a tran. non-coplanar conformation. As a result of these two con formations, the hydroxyphenyl moiety of the chromophore makes distinct interactions within the interior of the b-can. These data together with a quantum chemical model of the chromophore, suggest the cis coplanar conformation to be consistent with the fluorescent properties of HcRed, and the trans non-coplanar conformation to be consistent with non-fluorescent properties of hcCP, the chromoprotein parent of HcRed. Moreover, within the GFP-like family, it appears that where conformational freedom is permissible then flexibility in the chromophore conformation is possible. 2005 Elsevier Ltd. All rights reserved.

The crystal structure of a bacterial Class II ketol-acid reductolsomerase: Domain conservation and evolution

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class 1) found in fungi and most bacteria, and a long form (Class 11) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class 11 KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.

Data analysis in multiple-frequency bioelectrical impedance analysis

The performance of three analytical methods for multiple-frequency bioelectrical impedance analysis (MFBIA) data was assessed. The methods were the established method of Cole and Cole, the newly proposed method of Siconolfi and co-workers and a modification of this procedure. Method performance was assessed from the adequacy of the curve fitting techniques, as judged by the correlation coefficient and standard error of the estimate, and the accuracy of the different methods in determining the theoretical values of impedance parameters describing a set of model electrical circuits. The experimental data were well fitted by all curve-fitting procedures (r = 0.9 with SEE 0.3 to 3.5% or better for most circuit-procedure combinations). Cole-Cole modelling provided the most accurate estimates of circuit impedance values, generally within 1-2% of the theoretical values, followed by the Siconolfi procedure using a sixth-order polynomial regression (1-6% variation). None of the methods, however, accurately estimated circuit parameters when the measured impedances were low (<20 Omega) reflecting the electronic limits of the impedance meter used. These data suggest that Cole-Cole modelling remains the preferred method for the analysis of MFBIA data.