Wheel Plots Research Articles

Adaptive potential and productivity of two Salix viminalis L. clonal varieties grown in North Central Bulgaria

Salix viminalis L. is a broadly cultivated willow species that has been intensively used for breeding purposes, particularly in selection of clones and hybrids for biomass production. The present study aimed to explore and compare growth and adaptability of two basket willow (Salix viminalis L.) clonal varieties - rubra and purpurea - at the specific edaphic and climate conditions and cultivation practice presented in an experimental plantation in North Central Bulgaria. The influence of spacing, genotype, root age and rotation on their productivity were also investigated and allometric relationships for non-destructive estimation of woody plant biomass were suggested. The trial plantation was established in the spring of 2018 as 4 sectors of a Nelder wheel plot with 15 nearly-square spacings, ranging from 0.25 to 9.80 m2 (40000–1020 plants per hectare). The two varieties were arranged in 4 four-spoke sectors (2 sectors per clone). Data collection took place from 2018 to 2020 and comprised 1- and 2-year rotations. The study showed that Salix viminalis var. rubra was more susceptible to desiccation-causing adverse climatic changes, poorly adapted to the specific conditions of the study site and gradually died out during the second and the third year. Salix viminalis var. purpurea exhibited steady survival rate (97 - 77%) and annual biomass productivity varying from 3 to 17 Mg/ha/year across the densities, sectors and years. During the first year of growth purpurea variety had a significantly higher plant weight and sprout number than rubra variety. Growing space affected positively the number of shoots and tree dendromass, and sprouting was reinforced by coppicing that doubled the shoot number during the subsequent rotation. This was reflected in the development of the stump-level allometric models that were differentiated according to rotation. The stump-level allometric model of higher predictive power was based on the main shoot length and shoot number. An exponential relationship to the breast-height shoot diameter was also suggested for biomass estimation of each sprout separately.

Effect of density on Mediterranean pine seedlings using the Nelder wheel design: analysis of survival and early growth

Abstract There is growing interest in mixed-species forests but also a lack of studies that analyse them at the regeneration phases or any stage other than mature stands. Information is particularly scarce about relatively unproductive species such as Pinus pinaster and Pinus halepensis in Mediterranean ecosystems. The objective of this study was to investigate inter- and intra-specific interactions of both species at different tree densities during the first years of establishment. Five Nelder wheel plots were planted to test densities ranging from 1000 to 80 000 seedlings/ha and simulate establishment sub-processes at high densities. Further, P. pinaster and P. halepensis were mixed along the spokes, to obtain three mixture levels in which 100, 80 or 60 per cent of the seedlings were of the same species. Cox proportional-hazards models and binomial logistic regressions were fitted to analyse seedling survival. Early growth (basal diameter and height at one and four years after plantation) was analysed by fitting linear mixed-effects models. In this regard, P. halepensis showed higher survival rates and basal diameter increments but more time is needed to know how P. pinaster responds to density and mixture. Both competitive and facilitating seedling interactions were observed at higher densities, which fostered seedling survival but decreased early growth.

Density effect on growth variables of eucalypt clones in a Nelder wheel experiment

Nelder’s systematic design enables studies of tree plantation spacing in relatively small areas, which is of great interest for forest planning. However, dependence between observations can arise with this method, making it difficult to use classic statistics for evaluating an experiment. Therefore, our aims were to: evaluate the relationship between dendrometric variables (tree diameter, height and volume) and the available growing area; evaluate autocorrelation among the observations; and use multivariate analysis of the profiles of the clones for comparisons of the dendrometric variables. The data are from an experiment with four Nelder wheel plots of different Eucalyptus species clones of different ages, in the state of Tocantins, Brazil. Spearman’s rank correlation was applied to evaluate the relationship between the estimates of the studied variables and respective available growing area. Autocorrelation between the observations was evaluated with Moran’s I index. To analyse the profile behaviour of the clones, three questions were proposed: (1) Are there statistical differences between the estimations made for the different clones? (2) Are there differences between treatments applied in the experiment with the clones? (3) Is there interaction between clones and spacings? The main results show that multivariate profile analysis solved the autocorrelation problem in Nelder’s systematic design. Furthermore, we obtained answers as to differences between the growth variables diameter, volume and height among the clones at different spacings, whether there is a difference between the spacings for these variables, and whether there is an interaction between clones and spacings. The analysis method used for repeated measures is appropriate to evaluate trees in Nelder wheel trials.

Detecting vulnerability of humid tropical forests to multiple stressors

Detecting vulnerability of humid tropical forests to multiple stressors

Classification of the human THAP protein family identifies an evolutionarily conserved coiled coil region

BackgroundThe THAP (Thanatos Associated Proteins) protein family in humans is implicated in various important cellular processes like epigenetic regulation, maintenance of pluripotency, transposition and disorders like cancers and hemophilia. The human THAP protein family which consists of twelve members of different lengths has a well characterized amino terminal, zinc-coordinating, DNA-binding domain called the THAP domain. However, the carboxy terminus of most THAP proteins is yet to be structurally characterized. A coiled coil region is known to help in protein oligomerization in THAP1 and THAP11. It is not known if other human THAP proteins oligomerize. We have used bioinformatic tools to explore the possibility of dimerization of THAP proteins via a coiled coil region.ResultsClassification of human THAP protein into three size based groups led to the identification of an evolutionarily conserved alpha helical region, downstream of the amino terminal THAP domain. Secondary structure predictions, alpha helical wheel plots and protein models demonstrated the strong possibility of coiled coil formation in this conserved, leucine rich region of all THAP proteins except THAP10.ConclusionsThe identification of a predicted oligomerization region in the human THAP protein family opens new directions to investigate the members of this protein family.

Rill erosion in burned and salvage logged western montane forests: Effects of logging equipment type, traffic level, and slash treatment

Following wildfires, forest managers often consider salvage logging burned trees to recover monetary value of timber, reduce fuel loads, or to meet other objectives. Relatively little is known about the cumulative hydrologic effects of wildfire and subsequent timber harvest using logging equipment. We used controlled rill experiments in logged and unlogged (control) forests burned at high severity in northern Montana, eastern Washington, and southern British Columbia to quantify rill overland flow and sediment production rates (fluxes) after ground-based salvage logging. We tested different types of logging equipment—feller-bunchers, tracked and wheeled skidders, and wheeled forwarders—as well as traffic levels and the addition of slash to skid trails as a best management practice. Rill experiments were done at each location in the first year after the fire and repeated in subsequent years. Logging was completed in the first or second post-fire year. We found that ground-based logging using heavy equipment compacted soil, reduced soil water repellency, and reduced vegetation cover. Vegetation recovery rates were slower in most logged areas than the controls. Runoff rates were higher in the skidder and forwarder plots than their respective controls in the Montana and Washington sites in the year that logging occurred, and the difference in runoff between the skidder and control plots at the British Columbia site was nearly significant (p=0.089). Most of the significant increases in runoff in the logged plots persisted for subsequent years. The type of skidder, the addition of slash, and the amount of forwarder traffic did not significantly affect the runoff rates. Across the three sites, rill sediment fluxes were 5–1900% greater in logged plots than the controls in the year of logging, and the increases were significant for all logging treatments except the low use forwarder trails. There was no difference in the first-year sediment fluxes between the feller-buncher and tracked skidder plots, but the feller-buncher fluxes were lower than the values from the wheeled skidder plots. Manually adding slash after logging did not affect sediment flux rates. There were no significant changes in the control sediment fluxes over time, and none of the logging equipment impacted plots produced greater sediment fluxes than the controls in the second or third year after logging. Our results indicate that salvage logging increases the risk of sedimentation regardless of equipment type and amount of traffic, and that specific best management practices are needed to mitigate the hydrologic impacts of post-fire salvage logging.

Functional importance of the Gly cluster in transmembrane helix 2 of the Bordetella pertussis CyaA-hemolysin: Implications for toxin oligomerization and pore formation

Adenylate cyclase-hemolysin (CyaA) is a major virulence factor of Bordetella pertussis causing whooping cough in humans. We previously showed that two transmembrane helices (α2 and α3) in the hemolysin domain (CyaA-Hly) are crucially involved in hemolytic activity. Here, PCR-based substitutions were employed to investigate a potential involvement in hemolysis of a series of four Gly residues (Gly530, Gly533, Gly537 and Gly544) which map onto one face of a helical wheel plot of pore-lining helix 2. All CyaA-Hly mutant toxins were over-expressed in Escherichia coli as 126-kDa soluble proteins at levels comparable to the wild-type toxin. A drastic reduction in hemolytic activity against sheep erythrocytes was observed for three CyaA-Hly mutants, i.e. G530A, G533A and G537A, but not G544A, suggesting a functional importance of the Gly530_Gly533_Gly537 cluster. A homology-based structure of the α2-loop-α3 hairpin revealed that this crucial Gly cluster arranged as a GXXGXXXG motif is conceivably involved in helix–helix association. Furthermore, a plausible pore model comprising three α2-loop-α3 hairpins implicated that Gly530XXGly533XXXGly537 could function as an important framework for toxin oligomerization. Altogether, our present data signify for the first time that the Gly530_Gly533_Gly537 cluster in transmembrane helix 2 serves as a crucial constituent of the CyaA-Hly trimeric pore structure.

Military Vehicle Trafficking Impacts on Vegetation and Soil Bulk Density at Fort Benning, Georgia

Abstract. Potential increases in wind erosion that might be brought about by military vehicles traveling off-road during training are of concern to the U.S. military because wind erosion and vehicle dust emissions contribute to land and air quality degradation and can cause adverse effects on respiratory health. Field studies were conducted in the summer of 2012 at Fort Benning, Georgia, to assess the effects of military vehicle trafficking intensity on susceptibility to dust emissions. Quantitative data on soil and vegetation parameters are needed to make appropriate estimates of the susceptibility to dust generation from the soil surface and the magnitude of those emissions. The experiment consisted of making multiple trafficking passes with both tracked and wheeled vehicles and then measuring wind erosion parameters. A tracked (M1A1) and wheeled (HMMWV) vehicle were driven in a figure-8 pattern within replicated 40 m A— 80 m plots. On each plot, three levels of vehicle passes were made. On the tracked plots, the M1A1 was driven a cumulative total of 1, 5, and 10 passes. On the wheeled plots, the HMMWV was driven a cumulative total of 10, 25, and 50 passes. The vehicles were driven repeatedly over the same figure-8 path. Bulk density, aboveground biomass, and vegetative cover data were taken from the straight, curved, and cross-over sections of the vehicle tracks. Samples were also taken before the start of trafficking. Bulk density at three depths, total aboveground biomass, grass biomass, forb biomass, biomass by individual species, total cover, grass cover, and forb cover data were analyzed for differences between vehicles, vehicles passes, locations within the track sections, and their interactions. At the 5 cm depth, bulk density was significantly higher (p ≤ 0.05) than the control in both the M1A1 and HMMWV tracks. There was no significant evidence of soil compaction below 5 cm. At the end of all trafficking, grass and forb species biomass was reduced 65% to 100%. Vegetation cover showed strong response to vehicle type, trafficking intensity, location (within the vehicle tracks), and their interactions. Regression equations relating trafficking intensity by vehicle to reduction in cover and biomass were obtained.

Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum

TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.

Designing Nelder wheel plots for tree density experiments

The Nelder (Biometrics 18:283–307, 1962) wheel design allows a researcher to test multiple tree densities in a single plot. Because spatial relationships among planted trees are fundamental to a Nelder wheel, a researcher needs a specific set of layout parameters to establish a Nelder plot. While Nelder (Biometrics 18:283–307, 1962) provides calculus-based equations for determining the required layout parameters, the presentation focuses on derivation of these equations and not their application to forestry research. Other authors have outlined the design of Nelder plots for forestry research, but have done so using trigonometry-based equations. Existence of two layout methodologies in the literature is a source of confusion. In this paper, we present a straightforward means to determine the design parameters critical to the establishment of Nelder plots used within tree density research. The layout equations presented are expressed in terms that allow applied forestry researchers to easily answer the following question. Given the number and range of tree densities I want to evaluate, what are the required Nelder wheel layout parameters? Finally, we provide a step-by-step example of the design and installation of a Nelder plot for a scenario familiar to tree density research and discuss analysis of Nelder wheel experiments.

Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A

NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.

Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes

Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H(+)-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members.

NBCe1-A Transmembrane Segment 1 Lines the Ion Translocation Pathway

The electrogenic Na(+)/HCO(3)(-) cotransporter (NBCe1-A) transports sodium and bicarbonate across the basolateral membrane of the renal proximal tubule. In this study the structural requirement of transmembrane segment 1 (TM1) residues in mediating NBCe1-A transport was investigated. Twenty-five introduced cysteine mutants at positions Gln-424 to Gly-448 were tested for their sensitivity to the methanethiosulfonate reagents (2-sulfonatoethyl) methanethiosulfonate (MTSES), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and (2-aminoethyl) methanethiosulfonate (MTSEA). Two mutants, T442C and A435C, showed 100 and 70% sensitivity, respectively, to inhibition by all the three methanethiosulfonate (MTS) reagents, I441C had >50% sensitivity to MTSET and MTSEA, and A428C had 50% sensitivity to MTSEA inhibition. A helical wheel plot showed that mutants T442C, A435C, and A428C are clustered on one face of TM1 within a 100 degrees arc. Topology analysis of TM1 with biotin maleimide and 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) revealed Thr-442 marks the C-terminal end of TM1 and that extracellular FGGLLG stretch is in a small aqueous-accessible cavity. Functional studies indicated that Thr-442 resides in a narrow region of the ion translocation pore with strong delta(-) helical dipole influence. Analysis of the corresponding residue of NBCe1-A-Thr-442 in AE1 (Thr-422) shows it is functionally insensitive to MTSES and unlabeled with MTS-TAMRA, indicating that AE1-TM1 is oriented differently from NBCe1-A. In summary, we have identified residues Thr-442, Ala-435, and Ala-428 in TM1 lining the ion translocation pore of NBCe1-A. Our findings are suggestive of a delta(-) helical dipole at the C-terminal end of TM1 involving Thr-442 that plays a critical role in the function of the cotransporter.

CSSP(Consensus Secondary Structure Prediction): a web-based server for structural biologists

Sequence–structure correlation studies are important in deciphering the relationships between various structural aspects, which may shed light on the protein-folding problem. The first step of this process is the prediction of secondary structure for a protein sequence of unknown three-dimensional structure. To this end, a web server has been created to predict the consensus secondary structure using well known algorithms from the literature. Furthermore, the server allows users to see the occurrence of predicted secondary structural elements in other structure and sequence databases and to visualize predicted helices as a helical wheel plot. The web server is accessible at http://bioserver1.physics.iisc.ernet.in/cssp/.

지하철 곡선부소음과 차륜 마모와의 관계에 관한 연구

There are many curves in Seoul subway system. Therefore, the noise from subway system in curved line gives displeasure to passenger. The subway noise in curved line is affected not only by rail condition but also wheel condition and dynamic characteristics. The railway curving noise can be divided into 2 categories. The first is the noise due to stick-slip between wheel tread and rail head, and the second is one by wheel flange contact on rail side. Because of these phenomena - stick-slip and wheel flange contact - wheels are worn seriously. In this study the curving noise was reviewed by using eigen-mode of wheel and waterfall plot which shows noise level in time-frequency domain. And also those were reviewed in viewpoint of stick-slip noise and wheel flange contact noise. Finally, the relationship between curving noise and wheel wear was studied.

Characterization of biomimetc material, Mussel adhesive protein, using computer tools and servers.

In this paper six different mussel adhesion proteins (MAPs) retrieved from SwissProt database are analyzed using computer tools and servers. Primary analysis showed that all the MAPs are hydrophobic in nature due to the high content of non-polar amino acid residues. The sequence analysis showed the absence of sulphide bridges and non polar amino acid tryptophan in MAPs. Signal peptide is a common feature in MAPs. The aliphatic index computed by Expasy's Protparm infers that most of the MAPs are stable at a wide range of temperature. Secondary structure analysis showed that MAPs could results in a better interaction with water. The secondary structure analysis showed MAPs contain more βhelices and turns. The SOSUI server predicts one transmembarne region in Mytilus galprovincialis. The predicted transmembrane region was visualized and analyzed using helical wheel plots generated by EMBOSS PepWheel tool. The absence of disulphide bonds in MAPS was confirmed by SYC_REC tools and from 3 Dimensional structures created by Rasmol tool. The cystein position identified by Rasmol tool might be correct as the evaluated parameters (Rampage, CE and ProQ) are within the accepted limits for the model 3D structure. The signal peptide identified by SignalP server showed that they are secretary pathway in localization except in Mytilus edulus, where it is cytoplasmic. Based on the signal peptides reliability class the MAPs are classified into three groups. The phosphorylation sites of serine, threonoine and tyrosine are predicted by NetPhos server. Interestingly tyrosine is the most phosphorylated amino acid in the MAPs; hence it is inferred that MAPs can be purified by using antibodies.

Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

Potassium channels are tetrameric proteins that mediate K(+)-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (approximately 40 kDa) on SDS-PAGE. Addition of Ba(2+) (K(1/2) approximately 50 microm) prior to loading results in an additional tetramer band (approximately 160 kDa). Mutation A109C, at a residue located near the expected Ba(2+)-binding site, decreased tetramer stabilization by Ba(2+) (K(1/2) approximately 300 microm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba(2+). Neither mutation affected Ba(2+) block of channel activity (using (86)Rb(+) flux assay). In contrast to Ba(2+), Mg(2+) had no effect on tetramer stability (even though Mg(2+) was a potent blocker). Many studies have shown Cd(2+) block of K(+) channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd(2+) on KirBac1.1 with cysteine substitutions in M2. Cd(2+) block potency followed an alpha-helical pattern consistent with the crystal structure. Significantly, Cd(2+) strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba(2+), consistent with both ions simultaneously occupying the channel: Ba(2+) at the selectivity filter entrance and Cd(2+) coordinated by I138C side chains in the inner cavity.

In silico characterization of antifreeze proteins using computational tools and servers

In this paper, seventeen different fish Antifreeze Proteins (AFPs) retrieved from Swiss-Prot database are analysed and characterized using in silico tools. Primary structure analysis shows that most of the AFPs are hydrophobic in nature due to the high content of non-polar residues. The presence of 11 cysteines in the rainbow smelt fish and sea raven fish AFPs infer that these proteins may form disulphide (SS) bonds, which are regarded as a positive factor for stability. The aliphatic index computed by Ex-Pasy’s ProtParam infers that AFPs may be stable for a wide range of temperature. Secondary structure analysis shows that most of the fish AFPs have predominant α-helical structures and rest of the AFPs have mixed secondary structure. The very high coil structural content of rainbow smelt fish and sea raven fish AFPs are due to the rich content of more flexible glycine and hydrophobic proline amino acids. Proline has a special property of creating kinks in polypetide chains and disrupting ordered secondary structure. SOSUI server predicts one transmembrane region in winter flounder fish and atlantic cod and two transmembrane regions in yellowtail flounder fish AFP. The predicted transmembrane regions were visualized and analysed using helical wheel plots generated by EMBOSS pepwheel tool. The presence of disulphide (SS) bonds in the AFPs Q01758 and P05140 are predicted by CYS_REC tool and also identified from the three-dimensional structure using Rasmol tool. The disulphide bonds identified from the three-dimensional structure using the Rasmol tool might be correct as the evaluation parameters are within the acceptable limits for the modelled 3D structures.

BIOCOMPUTATIONAL ANALYSIS AND CHARACTERIZATION OF SOME ANTIFREEZE PROTEINS

Novel bioinformatic procedures and computational methods have been used to analyze, characterize and provide more detailed description of some selected fish antifreeze proteins (AFPs) retrieved from Swiss–Prot database. Analysis shows that AFPs are rich in non-polar residues and that the AFPs Q01758 and P05140 contain SS bonds. The aliphatic index computed by ExPasy's ProtParam infers that AFPs may be stable for a wide range of temperatures and the AFP P80961 is classified as an unstable protein. The very low GRAVY index of AFP P80961 infers its higher hydrosolubility. Secondary structure analysis shows that the flounder and sculpin fish AFPs are found to be of predominant ∝–helical structures and the rest of them are with mixed secondary structures. The average molecular weight of AFPs computed is 9584 Da. SOSUI server predicts one transmembrane region in P04002 (winter flounder fish) and two regions in P09031 (yellowtail flounder fish). The predicted transmembrane regions were visualized and analyzed using helical wheel plots generated by EMBOSS pepwheel tool. The residues A, L, G and N are identified as the antigenic sites by EMBOSS antigenic program. The presence of 11 Cys residues in AFPs Q01758 (rainbow smelt fish) and P05140 (sea raven fish) indicates the presence of disulfide bridges (SS bonds) in these AFPs, and it is also recognized by CYS_REC tool and well documented from the three dimensional structure using Rasmol tool.

Electron Paramagnetic Resonance Spectroscopy of Site-directed Spin Labels Reveals the Structural Heterogeneity in the N-terminal Domain of ApoA-I in Solution

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.