From protein structure to function with bioinformatics

It has long been known that amino acids are the building blocks for proteins and govern their folding into specific three-dimensional structures. However, the details of this process are still unknown and represent one of the main problems in structural bioinformatics, which is a highly active research area with the focus on the prediction of three-dimensional structure and its relationship to protein function. The protein structure prediction procedure encompasses several different steps from searches and analyses of sequences and structures, through sequence alignment to the creation of the structural model. Careful evaluation and analysis ultimately results in a hypothetical structure, which can be used to study biological phenomena in, for example, research at the molecular level, biotechnology and especially in drug discovery and development. In this thesis, the structures of five proteins were modeled with templatebased methods, which use proteins with known structures (templates) to model related or structurally similar proteins. The resulting models were an important asset for the interpretation and explanation of biological phenomena, such as amino acids and interaction networks that are essential for the function and/or ligand specificity of the studied proteins. The five proteins represent different case studies with their own challenges like varying template availability, which resulted in a different structure prediction process. This thesis presents the techniques and considerations, which should be taken into account in the modeling procedure to overcome limitations and produce a hypothetical and reliable three-dimensional structure. As each project shows, the reliability is highly dependent on the extensive incorporation of experimental data or known literature and, although experimental verification of in silico results is always desirable to increase the reliability, the presented projects show that also the experimental studies can greatly benefit from structural models. With the help of in silico studies, the experiments can be targeted and precisely designed, thereby saving both money and time. As the programs used in structural bioinformatics are constantly improved and the range of templates increases through structural genomics efforts, the mutual benefits between in silico and experimental studies become even more prominent. Hence, reliable models for protein three-dimensional structures achieved through careful planning and thoughtful executions are, and will continue to be, valuable and indispensable sources for structural information to be combined with functional data.

The relationship between stem-form, stand-closure and site -conditions : the influence of environmental conditions on tree allometry and forest structure in west-central Alberta

Changes in the configuration of a tree stern result insignificant differences in its total volume and in the proportion of that volume that is merchantable timber. Tree allometry, as represented by stem-fo~, is the result of the vertical force of gravity and the horizontal force of wind. The effect of wind force is demonstrated in the relationship between stem-form, standclosure and site-conditions. An increase in wind force on the individual tree due to a decrease in stand density should produce a more tapered tree. The density of the stand is determined by the conditions that the trees are growing under. The ability of the tree to respond to increased wind force may also be a function of these conditions . This stem-form/stand-closure/site-conditions relationship was examined using a pre-existing database from westcentral Alberta. This database consisted of environmental, vegetation, soils and timber data covering a wide range of sites. There were 653 sample trees with 82 variables that formed the basis of the analysis. There were eight tree species consisting of Pinus contorta, Picea mariana, Picea engelmannii x glauca, Abies lasiocarpa, Larix laricina, Populus tremuloides, Betula papyrifera and Populus balsamifera plus a comprehensive all-species data set. As the actual conformation of the stern is very individual, stem-fo~was represented by the diameter at breast height to total height r~tio. The four stand-closure variables, crown closure, total basal area, total volume and total number of stems were reduced to total basal area and total number of stems utilizing a bivariate correlation matrix by species. Site-conditions were subdivided into macro, meso and micro variables and reduced in number 3 using cross-tabulations, bivariate correlation and principal components analysis as screening tools. The stem-fo~/stand-closure relationship was examined using bivariate correlation coefficients for stem-fo~ with total number of stems and stem-fo~ with total basal area. The stem-fo~/site-conditions and the stand-closure/site- conditions relationships were examined using multiple correlation coefficients. The stem-form/stand-closure/site-conditions relationship was examined using multiple correlation coefficients in separate analyses for both total number of stems and total basal area. An increase in stand-closure produced a decrease in stem-form for both total number of stems and total basal area for most species. There was a significant relationship between stem-form and site-conditions and between stand-closure and site-conditions for both total number of stems and total basal area for most species. There was a significant relationship between the stemform and site-conditions, including the stand-closure, for most species; total number of stems was involved independently of the site-conditions in the prediction of stem-form and total basal area was not. Larix laricina and Betula papyrifera were the exceptions to the trends observed with most species. The influence of both stand-closure (total number of stems in particular) and site-conditions (elevation in particular) suggest that forest management practices should include these- ecological parameters in determining appropriate restocking levels.

Relationship between global structural parameters and Enzyme Commission hierarchy: Implications for function prediction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Relationship between global structural parameters and Enzyme Commission hierarchy: Implications for function prediction

In protein databases there is a substantial number of proteins structurally determined but without function annotation. Understanding the relationship between function and structure can be useful to predict function on a large scale. We have analyzed the similarities in global physicochemical parameters for a set of enzymes which were classified according to the four Enzyme Commission (EC) hierarchical levels. Using relevance theory we introduced a distance between proteins in the space of physicochemical characteristics. This was done by minimizing a cost function of the metric tensor built to reflect the EC classification system. Using an unsupervised clustering method on a set of 1025 enzymes, we obtained no relevant clustering formation compatible with EC classification. The distance distributions between enzymes from the same EC group and from different EC groups were compared by histograms. Such analysis was also performed using sequence alignment similarity as a distance. Our results suggest that global structure parameters are not sufficient to segregate enzymes according to EC hierarchy. This indicates that features essential for function are rather local than global. Consequently, methods for predicting function based on global attributes should not obtain high accuracy in main EC classes prediction without relying on similarities between enzymes from training and validation datasets. Furthermore, these results are consistent with a substantial number of studies suggesting that function evolves fundamentally by recruitment, i.e., a same protein motif or fold can be used to perform different enzymatic functions and a few specific amino acids (AAs) are actually responsible for enzyme activity. These essential amino acids should belong to active sites and an effective method for predicting function should be able to recognize them. (C) 2012 Elsevier Ltd. All rights reserved.

Cell surface galactosyltransferase structure and function during mesenchymal cell migration

In order to more fully understand the function of surface GalTase on mesenchymal cells, anti-GalTase IgG was used to (a) examine the role of surface GalTase during mouse mesenchymal cell migration on laminin and fibronectin; (b) define the plasma membrane distribution of GalTase by indirect immunofluorescence on migrating cells; (c) quantitate the level of surface GalTase on migrating cells; and (d) determine whether GalTase is associated with the cytoskeleton.^ Results show that anti-GalTase IgG was able to inhibit migration (48-80% as compared to basal rate) when cells were migrating on laminin-containing matrices. Monovalent Fab fragments inhibited migration on laminin by 90% after 4 hours. On the other hand, anti-GalTase IgG had no effect on cells migrating on fibronectin. This illustrates the substrate specificity of GalTase mediated-migration. When anti-GalTase IgG was used to localize surface GalTase on cells migratory on laminin, the enzyme was restricted to the leading and trailing edges of the cell. Assays indicate that GalTase is elevated approximately 3-fold when cells are migrating on laminin-containing matrices as compared to migratory cells on plastic or fibronectin, or as compared to stationary cells on any substrate. Laminin appears to recruit GalTase from preexisting intracellular pools to the growing lamellipodia.^ Double-label indirect immunofluorescence studies indicate that there is an apparent co-localization between some of the surface GalTase and some actin filaments. This relationship was explored by extracting cells prelabeled with anti-GalTase IgG and quantitated by radiolabeled second antibodies. Results show that 79% of the surface GalTase is associated with the cytoskeleton (as judged by detergent insolubility) when monovalent antibodies (Fab) are used. However virtually all (80-100%) of the surface GalTase can be induced to associate with the cytoskeleton when cross-linked with bivalent antibodies. Furthermore, when cells in suspension are incubated with divalent antibodies, an additional 66% of the surface GalTase can be induced to associate with the cytoskeleton. The elevated levels of surface GalTase detectable on cells migrating on laminin also appear to be associated with the cytoskeleton.^ Several lines of evidence suggest that GalTase is associated with F-actin. Data suggest that laminin induces the expression of surface GalTase to the growing lamellipodia where it becomes associated with the cytoskeleton leading to cell spreading and migration. (Abstract shortened with permission of author.) ^

Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense beta-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sense-strand globin mRNA.

Air Pollution and High Density Lipoprotein Structure and Function

Thesis (Ph.D.)--University of Washington, 2016-06

Relationship Between Orofacial Function, Dentofacial Morphology, And Bite Force In Young Subjects.

The aim was to evaluate the relationship between orofacial function, dentofacial morphology, and bite force in young subjects. Three hundred and sixteen subjects were divided according to dentition stage (early, intermediate, and late mixed and permanent dentition). Orofacial function was screened using the Nordic Orofacial Test-Screening (NOT-S). Orthodontic treatment need, bite force, lateral and frontal craniofacial dimensions and presence of sleep bruxism were also assessed. The results were submitted to descriptive statistics, normality and correlation tests, analysis of variance, and multiple linear regression to test the relationship between NOT-S scores and the studied independent variables. The variance of NOT-S scores between groups was not significant. The evaluation of the variables that significantly contributed to NOT-S scores variation showed that age and presence of bruxism related to higher NOT-S total scores, while the increase in overbite measurement and presence of closed lip posture related to lower scores. Bite force did not show a significant relationship with scores of orofacial dysfunction. No significant correlations between craniofacial dimensions and NOT-S scores were observed. Age and sleep bruxism were related to higher NOT-S scores, while the increase in overbite measurement and closed lip posture contributed to lower scores of orofacial dysfunction.

The Function and Three-Dimensional Structure of a Thromboxane A(2)/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD(7). In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the omega-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.

The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein

MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell division site of Escherichia coli. We have examined the self-association properties of MinE by analytical ultracentrifugation and by studies of hetero-oligomer formation in non-denaturing polyacrylamide gets. The self-association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C-terminal MinE(22-88) fragment forms hetero-oligomers with MinE(+) when the proteins are co-expressed. In contrast, the MinE(36-88) fragment does not form MinE(+)/MinE(36-88) hetero-oligomers, although MinE36-88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co-expressed with MinE(+) in wild-type cells. Therefore, hetero-oligomer formation is not necessary for the induction of mini-celling by expression of MinE(36-88) in wild-type cells. The interference with normal septal placement is ascribed to competition between MinE(36-88),nd the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.

Surface reflectance properties of Antarctic moss and their relationship to plant species, pigment composition and photosynthetic function

In this study the variations in surface reflectance properties and pigment concentrations of Antarctic moss over species, sites, microtopography and with water content were investigated. It was found that species had significantly different surface reflectance properties, particularly in the region of the red edge (approximately 700 nm), but this did not correlate strongly with pigment concentrations. Surface reflectance of moss also varied in the visible region and in the characteristics of the red edge over different sites. Reflectance parameters, such as the photochemical reflectance index (PRI) and cold hard band were useful discriminators of site, microtopographic position and water content. The PRI was correlated both with the concentrations of active xanthophyll-cycle pigments and the photosynthetic light use efficiency, F-v/F-m, measured using chlorophyll fluorescence. Water content of moss strongly influenced the amplitude and position of the red-edge as well as the PRI, and may be responsible for observed differences in reflectance properties for different species and sites. All moss showed sustained high levels of photoprotective xanthophyll pigments, especially at exposed sites, indicating moss is experiencing continual high levels of photochemical stress.

Renal structure and function evaluation of rats from dams that received increased sodium intake during pregnancy and lactation submitted or not to 5/6 nephrectomy

Adult rats submitted to perinatal salt overload presented renin-angiotensin system (RAS) functional disturbances. The RAS contributes to the renal development and renal damage in a 5/6 nephrectomy model. The aim of the present study was to analyze the renal structure and function of offspring from dams that received a high-salt intake during pregnancy and lactation. We also evaluated the influence of the prenatal high-salt intake on the evolution of 5/6 nephrectomy in adult rats. A total of 111 sixty-day-old rat pups from dams that received saline or water during pregnancy and lactation were submitted to 5/6 nephrectomy (nephrectomized) or to a sham operation (sham). The animals were killed 120 days after surgery, and the kidneys were removed for immunohistochemical and histological analysis. Systolic blood pressure (SBP), albuminuria, and glomerular filtration rate (GFR) were evaluated. Increased SBP, albuminuria, and decreased GFR were observed in the rats from dams submitted to high-sodium intake before surgery. However, there was no difference in these parameters between the groups after the 5/6 nephrectomy. The scores for tubulointerstitial lesions and glomerulosclerosis were higher in the rats from the sham saline group compared to the same age control rats, but there was no difference in the histological findings between the groups of nephrectomized rats. In conclusion, our data showed that the high-salt intake during pregnancy and lactation in rats leads to structural changes in the kidney of adult offspring. However, the progression of the renal lesions after 5/6 nephrectomy was similar in both groups.