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Role of the Rubisco small subunit. Final report for period May 1, 1997--April 30,2000

Description: CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesis is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.
Date: October 4, 2000
Creator: Spreitzer, Robert J.
Partner: UNT Libraries Government Documents Department

Use of Microarray Test Data for Toxicogenomic Prediction-Multi-Intelligent Systems for Toxicogenomic Applications (MISTA)

Description: The YAHSGS LLC and Oak Ridge National Laboratory established a CRADA to develop a computational neural network and wavelets software to facilitate providing national needs for toxicity prediction and overcome the voracious drain of resources (money and time) being directed to the development of pharmaceutical agents. The research project was supported through a STTR Phase I task by NIEHS in 2004. The research deploys state-of-the-art computational neural networks and wavelets to make toxicity prediction on three independent bases: (1) quantitative structure-activity relationships, (2) microarray data, and (3) Massively Parallel Signature Sequencing technology. Upon completion of Phase I, a prototype software Multi-Intelligent System for Toxicogenomic and Applications (MISTA) was developed, the utility's feasibility was demonstrated, and a Phase II proposal was jointly prepared and submitted to NIEHS for funding evaluation. The goals and objectives of the program have been achieved.
Date: September 12, 2005
Creator: Wasson, J.S. & Lu, P.-Y.
Partner: UNT Libraries Government Documents Department

From photons to protons in the photocycle of bacterial reaction center

Description: The detailed knowledge of the atomic coordinates of the bacterial reaction center (RC) has permitted a close scrutiny of structure/function relationships not only of the quinones but of the protein itself with its internal water structure. Protonatable groups were identified as intrinsic part of the redox reactions, providing charge compensation and forming channels for the movement of hydrogen ions to QB2-. The nature and position of these groups give rise to electrostatic profiles that determine the kinetics and energetics of proton transport. Fine tuning or dramatic variations of protein delivery pathways can adapt the photocycle to changes in bulk phase pH value, buffering capacities and primary structure of the RC.
Date: December 31, 1995
Creator: Maroti, P., Osvath, S., Tapai, C., Hanson, D.K.
Partner: UNT Libraries Government Documents Department

Improved Evolutionary Hybrids for Flexible Ligand Docking in Autodock

Description: In this paper we evaluate the design of the hybrid evolutionary algorithms (EAs) that are currently used to perform flexible ligand binding in the Autodock docking software. Hybrid EAs incorporate specialized operators that exploit domain-specific features to accelerate an EA's search. We consider hybrid EAs that use an integrated local search operator to reline individuals within each iteration of the search. We evaluate several factors that impact the efficacy of a hybrid EA, and we propose new hybrid EAs that provide more robust convergence to low-energy docking configurations than the methods currently available in Autodock.
Date: January 27, 1999
Creator: Belew, R.K.; Hart, W.E.; Morris, G.M. & Rosin, C.
Partner: UNT Libraries Government Documents Department

Structural mechanisms of nonplanar hemes in proteins

Description: The objective is to assess the occurrence of nonplanar distortions of hemes and other tetrapyrroles in proteins and to determine the biological function of these distortions. Recently, these distortions were found by us to be conserved among proteins belonging to a functional class. Conservation of the conformation of the heme indicates a possible functional role. Researchers have suggested possible mechanisms by which heme distortions might influence biological properties; however, no heme distortion has yet been shown conclusively to participate in a structural mechanism of hemoprotein function. The specific aims of the proposed work are: (1) to characterize and quantify the distortions of the hemes in all of the more than 300 hemoprotein X-ray crystal structures in terms of displacements along the lowest-frequency normal coordinates, (2) to determine the structural features of the protein component that generate and control these nonplanar distortions by using spectroscopic studies and molecular-mechanics calculations for the native proteins, their mutants and heme-peptide fragments, and model porphyrins, (3) to determine spectroscopic markers for the various types of distortion, and, finally, (4) to discover the functional significance of the nonplanar distortions by correlating function with porphyrin conformation for proteins and model porphyrins.
Date: May 1, 1997
Creator: Shelnutt, J.A.
Partner: UNT Libraries Government Documents Department

Structure-function correlation for ras p21 and the molecular origin of cancer

Description: In the past five years the authors followed different routes in correlating the structure and function of p21{sup ras} on an atomic level. The main project focused on understanding the GTPase mechanism catalyzed by p21{sup ras} and other GTP-binding proteins. The progress on this front is summarized. The starting point was the crystal structure of p21{sup ras} that was solved by the Kim group and the Wittinghofer group and paved the way for any attempt of understanding the hydrolysis mechanism in this protein. The crystallographic analysis has identified a water molecule (Wat175) in a position that makes it likely to be able to act as the nucleophile in the hydrolysis reaction. This water is directly located between the {gamma}-phosphate and the side chain of Gln61 in one of its possible orientations. This arrangement and the fact that mutations of Gln61 decrease the GPTase reaction rate led to the suggestion that this residue plays an important role in catalysis by acting as the general base for the nucleophilic water molecule and that it is assisted by Glu63.
Date: December 1, 1997
Partner: UNT Libraries Government Documents Department

Workshop on stems and trunks in plant form and function. Final report

Description: This document is the final report on the workshop on stems and trunks in plant form and function relating to DOE grant DE-FG06-93ER20128 which took place at Oregon State University in February 1994. The resulting book is organized into four sections and a synthesis: roles of stem architecture in plant performance, roles of stems in transport and storage of water, roles of live stem cells in plant performance, and the roles of stems in preventing or reacting to response to plant injury. The synthesis stemmed from debated and discussion by the authors and a few dozen other workshop participants. The authors cover many stem functions, although the list is not exhaustive, and the focus is on terrestrial woody tree stems, primarily of temperate and boreal zones. More research on trunks, branches and twigs is important for a baseline understanding of plant biology. In the face of anticipated human-caused changes to most environments, we need not only have a baseline understanding of whole-plant biology, but also predictive capabilities for how plants will react to perturbations.
Date: March 1, 1995
Creator: Gartner, B.L.
Partner: UNT Libraries Government Documents Department

Three-dimensional model of a selective theophylline-binding RNA molecule

Description: We propose a three-dimensional (3D) model for an RNA molecule that selectively binds theophylline but not caffeine. This RNA, which was found using SELEX [Jenison, R.D., et al., Science (1994) 263:1425] is 10,000 times more specific for theophylline (Kd=320 nM) than for caffeine (Kd=3.5 mM), although the two ligands are identical except for a methyl group substituted at N7 (present only in caffeine). The binding affinity for ten xanthine-based ligands was used to derive a Comparative Molecular Field Analysis (CoMFA) model (R{sup 2} = 0.93 for 3 components, with cross-validated R{sup 2} of 0.73), using the SYBYL and GOLPE programs. A pharmacophoric map was generated to locate steric and electrostatic interactions between theophylline and the RNA binding site. This information was used to identify putative functional groups of the binding pocket and to generate distance constraints. Based on a model for the secondary structure (Jenison et al., idem), the 3D structure of this RNA was then generated using the following method: each helical region of the RNA molecule was treated as a rigid body; single-stranded loops with specific end-to-end distances were generated. The structures of RNA-xanthine complexes were studied using a modified Monte Carlo algorithm. The detailed structure of an RNA-ligand complex model, as well as possible explanations for the theophylline selectivity will be discussed.
Date: July 1, 1995
Creator: Tung, Chang-Shung; Oprea, T.I.; Hummer, G. & Garcia, A.E.
Partner: UNT Libraries Government Documents Department

Discovery, SAR, and Radiolabeling of Halogenated Benzimidazole Carboxamide Antagonists as Useful Tools for (alpha)4(beta)1 Integrin Expressed on T- and B-cell Lymphomas

Description: The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin is an attractive yet poorly understood target for selective diagnosis and treatment of T- and B-cell lymphomas. This report focuses on the rapid microwave preparation of medicinally pertinent benzimidazole heterocycles, structure-activity relationships (SAR) of novel halobenzimidazole carboxamide antagonists 3-6, and preliminary biological evaluation of radioiodinated agents 7, 8, and 18. The I-125 derivative 18 had good tumor uptake (12 {+-} 1% ID/g at 24 h; 4.5 {+-} 1% ID/g at 48 h) and tumor:kidney ratio ({approx}4:1 at 24 h; 2.5:1 at 48 h) in xenograft murine models of B-cell lymphoma. Molecular homology models of {alpha}{sub 4}{beta}{sub 1} integrin have predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. These high affinity ({approx} pM binding) halogenated ligands are attractive tools for medicinal and biological use; the fluoro and iodo derivatives are potential radiodiagnostic ({sup 18}F) or radiotherapeutic ({sup 131}I) agents, whereas the chloro and bromo analogues could provide structural insight into integrin-ligand interactions through photoaffinity cross-linking/mass spectroscopy experiments, as well as co-crystallization X-ray studies.
Date: February 8, 2010
Creator: Carpenter, R D; Natarajan, A; Lau, E Y; Andrei, M; Solano, D M; Lightstone, F C et al.
Partner: UNT Libraries Government Documents Department

Effects of cavities in the bacterial reaction center

Description: A site-specific double mutant of Rhodobacter capsulatus, in which the large aromatic residues M208Tyr and L181Phe in the interior of the photosynthetic reaction center (RC) complex were replaced by smaller theonine residues, showed a dramatic reduction in the number of assembled complexes and was incapable of photosynthetic growth. The cavity created by the smaller side chains interferes mostly with the assembly of the complex. Phenotypic revertants were recovered in which a spontaneous second-site mutation restored photocompetence in the presence of the original site-specific mutations. In these strains, an Ala to Pro substitution in neighboring transmembrane helix (at M271) resulted in an increased yield of RC complexes. To test the hypothesis that the original phenotype was due to a cavity, other mutants were constructed where L180Phe and M207Leu were replaced with alanines that created similar-sized voids at other positions in the membrane-spanning interior. The L180Ala-M207A mutant had the same phenotype. Coupling of the above proline substitution to these new cavity mutants also resulted in photocompetant strains that carry increased levels of RC complexes. Therefore, the proline substitution at M271 serves as a global suppressor of the phenotype caused by these internal cavities.
Date: December 31, 1995
Creator: Schiffer, M.; Deng, Y.-L.; Marrufo, A. & Hanson, D.K.
Partner: UNT Libraries Government Documents Department

[Enhancement of photoassimilate utilization by manipulation of ADP-glucose pyrophosphorylase gene]. Final progress report

Description: Part 1 of this research focuses on patterns of gene expression of ADPG-pyrophosphorylase in native and transgenic potato plants. To elucidate the mechanism controlling AGP expression during plant development, the expression of the potato tuber AGP small subunit (sAGP) gene was analyzed in transgenic potato plants using a promoter-{beta}-glucuronidase expression system. Part II evaluated the structure-function relationships of AGP.
Date: April 1, 1999
Creator: Okita, T.W.
Partner: UNT Libraries Government Documents Department

Role of zein proteins in structure and assembly of protein bodies and endosperm texture. Progress report and appendix 1 - preliminary data

Description: Although funding for this project was initiated less than two years ago, we have made significant progress with our research objectives. We have cloned the gene responsible for the fl2 mutation. In fl2, the mutant phenotype appears to result from a defective signal peptide in an alpha-zein protein. As a consequence, the signal peptide remains attached when the protein accumulates in the protein body. A mutation like fl2 could explain other semidominant and dominant opaque mutants on the basis of abnormal zein polypeptides. A manuscript describing the research that led to the cloning of fl2 is in press, and a second manuscript on the characterization of this gene has been prepared for publication. We found that increased amounts of the 27-kD gamma-zein protein enlarge the proportion of vitreous endosperm and increases the hardness of o2 mutants. This protein also enhances these properties in wild type seeds. The mechanism by which the gamma-zein protein brings about these changes is unclear, and is under investigation. We have found and characterized several mutants that reduce gamma-zein synthesis. The mutations do not significantly affect synthesis of any other type of zein protein. They appear to create an opaque phenotype by reducing the number rather than the size of protein bodies. Interestingly, the mutant seeds fail to germinate. A manuscript describing one of these mutants, o15, has been prepared for publication. We have created a number of transgenic tobacco plants that can produce alpha-, beta-, gamma(27-kD)-, or delta-zeins, as well as combinations of these proteins. Analysis of seeds from these plants and crosses of these plants has shown that tobacco endosperm can serve as a heterologous system to study zein interactions. We have obtained evidence that interactions between alpha- and gamma-zein proteins are required for stable accumulation of alpha-zeins in the endosperm. These and other ...
Date: May 1, 1997
Creator: Larkins, B.
Partner: UNT Libraries Government Documents Department

Histone-DNA contacts in structure/function relationships of nucleosomes as revealed by crosslinking

Description: The magnitude of the problem of understanding the structure/function relationships of eukaryotic chromosomes can be appreciated from the fact that the human diploid genome contains more than 2 meters of DNA packaged into 46 chromosomes, each at metaphase being several microns in length. Each chromatid of a chromosome contains a single DNA molecule several centimeters in length. In addition to the DNA, chromosomes contain an equal weight of histones and an equal weight of non-histone chromosomal proteins. These histones are the major chromosomal structural proteins. The non-histone chromosomal proteins are involved in the DNA processes of transcription and replication, in chromosome organization and in nuclear architecture. Polytene chromosomes with their bands and interbands and puffs of active genetic loci provide visual evidence for long range order as do the bands and interbands of mammalian metaphase chromosomes. The gentle removal of histones and all but the most tightly bound 2--3% of non-histone proteins from metaphase chromosomes revealed by electron microscopy a residual protein scaffold constraining a halo of DNA loops extending out from the scaffold.
Date: December 31, 1998
Creator: Usachenko, S.I. & Bradbury, E.M.
Partner: UNT Libraries Government Documents Department

Studies of protein structure in solution and protein folding using synchrotron small-angle x-ray scattering

Description: Synchrotron small angle x-ray scattering (SAXS) has been applied to the structural study of several biological systems, including the nitrogenase complex, the heat shock cognate protein (hsc70), and lysozyme folding. The structural information revealed from the SAXS experiments is complementary to information obtained by other physical and biochemical methods, and adds to our knowledge and understanding of these systems.
Date: April 1, 1996
Creator: Chen, Lingling
Partner: UNT Libraries Government Documents Department

International symposium on cellular and molecular biology of phosphate and phosphorylated compounds in microorganisms: Proceedings

Description: This report contains the abstracts of papers presented at the conference. Attention is focused on the following topics: regulation of phosphate metabolism in bacteria; structure-function of alkaline phosphatase; regulation of phosphate metabolism in yeast; transport of phosphate and phosphorylated compounds; and phosphate regulation in pathogenesis and secondary metabolism.
Date: December 31, 1993
Partner: UNT Libraries Government Documents Department

Structure and thermodynamics of surface recognition

Description: This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Interactions of the surface glycoprotein, gp120, with the receptors of host cells define the pathogenesis of HIV-1, the virus that causes AIDS. gp120 is made of several disulfide-bridged loops--the amino acid sequences of some of these loops are fairly conserved whereas the rest are variable. The third variable (V3) loop has been the target of vaccine design for quite some time since this loop is involved in various steps of viral pathogenesis. However, this loop also happens to be the most variable one. The authors have carried out structural and immunological studies to determine the sequence-structure-antigenicity correlations of the HIV-1 V3 loops. This resulted in the identification of a secondary structure at the tip of the V3 loop that remains invariant in spite of the sequence variation. The authors designed a multi-valent V3-based antigen that presents multiple copies of the same tip element several times in the same structure. During the course of this project, they realized that the protective epitopes of gp120 should be judged in the context of the native structure. Therefore, the authors developed a method to obtain a model of gp120 that is consistent with all the immunology and virology data. This model is useful in choosing or designing gp120 subdomains for vaccine development.
Date: November 1998
Creator: Gupta, G.
Partner: UNT Libraries Government Documents Department

Solution assembly of cytokine receptor ectodomain complexes

Description: For the majority of single transmembrane-spanning cell surface receptors, signal transmission across the lipid bilayer barrier involves several discrete components of molecular recognition. The interaction between ligand and the extracellular segment of its cognate receptor (ectodomain) initiates either homomeric or heteromeric association of receptor subunits. Specific recognition among these subunits may then occur between ectodomain regions, within the membrane by interhelical contact or inside the cell between cytoplasmic domains. Any or all of these interactions may contribute to the stability of the signaling complex. It is the characteristics of ligand binding by the ectodomains of these receptors that controls the heteromeric or homomeric nature and the stoichiometry of the complex. Cytokines and their receptors belong to a growing family of macromolecular systems that exhibit these functional features and share many structural similarities as well. Interleukin-2 is a multifunctional cytokine that represents, perhaps, the most complex example to date of ligand recognition among the hematopoietin receptor family. It is the cooperative binding of IL-2 by all three proteins on the surface of activated T-lymphocytes, however, that ultimately results in crosslinking of the {beta}- and {gamma}-subunits and signaling via association of their cytoplasmic domains. Although the high-affinity IL-2R functions as a heterotrimer, heterodimers of the receptor subunits are also physiologically important. The {alpha}/{beta} heterodimer or {open_quotes}pseudo-high affinity{close_quotes} receptor captures IL-2 as a preformed cell surface complex while the {beta}/{gamma} intermediate affinity site exists, in the absence of the {alpha} subunit, on the majority of natural killer cells. We have begun to study stable complexes of cytokine receptor ectodomains of defined composition and that mimic the ligand binding characteristics of the equivalent cell surface receptor sites.
Date: September 1, 1995
Creator: Wu, Zining; Ciardelli, T.L. & Johnson, K.W.
Partner: UNT Libraries Government Documents Department

Dynamics of initial ionization events in biological molecules: Formation and fate of free radicals. Final technical report, May 1, 1994--December 31, 1995

Description: Study of early time events following the absorption of electromagnetic radiation in biological systems has potentially significant impact on several areas of importance. In this context, the studies being conducted under this program provided insight into the conformational changes as well as the reactions leading to a variety of transformations that culminate from hydrogen atom and proton transfer events. These studies enabled an investigation of molecular details of structure-function relationships. In a second aspect of the program, investigations were conducted to provide basic underpinning research that contributed to a quantification of the behavior of radionuclides and pollutants associated with advanced energy activities after these materials emanate from their source and become transferred through the environment to the biota and human receptor. The approach to elucidating factors governing the difference between reactions in the gas and condensed phase was to study the initiating steps at progressively higher degrees of cluster aggregation. The author employed ultrafast laser techniques, in combination with selected molecules, carefully prepared in tailored compositions, to investigation the primary mechanisms involved in various molecular functional groups following the absorption of electromagnetic radiation. He also studied various molecules representing chromophores in such biologically important molecules as tyrosine and amines.
Date: August 1, 1997
Creator: Castleman, A. W., Jr.
Partner: UNT Libraries Government Documents Department

Nanometer scale exciton/photon dynamic spectrochemical imaging for DNA structure-activity relations and radiation signatures. Final progress report, December 24, 1993--December 23, 1996

Description: This report is a summary of the work conducted during the period from December 24, 1993--December 23, 1996. Research is divided into five major areas: Near-field imaging of DNA clusters, chromosomes, etc.; Femtosecond near-field optical microscopy; Ultrafast and ultrasmall fluorescent chemical sensors; Feasibility study for nonlinear optics studies in the near-field; and Three-dimensional pH microprobing with a laser tweezer manipulated fluorescent particle. Brief summaries are given of each project and a series of photos are included.
Date: June 1, 1997
Creator: Kopelman, R.
Partner: UNT Libraries Government Documents Department

Comparative evolution of the recA gene of surface and deep subsurface microorganisms (an evolutionary clock of intermediate rate). Final report

Description: Because of the ability of the recA protein product to maintain both DNA integrity and increase genetic diversity, this gene may be essential to the survival of microorganisms following the damaging effects of numerous environmental stresses such as exposure to solar UV radiation, exposure to gamma radiation, starvation, and changing environments. While the various activities and amino-acid sequence of recA have been highly conserved among the eubacteria and archaea, little is known as to whether a strict structure-function relationship has been conserved. In other words, are the same regions of this highly plastic, functionally heterogeneous protein involved in the same catalytic capacities throughout the bacterial kingdom? While it is reasonable to assume that this type of conservation has also occurred, we felt it necessary to test the assumption by demonstrating that mutations in different genera of bacteria which eliminate similar functions (i.e., lead to similar phenotypes) are caused by changes in the amino-acid sequence in the same regions of their recA proteins. Therefore, we located the changes in nucleotide sequence in two recA mutants of P. aeruginosa which displayed mutant phenotypes in recombination and UV resistance. Our assumption was that if structure-function relationships held, these mutations would be found in areas already identified as essential for the function of the E. coli recA protein.
Date: April 1, 1998
Creator: Miller, R.V.
Partner: UNT Libraries Government Documents Department

Structural biology of disease-associated repetitive DNA sequences and protein-DNA complexes involved in DNA damage and repair

Description: This project is aimed at formulating the sequence-structure-function correlations of various microsatellites in the human (and other eukaryotic) genomes. Here the authors have been able to develop and apply structure biology tools to understand the following: the molecular mechanism of length polymorphism microsatellites; the molecular mechanism by which the microsatellites in the noncoding regions alter the regulation of the associated gene; and finally, the molecular mechanism by which the expansion of these microsatellites impairs gene expression and causes the disease. Their multidisciplinary structural biology approach is quantitative and can be applied to all coding and noncoding DNA sequences associated with any gene. Both NIH and DOE are interested in developing quantitative tools for understanding the function of various human genes for prevention against diseases caused by genetic and environmental effects.
Date: July 1, 1997
Creator: Gupta, G.; Santhana Mariappan, S.V.; Chen, X.; Catasti, P.; Silks, L.A. III; Moyzis, R.K. et al.
Partner: UNT Libraries Government Documents Department

[Mechanisms of proton pumping in bacteriorhodopsin]. Progress report

Description: This report consists of two parts namely a brief statement of the progress made during the past four years of the project and more extensive discussion of the current state of understanding of molecular mechanisms controlling the proton pump (bacteriorhodopsin). Detailed descriptions are provided of how the protein undergoes conformational changes on absorbing a photon. Studies are described where the protein structure has been manipulated and the biochemical properties are assessed.
Date: December 31, 1995
Creator: Ebrey, T.G.
Partner: UNT Libraries Government Documents Department