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At least 433 records · Page 24
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Structural insights into binding of polyglutamylated tetrahydrofolate by serine hydroxymethyltransferase 8 from soybean

Tetrahydrofolate and its derivatives participate in one-carbon transfer reactions in all organisms. The cellular form of tetrahydrofolate (THF) is modified by multiple glutamate residues and polyglutamylation plays a key role in organellar and cellular folate homeostasis. In addition, polyglutamylation of THF is known to increase the binding affinity to enzymes in the folate cycle, many of which can utilize polyglutamylated THF as a substrate. Here, we use X-ray crystallography to provide a high-resolution view of interactions between the enzyme serine hydroxymethyltransferase (SHMT), which provides one carbon precursors for the folate cycle, and a polyglutamylated form of THF. Our 1.7 Å crystal structure of soybean SHMT8 in complex with diglutamylated 5-formyl-THF reveals, for the first time, a structural rearrangement of a loop at the entrance to the folate binding site accompanied by the formation of novel specific interactions between the enzyme and the diglutamyl tail of the ligand. Biochemical assays show that additional glutamate moieties on the folate ligand increase both enzyme stability and binding affinity. Together these studies provide new information on SHMT structure and function and inform the design of anti-folate agents.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

On the Rapid Calculation of Binding Affinities for Antigen and Antibody Design and Affinity Maturation Simulations

The accurate and efficient calculation of protein-protein binding affinities is an essential component in antibody and antigen design and optimization, and in computer modeling of antibody affinity maturation. Such calculations remain challenging despite advances in computer hardware and algorithms, primarily because proteins are flexible molecules, and thus, require explicit or implicit incorporation of multiple conformational states into the computational procedure. The astronomical size of the amino acid sequence space further compounds the challenge by requiring predictions to be computed within a short time so that many sequence variants can be tested. In this study, we compare three classes of methods for antibody/antigen (Ab/Ag) binding affinity calculations: (i) a method that relies on the physical separation of the Ab/Ag complex in equilibrium molecular dynamics (MD) simulations, (ii) a collection of 18 scoring functions that act on an ensemble of structures created using homology modeling software, and (iii) methods based on the molecular mechanics-generalized Born surface area (MM-GBSA) energy decomposition, in which the individual contributions of the energy terms are scaled to optimize agreement with the experiment. When applied to a set of 49 antibody mutations in two Ab/HIV gp120 complexes, all of the methods are found to have modest accuracy, with the highest Pearson correlations reaching about 0.6. In particular, the most computationally intensive method, i.e., MD simulation, did not outperform several scoring functions. The optimized energy decomposition methods provided marginally higher accuracy, but at the expense of requiring experimental data for parametrization. Within each method class, we examined the effect of the number of independent computational replicates, i.e., modeled structures or reinitialized MD simulations, on the prediction accuracy. We suggest using about ten modeled structures for scoring methods, and about five simulation replicates for MD simulations as a rule of thumb for obtaining reasonable convergence. We anticipate that our study will be a useful resource for practitioners working to incorporate binding affinity calculations within their protein design and optimization process.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Identification of Exported Plasmodium falciparum Proteins That Bind to the Erythrocyte Cytoskeleton

Plasmodium proteins are exported to the erythrocyte cytoplasm to create an environment that supports parasite replication. Although hundreds of proteins are predicted to be exported through Plasmodium export element (PEXEL)-dependent and -independent mechanisms, the functions of exported proteins are largely uncharacterized. In this study, we used a biochemical screening approach to identify putative exported P. falciparum proteins that bound to inside-out vesicles prepared from erythrocytes. Out of 69 P. falciparum PEXEL-motif proteins tested, 18 bound to inside-out vesicles (IOVs) in two or more independent assays. Using co-affinity purifications followed by mass spectrometry, pairwise co-purification experiments, and the split-luciferase assay, we identified 31 putative protein–protein interactions between erythrocyte cytoskeletal proteins and predicted exported P. falciparum proteins. We further showed that PF3D7_1401600 binds to the spectrin-binding domain of erythrocyte ankyrin via its MESA erythrocyte cytoskeleton binding (MEC) motif and to the N-terminal domains of ankyrin and 4.1R through a fragment that required an intact Plasmodium helical interspersed sub-telomeric (PHIST) domain. Introduction of PF3D7_1401600 into erythrocyte ghosts increased retention in the microsphiltration assay, consistent with previous data that reported a reduction of rigidity in red blood cells infected with PF3D7_1401600-deficient parasites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (‘fuzzy’ binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Novel sterol binding domains in bacteria

Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of M. capsulatus, and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1974

Analytic variational calculation of the ground-state binding energy of hydrogen in intermediate and intense magnetic fields

The present work investigates analytically the effect of an intermediate or intense magnetic field, such as probably exist in white dwarfs and near pulsars, on the binding energy of the hydrogen ground state. A wave-function 'prescription' is given for an analytic variational calculation of the binding energy. The calculation still gives a smooth transition between intermediate and intense fields. An explicit calculation of the ground-state binding energy as B goes to infinity is provided for the Yafet et al. (1956) trial function.

Wilson, L. W.↗
NASA NTRS1981

Evolving nucleotide binding surfaces

An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

Kieber-Emmons, T.↗
NASA NTRS1984

Increased cytosolic androgen receptor binding in rat striated muscle following denervation and disuse

The effects of denervation and disuse on cytosolic androgen receptor binding by rat striated muscle are investigated. Denervation of the extensor digitorum longus and tibialis anterior muscles caused by a 40-50-percent increase in cytosolic androgen receptor concentration with no change in apparent binding affinity. This effect was evident at 6 h postdenervation, maximal at 24 h, and declined to 120 percent of the control level 72 h after denervation. A 40-percent increase in cytosolic androgen receptor concentration was also noted 24 hr after denervation of the hormone-sensitive levator ani muscle. The effect of denervation on androgen receptors was blocked by in vivo injection of cycloheximide; therefore, de novo receptor synthesis probably is not involved in the observed increase. Disuse, produced by subperineurial injection of tetrodotoxin into the tibial and common peroneal branches of the sciatic nerve, mimicked the effect of denervation on androgen receptor binding, suggesting that neuromuscular activity is important in regulation of receptor concentration. Possible mechanisms subserving this effect are discussed.

Bernard, P. A.↗
NASA NTRS1989

Theoretical investigations of the structures and binding energies of Be(sub n) and Mg(sub n) (n = 3-5) clusters

Researchers determined the equilibrium geometries and binding energies of Be and Mg trimers, tetramers and pentamers using single and double excitation coupled cluster (CCSD) and complete active space self-consistent-field (CASSCF) multireference configuration interaction (MRCI) wave functions in conjunction with extended atomic basis sets. Best estimates of the cluster binding energies are 24, 83 and 110 kcal/mole for Be3, Be4 and Be5; and 9, 31 and 41 kcal/mole for Mg3, Mg4 and Mg5, respectively. A comparison of the MRCI and CCSD results shows that even the best single-reference approach (limited to single and double excitations) is not capable of quantitative accuracy in determining the binding energies of Be and Mg clusters.

Lee, Timothy J.↗
NASA NTRS1990

Theoretical investigations of the structures and binding energies of Be(n) and Mg(n) (n = 3-5) clusters

The equilibrium geometries and binding energies of Be and Mg trimers, tetramers and pentamers have been determined using single and double excitation coupled cluster (CCSD) and complete active space self-consistent-field (CASSCF) multireference configuration interaction (MRCI) wave functions in conjunction with extended atomic basis sets. The best estimates of the cluster binding energies are 24, 83, and 110 kcal/mol for Be3, Be4, and Be5; and 9, 31, and 41 kcal/mol for Mg3, Mg4, and Mg5, respectively. A comparison of the MRCI and CCSD results shows that even the best single-reference approach (limited to single and double excitations) is not capable of quantitative accuracy in determining the binding energies of Be and Mg clusters.

Lee, Timothy J.↗
NASA NTRS1990

The volume- and surface-binding energies of ice systems containing CO, CO2, and H2O

Laboratory-measured, temperature-dependent sticking efficiencies are presently used to derive the surface-binding energies of CO and CO2 on H2O-rich ices, with a view to determining the condensation and vaporization properties of these systems as well as to the measured energies' implications for both cometary behavior and the evolution of interstellar ices. The molecular volume and the surface binding energies are not found to be necessarily related on the basis of simple nearest-neighbor scaling in surface and bulk sites; this may be due to the physical constraints associated with matrix structure-associated physical constraints, which sometimes dominate the volume-binding energies.

Sandford, Scott A.↗
NASA NTRS1988

Ada/POSIX binding: A focused Ada investigation

NASA is seeking an operating system interface definition (OSID) for the Space Station Program (SSP) in order to take advantage of the commercial off-the-shelf (COTS) products available today and the many that are expected in the future. NASA would also like to avoid the reliance on any one source for operating systems, information system, communication system, or instruction set architecture. The use of the Portable Operating System Interface for Computer Environments (POSIX) is examined as a possible solution to this problem. Since Ada is already the language of choice for SSP, the question of an Ada/POSIX binding is addressed. The intent of the binding is to provide access to the POSIX standard operation system (OS) interface and environment, by which application portability of Ada applications will be supported at the source code level. A guiding principle of Ada/POSIX binding development is a clear conformance of the Ada interface with the functional definition of POSIX. The interface is intended to be used by both application developers and system implementors. The objective is to provide a standard that allows a strictly conforming application source program that can be compiled to execute on any conforming implementation. Special emphasis is placed on first providing those functions and facilities that are needed in a wide variety of commercial applications

Legrand, Sue↗
NASA NTRS1992

Calculation of Mg(+)-ligand relative binding energies

The calculated relative binding energies of 16 organic molecules to Mg(+) are compared with experimental results where available. The geometries of the ligands and the Mg(+)-ligand complexes arc optimized at the self-consistent field level using a 6-31G* basis set. The Mg(+) binding energies are evaluated using second-order perturbation theory and basis sets of triple-sigma quality augmented with two sets of polarization functions. This level of theory is calibrated against higher levels of theory for selected systems. The computed binding energies are accurate to about 2 kcal/mol.

Partridge, Harry↗
NASA NTRS1995

Collagen and Stretch Modulate Autocrine Secretion of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Proteins from Differentiated Skeletal Muscle Cells

Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micro-g of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.

Perrone, Carmen E.↗
NASA NTRS2004

Intricate Crystal Structure of Dihydrolipoamide Dehydrogenase (E3) with its Binding Protein: Multiple Copies, Dynamic and Static Disorders

Human E3 and binding protein E3BP are two components of the pyruvate dehydrogenase complex. Crystallization of E3 with 221-amino acid fragment of E3BP (E3BPdd) led to crystals that diffracted to a resolution of 2.6 Angstroms. Structure determination involved molecular replacement using a dimer of E3 homolog as a search model and de novo building of the E3BPdd peptide. Solution was achieved by inclusion of one E3 dimer at a time, followed by refinement until five E3 dimers were located. This complete content of E3 provided electron density maps suitable for tracing nine peptide chains of E3BPdd, eight of them being identified with partial occupancies. Final content of the asymmetric unit consists of five E3 dimers, each binding one E3BPdd molecule. In four of these molecular complexes, E3BPdd is in static disorder resulting in E3BPdd binding to either one or the other monomer of the E3 dimer. However, E3BPdd of the fifth E3 dimer forms specific contacts that lock it at one monomer. In addition to this static disorder, E3BPdd reveals high mobility in the limited space of the crystal lattice. Support from NIH and NASA.

Makal, A.↗
NASA NTRS1987

Water binding in legume seeds

The physical status of water in seeds has a pivotal role in determining the physiological reactions that can take place in the dry state. Using water sorption isotherms from cotyledon and axis tissue of five leguminous seeds, the strength of water binding and the numbers of binding sites have been estimated using van't Hoff analyses and the D'Arcy/Watt equation. These parameters of water sorption are calculated for each of the three regions of water binding and for a range of temperatures. Water sorption characteristics are reflective of the chemical composition of the biological materials as well as the temperature at which hydration takes place. Changes in the sorption characteristics with temperature and hydration level may suggest hydration-induced structural changes in cellular components.

NASA Discipline Plant Biology↗
NASA NTRS1990

Binding of isolated plant lectin by rhizobia during episodes of reduced gravity obtained by parabolic flight

Development of a legume root nodule is a complex process culminating in a plant/bacterial symbiosis possessing the capacity for biological dinitrogen fixation. Formation of root nodules is initiated by the binding and stabilization of rhizobia to plant root hairs, mediated in part by a receptor/ligand recognition system composed of lectins on the plant root surface and lectin-binding sites on the rhizobial cell surface. The dinitrogen fixation activity of these root nodules may be an important feature of enclosed, space-based life support systems, and may provide an ecological method to recycle nitrogen for amino acid production. However, the effects on nodule development of varied gravitational fields, or of root nutrient delivery hardware, remain unknown. We have investigated the effects of microgravity on root nodule formation, with preliminary experiments focused upon the receptor/ligand component. Microgravity, obtained during parabolic flight aboard NASA 930, has no apparent effect on the binding of purified lectin to rhizobia, a result that will facilitate forthcoming experiments using intact root tissues.

manned↗
NASA NTRS1989

Purification and identification of the fusicoccin binding protein from oat root plasma membrane

Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.

NASA Discipline Number 29-20↗