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At least 19 records

Isoelectric focusing of proteins and peptides

Egg-white solution was chosen as the reference solution in order to assess the effects of operational parameters (voltage, flow rate, ampholine pH range and concentration, and protein concentration) of the RIEF apparatus on protein resolution. Topics of discussion include: (1) comparison of RIEF apparatus to conventional IEF techniques (column and PAG) with respect to resolution and throughput; (2) peptide and protein separation (AHF, Thymosin - Fraction 5, vasoactive peptide, L-asparaginase and ACP); and (3) detection of peptides - dansyl derivatives of amino acids and peptides, post-focusing fluorescent labeling of amino acids, peptides and proteins, and ampholine extraction from focused gels.

Egen, N.

Rate constants measured for hydrated electron reactions with peptides and proteins

Effects of ionizing radiation on the amino acids of proteins and the reactivity of the protonated amino group depends upon the pK subscript a of the group. Estimates of the rate constants for reactions involving the amino acid side chains are presented. These rate constants gave an approximate rate constant for three different protein molecules.

Braams, R.

Molecular Simulations in Astrobiology

One of the main goals of astrobiology is to understand the origin of cellular life. In the absence of any record of the earliest ancestors of contemporary cells, protocells, the most direct way to test our understanding of their characteristics is to construct laboratory models of protocells. Such efforts, currently underway in the NASA Astrobiology Program, are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures and developing designs of molecules that are capable of performing protocellular functions. Many of these functions, such as importing nutrients, capturing and storing energy, and responding to changes in the environment, are carried out by proteins bound to membranes. We use computer simulations to address the following, questions about these proteins: (1) How do small proteins (peptides) organize themselves into ordered structures at water-membrane interfaces and insert into membranes? (2) How do peptides aggregate to form membrane-spannin(y structures (e.g., channels)? (3) By what mechanisms do such aggregates perform their functions? The simulations are performed using the molecular dynamics (MD) method. In this method, Newton's equations of motion for each atom in the system are solved iteratively. At each time step, the forces exerted on each atom by the remaining atoms are evaluated by dividing them into two parts. Short-range forces are calculated directly in real space while long-range forces are evaluated in reciprocal space, usually using a particle-mesh algorithm which is of order O(NlnN). Currently, a time step of 2 femtoseconds is typically used, thereby making studies of problems occurring on multi-nanosecond time scales (10(exp 6) - 10(exp 8) time steps) accessible. To address a broader range of problems, simulations need to be extended by three orders of magnitude. Such an extension requires both algorithmic improvements and codes scalable to a large number of parallel processors. Work in this direction is in progress. Two specific series of simulations that demonstrate how peptides self-organize and function in membranes are discussed. In one series of simulations, it was shown that nonpolar peptides, disordered in water, translocate to the nonpolar interior of the membrane and, simultaneously, fold into two different helical structures, which remain in equilibrium. Once in the membrane, the peptides can readily change their orientation, especially in response to local electric fields. This structural and orientational flexibility of peptides with changing conditions may have provided a mechanism of transmitting signals between the environment and the interior of the protocell. In another series of simulations, the mechanism by which a simple protein channel efficiently mediates proton transport across membranes was investigated. This process is a key step in cellular bioenergetics. In the channel under study, proton transport is gated by four histidines that occlude the channel pore. The simulations demonstrate that protons move through the gate by a "shuttle" mechanism, wherein one histidine is protonated on the extracellular side and, subsequently, the proton bound on the opposite side is released.

Pohorille, Andrew

Emerging protein sequencing technologies: proteomics without mass spectrometry?

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been a leading method for proteomics for 30 years. Advantages provided by LC-MS/MS are offset by significant disadvantages, including cost. Recently, several non-mass spectrometric methods have emerged, but little information is available about their capacity to analyze the complex mixtures routine for mass spectrometry. Areas Covered: We review recent non-mass-spectrometric methods for sequencing proteins and peptides, including those using nanopores, sequencing by degradation, reverse translation, and short-epitope mapping, with comments on bioinformatics challenges, fundamental limitations, and areas where new technologies will be more or less competitive with LC-MS/MS. In addition to conventional literature searches, instrument vendor websites, patents, webinars, and preprints were also consulted to give a more up-to-date picture. Expert Opinion: Many new technologies are promising. However, demonstrations that they outperform mass spectrometry in terms of peptides and proteins identified have not yet been published, and astute observers note important disadvantages, especially relating to the dynamic range of single-molecule measurements of complex mixtures. Still, even if the performance of emerging methods proves inferior to LC-MS/MS, their low cost could create a different kind of revolution: a dramatic increase in the number of biology laboratories engaging in new forms of proteomics research.

59 BASIC BIOLOGICAL SCIENCES

Inhibition of browning in foodstuffs

Addition of water-soluble sulfur-containing compounds, thio compounds or potential thio compounds, to a mixture of carbohydrates, and either proteins, peptides, or amino acids can retard or completely eliminate the browning process. Determining factor is dependent upon the concentration of the anti-browning agent in the aqueous media.

Rosenthal, N. A.

Recycling isoelectric focusing with computer controlled data acquisition system

Isoelectric focusing is a high-resolution technique for separating and purifying large peptides, proteins, and other biomolecules. The apparatus described in the present paper constitutes a new approach to fluid stabilization and increased throughput. Stabilization is achieved by flowing the process fluid uniformly through an array of closely spaced filter elements oriented parallel both to the electrodes and the direction of the flow. This seems to overcome the major difficulties of parabolic flow and electroosmosis at the walls, while limiting the convection to chamber compartments defined by adjacent spacers. Increased throughput is achieved by recirculating the process fluid through external heat exchange reservoirs, where the Joule heat is dissipated.

Egen, N. B.

Simulation of isoelectro focusing processes

This paper presents the computer implementation of a model for the stationary electrolysis of two or more charged species. This has specific application to the technique of isoelectric focussing, in which the stationary electrolysis of ampholytes is used to generate a pH gradient useful for the separation of proteins, peptides and other biomolecules. The fundamental equations describing the process are given. These equations are transformed to a form suitable for digital computer implementation. Some results of computer simulation are described and compared to data obtained in the laboratory.

Palusinski, O. A.

Isoelectric focusing in space

The potential of space electrophoresis is conditioned by the fact that all electrophoretic techniques require the suppression of gravity-caused convection. Isoelectric focusing (IEF) is a powerful variant of electrophoresis, in which amphoteric substances are separated in a pH gradient according to their isoelectric points. A new apparatus for large scale IEF, utilizing a recycling principle, has been developed. In the ground-based prototype, laminar flow is provided by a series of parallel filter elements. The operation of the apparatus is monitored by an automated array of pH and ultraviolet absorption sensors under control of a desk-top computer. The apparatus has proven to be useful for the purification of a variety of enzymes, snake venom proteins, peptide hormones, and other biologicals, including interferon produced by genetic engineering techniques. In planning for a possible space apparatus, a crucial question regarding electroosmosis needs to be addressed To solve this problem, simple focusing test modules are planned for inclusion in an early Shuttle flight.

Bier, M.

Computer Modeling of the Earliest Cellular Structures and Functions

In the absence of extinct or extant record of protocells (the earliest ancestors of contemporary cells). the most direct way to test our understanding of the origin of cellular life is to construct laboratory models of protocells. Such efforts are currently underway in the NASA Astrobiology Program. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures and developing designs for molecules that perform proto-cellular functions. Many of these functions, such as import of nutrients, capture and storage of energy. and response to changes in the environment are carried out by proteins bound to membrane< We will discuss a series of large-scale, molecular-level computer simulations which demonstrate (a) how small proteins (peptides) organize themselves into ordered structures at water-membrane interfaces and insert into membranes, (b) how these peptides aggregate to form membrane-spanning structures (eg. channels), and (c) by what mechanisms such aggregates perform essential proto-cellular functions, such as proton transport of protons across cell walls, a key step in cellular bioenergetics. The simulations were performed using the molecular dynamics method, in which Newton's equations of motion for each item in the system are solved iteratively. The problems of interest required simulations on multi-nanosecond time scales, which corresponded to 10(exp 6)-10(exp 8) time steps.

Pohorille, Andrew

BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields

We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

Non-NASA Center

Sequence Programmable Order–Disorder Transitions in Supramolecular Assembly of Peptide Nanofibers

Protein–protein interactions determine the assembly of complexes that are responsible for numerous key biological processes. The assembly of many natural protein complexes is mediated by post-translational structural changes and environmental stimuli. In this study, we show that incorporation of adjacent lysine residues results in the pH-tunable stability of peptide secondary structure and assembly, allowing for the incorporation of complementary order-inducing motifs. The strategic placement of cysteine pairs in the same peptide sequence results in redox-dependent disulfide staple formation, inducing a transition from random coil to β-sheet conformation and subsequent supramolecular nanofiber assembly from otherwise disordered peptide monomers. Spectroscopic, imaging, molecular dynamics, and kinetic studies highlight the critical role of sequence motif location, oligomerization, and the competitive interplay between intra- and interpeptide disulfide bonding in determining assembly outcomes. We extend this approach to demonstrate phosphorylation-dependent assembly from the design of the same parent peptide sequence, suggesting a general approach to the design of diverse stimulus-responsive peptide sequences for supramolecular assembly. Furthermore, these findings also provide a framework for investigating sequence-dependent pathways in amyloid fiber formation with potential implications for neurodegenerative disease research.

Disulfides

The effects of orbital spaceflight on bone histomorphometry and messenger ribonucleic acid levels for bone matrix proteins and skeletal signaling peptides in ovariectomized growing rats

A 14-day orbital spaceflight was performed using ovariectomized Fisher 344 rats to determine the combined effects of estrogen deficiency and near weightlessness on tibia radial bone growth and cancellous bone turnover. Twelve ovariectomized rats with established cancellous osteopenia were flown aboard the space shuttle Columbia (STS-62). Thirty ovariectomized rats were housed on earth as ground controls: 12 in animal enclosure modules, 12 in vivarium cages, and 6 killed the day of launch for baseline measurements. An additional 18 ovary-intact rats were housed in vivarium cages as ground controls: 8 rats were killed as baseline controls and the remaining 10 rats were killed 14 days later. Ovariectomy increased periosteal bone formation at the tibia-fibula synostosis; cancellous bone resorption and formation in the secondary spongiosa of the proximal tibial metaphysis; and messenger RNA (mRNA) levels for the prepro-alpha2(1) subunit of type 1 collagen, osteocalcin, transforming growth factor-beta, and insulin-like growth factor I in the contralateral proximal tibial metaphysis and for the collagen subunit in periosteum pooled from tibiae and femora and decreased cancellous bone area. Compared to ovariectomized weight-bearing rats, the flight group experienced decreases in periosteal bone formation, collagen subunit mRNA levels, and cancellous bone area. The flight rats had a small decrease in the cancellous mineral apposition rate, but no change in the calculated bone formation rate. Also, spaceflight had no effect on cancellous osteoblast and osteoclast perimeters or on mRNA levels for bone matrix proteins and signaling peptides. On the other hand, spaceflight resulted in an increase in bone resorption, as ascertained from the diminished retention of a preflight fluorochrome label. This latter finding suggests that osteoclast activity was increased. In a follow-up ground-based experiment, unilateral sciatic neurotomy of ovariectomized rats resulted in cancellous bone loss in the unloaded limb in excess of that induced by gonadal hormone deficiency. This additional bone loss was arrested by estrogen replacement. We conclude from these studies that estrogen alters the expression of signaling peptides believed to mediate skeletal adaptation to changes in mechanical usage and likewise modifies the skeletal response to mechanical unloading.

NASA Program Space Physiology and Countermeasures

Cyclic Peptides for Lanthanide Binding

Lanthanide ions are difficult to separate from one another due to their similar chemical properties. The discovery of lanthanide-binding peptides and proteins in nature has led to an increased interest in the possibility of utilizing the strong binding of peptides to lanthanide ions for their separations; as such, there has been an effort to identify or design peptides with improved lanthanide binding and selectivity toward particular lanthanide ions. Here, in this study, we designed and characterized lanthanide-binding cyclic peptides (LBCPs) with molecular dynamics simulations, electronic structure calculations, and emission spectroscopy. Luminescent decay measurements were done to determine the number of water molecules coordinated to the Eu 3+ ion in Eu-LBCP complexes and compare to the predicted number of water molecules by computation to assess the lanthanide-binding affinity of LBCPs. Measured stability constants show binding of the LBCPs to the Eu 3+ ion with stronger than micromolar affinity. We were able to identify multiple peptides that selectively bind to middle lanthanides. We describe the structural basis of the lanthanide-binding selectivity trend with strongest binding to the middle lanthanides, followed by the heavier lanthanides, and finally to the lighter ions.

ions

The Biogeochemistry of Sulfur in Hydrothermal Systems

The incorporation of sulfur into many biomolecules likely dates back to the development of the earliest metabolic strategies. Sulfur is common in enzymes and co-enzymes and is an indispensable structural component in many peptides and proteins. Early metabolism may have been heavily influenced by the abundance of sulfide minerals in hydrothermal systems. The incorporation of sulfur into many biomolecules likely dates back to the development of the earliest metabolic strategies. Sulfur is common in enzymes and co-enzymes and is an indispensable structural component in many peptides and proteins. Early metabolism may have been heavily influenced by the abundance of sulfide minerals in hydrothermal systems. Understanding how sulfur became prevalent in biochemical processes and many biomolecules requires knowledge of the reaction properties of sulfur-bearing compounds. We have previously estimated thermodynamic data for thiols, the simplest organic sulfur compounds, at elevated temperatures and pressures. If life began in hydrothermal environments, it is especially important to understand reactions at elevated temperatures among sulfur-bearing compounds and other organic molecules essential for the origin and persistence of life. Here we examine reactions that may have formed amino acids with thiols as reaction intermediates in hypothetical early Earth hydrothermal environments. (There are two amino acids, cysteine and methionine, that contain sulfur.) Our calculations suggest that significant amounts of some amino acids were produced in early Earth hydrothermal fluids, given reasonable concentrations H2, NH3, H2S and CO. For example, preliminary results indicate that glycine activities as high as 1 mmol can be reached in these systems at 100 C. Alanine formation from propanethiol is also a favorable reaction. On the other hand, the calculated equilibrium log activities of cysteine and serine from propanethiol are -21 and -19, respectively, at 100 C. These results indicate that while amino acid formation with thiols as intermediates is favored in some cases, other mechanisms may have been necessary to produce significant amounts of other amino acids. Coupled with our previous results for thiols, these studies imply that sulfur may have been easily incorporated into the organic geochemistry of early Earth hydrothermal systems, leading to its widespread use in biomolecules. Formation of more complex biomolecules in hydrothermal systems may have required sulfur-bearing organic compounds as reaction intermediates.

Schulte, Mitchell

Transgenic plants with increased calcium stores

The present invention provides transgenic plants over-expressing a transgene encoding a calcium-binding protein or peptide (CaBP). Preferably, the CaBP is a calcium storage protein and over-expression thereof does not have undue adverse effects on calcium homeostasis or biochemical pathways that are regulated by calcium. In preferred embodiments, the CaBP is calreticulin (CRT) or calsequestrin. In more preferred embodiments, the CaBP is the C-domain of CRT, a fragment of the C-domain, or multimers of the foregoing. In other preferred embodiments, the CaBP is localized to the endoplasmic reticulum by operatively associating the transgene encoding the CaBP with an endoplasmic reticulum localization peptide. Alternatively, the CaBP is targeted to any other sub-cellular compartment that permits the calcium to be stored in a form that is biologically available to the plant. Also provided are methods of producing plants with desirable phenotypic traits by transformation of the plant with a transgene encoding a CaBP. Such phenotypic traits include increased calcium storage, enhanced resistance to calcium-limiting conditions, enhanced growth and viability, increased disease and stress resistance, enhanced flower and fruit production, reduced senescence, and a decreased need for fertilizer production. Further provided are plants with enhanced nutritional value as human food or animal feed.

Wyatt, Sarah

Circular Permutation of a Chaperonin Protein: Biophysics and Application to Nanotechnology

We have designed five circular permutants of a chaperonin protein derived from the hyperthermophilic organism Sulfolobus shibatae. These permuted proteins were expressed in E. coli and are well-folded. Furthermore, all the permutants assemble into 18-mer double rings of the same form as the wild-type protein. We characterized the thermodynamics of folding for each permutant by both guanidine denaturation and differential scanning calorimetry. We also examined the assembly of chaperonin rings into higher order structures that may be used as nanoscale templates. The results show that circular permutation can be used to tune the thermodynamic properties of a protein template as well as facilitating the fusion of peptides, binding proteins or enzymes onto nanostructured templates.

Paavola, Chad

Native Chemical Ligation of Peptoid Oligomers

Bioorganic chemists are inspired by natural biopolymers to design peptidomimetic oligomers that can exhibit sequence-structure-function relationships. Biomimetic polymers can be synthesized to incorporate a specific sequence of nonbiological monomer units using a variety of iterative solution-phase or solid-phase reaction schemes. These protocols generally provide access to a vast diversity of oligomeric compounds but are limited with respect to their ability to attain protein-like chain lengths. This constraint can preclude access to sequence-defined synthetic macromolecules with sufficient sizes required to exhibit tertiary structure and other protein-mimetic attributes. In contrast, peptide chemists have overcome this limitation by developing convergent synthetic methods, such as native chemical ligation, to join individual, smaller peptide chains together to make larger peptides or full proteins. A similar convergent approach is needed to establish efficient synthetic routes to non-natural sequence-defined macromolecules. Herein, we adapt the peptide native chemical ligation method to peptoid oligomers, demonstrating how short chains can be conjoined to create sequence-defined peptoid macromolecules. Nanosheet-forming peptoid polymers with distinct surface loop display domains were generated by sequential ligation of several discrete fragments. This method provides a reliable convergent ligation route for sequence-defined polypeptoids that results in a native amide bond joining the fragments. We envision that this strategy will be useful in synthesizing peptoid-based proteomimetics that incorporate diverse chemical features.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH