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At least 577 records · Page 32

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NASA NTRS1987

Use of hydrophilic polymer coatings for control of electroosmosis and protein adsorption

The purpose of this project was to examine the utility of polyethylene glycol (PEG) and dextran coatings for control of electroosmosis and protein adsorption; electroosmosis is an important, deleterious process affecting electrophoretic separations, and protein adsorption is a factor which needs to be controlled during protein crystal growth to avoid multiple nucleation sites. Performance of the project required use of X-ray photoelectron spectroscopy to refine previously developed synthetic methods. The results of this spectroscopic examination are reported. Measurements of electroosmotic mobility of charged particles in appropriately coated capillaries reveals that a new, one-step route to coating capillaries gives a surface in which electroosmosis is dramatically reduced. Similarly, both PEG and dextran coatings were shown by protein adsorption measurements to be highly effective at reducing protein adsorption on solid surfaces. These results should have impact on future low-g electrophoretic and protein crystal growth experiments.

Harris, J. Milton↗

NASA NTRS1988

Two puzzling aspects of protein crystal growth

A study is presented of several mechanisms which may reduce crystal growth rates and or terminate crystal growth. It is found that salt gradients which change the local chemical potential of the protein are insufficient to account for the slow crystal growth rates which have been reported. Contaminants which adsorb protein from solution may reduce the effective protein concentration, but the impurity's concentration and its affinity for protein are unknown. Association of protein molecules in bulk solution can reduce the monomer concentration significantly, but extant theory and experiment are not sensitive enough to determine the actual concentration of aggregates in solution. For systems of interest, shear-induced effects were found to be too weak to interfere with normal binding of incoming protein molecules. Although we found that most crystal growth occurs in a regime where both interfacial kinetics and diffusion influence crystal growth, the role of mass transfer rates on the terminal size of crystals is unknown, primarily because no data exist which cover the size range of interest (0.1 mm to 1 mm in length).

Grant, M. L.↗

NASA NTRS1988

An automated protein crystal growth facility on the space station

The need is addressed for an automated Protein Crystal Growth experiment on the Space Station and how robotics will be integrated into the system design. This automated laboratory system will enable several hundred protein crystals to grow simultaneously in microgravity and will allow the major variables in protein crystal growth to be monitored and controlled during the experiment. Growing good quality crystals is important in determining the complete structure of the protein by X-ray diffraction. This information is useful in the research and development of medicines and other important medical and biotechnological products. Previous Protein Crystal Growth experiments indicate that the microgravity environment of space allows larger crystals of higher quality to be grown as compared to the same crystals grown on the ground. It is therefore important to have a laboratory in space where protein crystals can be grown under carefully controlled conditions so that a crystal type can be reproduced as needed.

Herrmann, Melody↗

NASA NTRS1990

Crystallization of proteins by dynamic control of supersaturation

The growth of protein crystals is known to be the limiting factor in the determination of the three-dimensional structures of most proteins. It is expected that the kinetics of supersaturation, which is directly related to solvent evaporation, will affect protein crystal growth and nucleation and accordingly determine the quality, number, size, and morphology of the crystals. With a technique that controls the evaporation of solvent from a protein solution with N2(g) it is possible to determine the effect of different evaporation profiles on hen egg white lysozyme crystals. Hen egg white lysozyme was chosen as the model protein because it crystallizes easily and has solubility data available for most salt, pH, and temperature ranges. Commercially available lysozyme was further purified by a number of methods. Crystals grown with the purified lysozyme and with the unpurified lysozyme in citrate buffer were different shapes but were found to be of the same symmetry space group by precession photos. Differences were seen in the lysozyme crystals grown using different evaporation rates. At three of the four initial conditions for lysozyme crystal growth, longer evaporation times yielded better crystals. The evaporation times required to see a change in the appearance of the crystals was much longer than expected. The number of rates studied so far represent only a small fraction of the ones now available with the gas evaporation device. The technique also provides for control of both solution pH and temperature which are related to the solubilities of proteins.

Wilson, Lori June↗

NASA NTRS1992

Size effects in models for mechanically-stressed protein crystals and aggregates

As protein aggregates increase in size, they become easier to disrupt mechanically. Using the scaling properties of models proposed to govern protein aggregation, the effect of thermal vibrations and gravity are investigated as deforming forces. For typical protein assemblies made of 30 A proteins, the assembled diameter must remain less than 100-10,000 times the molecular radius to survive in finite thermal and gravity fields. The analysis predicts the following experimental outcomes: (1) reductions in gravitational strain should favor larger protein aggregates; (2) in comparing the aggregate stability of different proteins, the addition of peptide chains should stabilize against thermal strain, but should not affect gravitational strain; (3) critical aggregate sizes should show significant (exponential) sensitivity to cluster geometry, solution preparation and growth conditions. The analysis is extended to consider qualitative size effects in crystal damage during X-ray exposure.

Noever, David A.↗

NASA NTRS1996

A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

Wang, W.↗

NASA NTRS1995

Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

Patil, Shameekumar↗

NASA NTRS1996

Thermal Optimization of Growth and Quality in Protein Crystals

Experimental evidence suggests that larger and higher quality crystals can be attained in the microgravity of space; however, the effect of growth rate on protein crystal quality is not well documented. This research is the first step towards providing strategies to grow crystals under constant rates of growth. Controlling growth rates at a constant value allows for direct one-to-one comparison of results obtained in microgravity and on earth. The overall goal of the project was to control supersaturation at a constant value during protein crystal growth by varying temperature in a predetermined manner. Applying appropriate theory requires knowledge of specific physicochemical properties of the protein solution including the effect of supersaturation on growth rates and the effect of temperature on protein solubility. Such measurements typically require gram quantities of protein and many months of data acquisition. A second goal of the project applied microcalorimetry for the rapid determination of these physicochemical properties using a minimum amount of protein. These two goals were successfully implemented on hen egg-white lysozyme. Results of these studies are described in the attached reprints.

Wiencek, John M.↗

NASA NTRS1998

Nucleation and Convection Effects in Protein Crystal Growth

Our work under this grant has significantly contributed to the goals of the NASA supported protein crystallization program. We have achieved the main objectives of the proposed work, as outlined in the original proposal: (1) We have provided important insight into protein nucleation and crystal growth mechanisms to facilitate a rational approach to protein crystallization; (2) We have delineated the factors that currently limit the x-ray diffraction resolution of protein crystals, and their correlation to crystallization conditions; (3) We have developed novel technologies to study and monitor protein crystal nucleation and growth processes, in order to increase the reproducibility and yield of protein crystallization. We have published 17 papers in peer-reviewed scientific journals and books and made more than 15 invited and 9 contributed presentations of our results at international and national scientific meetings.

Vekilow, Peter G.↗

NASA NTRS1997

A Critical Assessment of Protein Crystal Growth in Microgravity

Experiments to grow higher diffraction quality protein crystals in the microgravity environment of an orbiting spacecraft are one of the most frequently flown space experiments. Ground-based research has shown that convective flows occur even about protein crystals growing in the Earth's gravitational field. Further, this research has shown that the resultant flow velocities can cause growth cessation, and probably affect the measured X-ray data quality obtained. How flow deleteriously affects protein crystal growth (PCG) is still not known, and is the subject of ongoing research. Failing a rational method for ameliorating flow effects on Earth, one can, through NASA and other nations space agency sponsored programs, carry out protein crystal growth in the microgravity environment of an orbiting spacecraft. Early first generation PCG hardware was characterized by a very low success rate and a steep design learning curve. Subsequent hardware designs have improved upon their predecessors. Now the crystal grower has a wide variety of hardware configurations and crystal growth protocols to choose from, many of which implement "standard" laboratory protein crystal growth methods. While many of these are first or early second generation hardware the success rate, defined as growing crystals giving data better than has been obtained on Earth, is at least 20% overall and may be considerably higher if one only considers latter experiments. There are a large number of protein crystals grown every year, with hundreds of structures determined. Those crystallized in microgravity represent a small proportion of this total, and there is concern that the costs of the microgravity PCG program(s) do not justify such limited returns. Empirical evidence suggests that optimum crystal growth conditions in microgravity differ from those determined on Earth, further exacerbating the chances of success. Microgravity PCG is probably best suited for "mature" crystallizations, where one has considerable experience with the particular macromolecular system and has reached an apparent limit in diffraction resolution. Under such circumstances the cost of one or two successful flight experiments may more than offset the equivalent cost of several years effort to obtain the same improvement in data quality.

Pusey, Marc↗

NASA NTRS1999

Fluorescence Studies of Protein Crystallization Interactions

We are investigating protein-protein interactions in under- and over-saturated crystallization solution conditions using fluorescence methods. The use of fluorescence requires fluorescent derivatives where the probe does not markedly affect the crystal packing. A number of chicken egg white lysozyme (CEWL) derivatives have been prepared, with the probes covalently attached to one of two different sites on the protein molecule; the side chain carboxyl of ASP 101, within the active site cleft, and the N-terminal amine. The ASP 101 derivatives crystallize while the N-terminal amine derivatives do not. However, the N-terminal amine is part of the contact region between adjacent 43 helix chains, and blocking this site does would not interfere with formation of these structures in solution. Preliminary FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C, using the N-terminal bound pyrene acetic acid (PAA, Ex 340 nm, Em 376 nm) and ASP 101 bound Lucifer Yellow (LY, Ex 425 nm, Em 525 nm) probe combination. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10 (exp 5) M respectively), and all experiments were carried out at approximately Csat or lower total protein concentration. The data at both salt concentrations show a consistent trend of decreasing fluorescence yield of the donor species (PAA) with increasing total protein concentration. This decrease is apparently more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations (reflected in the lower solubility). The estimated average distance between protein molecules at 5 x 10 (exp 6) M is approximately 70 nm, well beyond the range where any FRET can be expected. The calculated RO, where 50% of the donor energy is transferred to the acceptor, for the PAA-CEWL * LY-CEWL system is 3.28 nm, based upon a PAA-CEWL quantum efficiency of 0.41.

Pusey, Marc L.↗

NASA NTRS2001

Lab-on-a-Chip Based Protein Crystallization

We are developing a novel technique with which we will grow protein crystals in very small volumes, utilizing chip-based, microfluidic ("LabChip") technology. This development, which is a collaborative effort between NASA's Marshall Space Flight Center and Caliper Technologies Corporation, promises a breakthrough in the field of protein crystal growth. Our initial results obtained from two model proteins, Lysozyme and Thaumatin, show that it is feasible to dispense and adequately mix protein and precipitant solutions on a nano-liter scale. The mixtures have shown crystal growth in volumes in the range of 10 nanoliters to 5 microliters. In addition, large diffraction quality crystals were obtained by this method. X-ray data from these crystals were shown to be of excellent quality. Our future efforts will include the further development of protein crystal growth with LabChip(trademark) technology for more complex systems. We will initially address the batch growth method, followed by the vapor diffusion method and the liquid-liquid diffusion method. The culmination of these chip developments is to lead to an on orbit protein crystallization facility on the International Space Station. Structural biologists will be invited to utilize the on orbit Iterative Biological Crystallization facility to grow high quality macromolecular crystals in microgravity.

vanderWoerd, Mark J.↗

NASA NTRS2001

The Role of Stress Proteins in Cell Stabilization: A Perspective from an Extremophile

The existence of organisms that live at near boiling temperatures is living proof that all of the complex biochemical machinery of life can be adapted to function under these harsh conditions. The purpose of our research is to elucidate the role of a group of proteins known as heat shock proteins or HSP60s in this adaptation to high temperatures. HSP60s are found in all organisms and they are among the most highly conserved proteins known. We are investigating HSP60s in an organism growing at 80 C and pH 2.0 (Sulfolobus shibatae). This organism produces three closely-related HSP60 proteins, referred to as HSP60 alpha, beta, and gamma. Our DOE-funded research during the last two years has focused on clarifying the role of FiSP60 alpha and beta. These are among the two most abundant proteins in S. shibatae grown at high temperatures and significantly increase in abundance when the cells are exposed to near-lethal temperatures. We have demonstrated that these proteins protect the cells from lethal temperatures by stabilizing their membranes. During this last year we have been studying gamma, which was discovered by genome sequence analysis but nothing was known about its function. We have determined that gamma is only expressed at low temperatures. that it interacts with alpha and beta, and that it influences their ability to form higher-order structures critical to their function. We propose that gamma modulates HSP60 function at low temperatures.

Trent, Jonathan↗

NASA NTRS2002

Building Muscles, Keeping Muscles: Protein Turnover During Space Flight

As we age we lose muscle mass and strength. The problem is a matter of use it or lose it and more - a fact to which any active senior can attest. An imbalance in the natural cycle of protein turnover may be a contributing factor to decreased muscle mass. But the answer is not so simple, since aging is associated with changes in hormones, activity levels, nutrition, and often, disease. The human body constantly uses amino acids to build muscle protein, which then breaks down and must be replaced. When protein turnover gets out of balance, so that more protein breaks down than the body can replace, the result is muscle loss. This is not just the bane of aging, however. Severely burned people may have difficulty building new muscle long after the burned skin has been repaired. Answers to why we lose muscle mass and strength - and how doctors can fix it - may come from space. Astronauts usually eat a well-balanced diet and maintain an exercise routine to stay in top health. During long-duration flight, they exercise regularly to reduce the muscle loss that results from being in a near-weightless environment. Despite these precautions, astronauts lose muscle mass and strength during most missions. They quickly recover after returning to Earth - this is a temporary condition in an otherwise healthy population. Members of the STS-107 crew are participating in a study of the effects of space flight, hormone levels, and stress on protein turnover. When we are under stress, the body responds with a change in hormone levels. Researchers hypothesize that this stress-induced change in hormones along with the near-weightlessness might result in the body synthesizing less muscle protein, causing muscles to lose their strength and size. Astronauts, who must perform numerous duties in a confined and unusual environment, experience some stress during their flight, making them excellent candidates for testing the researchers' hypothesis.

Arny Ferrando↗

NASA NTRS2003

Structure, Function, Self-Assembly and Origin of Simple Membrane Proteins

Integral membrane proteins perform such essential cellular functions as transport of ions, nutrients and waste products across cell walls, transduction of environmental signals, regulation of cell fusion, recognition of other cells, energy capture and its conversion into high-energy compounds. In fact, 30-40% of genes in modem organisms codes for membrane proteins. Although contemporary membrane proteins or their functional assemblies can be quite complex, their transmembrane fragments are usually remarkably simple. The most common structural motif for these fragments is a bundle of alpha-helices, but occasionally it could be a beta-barrel. In a series of molecular dynamics computer simulations we investigated self-organizing properties of simple membrane proteins based on these structural motifs. Specifically, we studied folding and insertion into membranes of short, nonpolar or amphiphatic peptides. We also investigated glycophorin A, a peptide that forms sequence-specific dimers, and a transmembrane aggregate of four identical alpha-helices that forms an efficient and selective voltage-gated proton channel was investigated. Many peptides are attracted to water-membrane interfaces. Once at the interface, nonpolar peptides spontaneously fold to a-helices. Whenever the sequence permits, peptides that contain both polar and nonpolar amino also adopt helical structures, in which polar and nonpolar amino acid side chains are immersed in water and membrane, respectively. Specific identity of side chains is less important. Helical peptides at the interface could insert into the membrane and adopt a transmembrane conformation. However, insertion of a single helix is unfavorable because polar groups in the peptide become completely dehydrated upon insertion. The unfavorable free energy of insertion can be regained by spontaneous association of peptides in the membrane. The first step in this process is the formation of dimers, although the most common are aggregates of 4-7 helices. The helices could arrange themselves such that they formed pores capable of transporting ions and small molecules across membranes. Stability of transmembrane aggregates of simple proteins is often only marginal and, therefore, it can be regulated by environmental signals or small sequence modifications in the region of interhelical interactions. A key step in the earliest evolution of membrane proteins was the emergence of selectivity for specific substrates. Many channels could become selective if one or only a few properly chosen amino acids are properly placed along the channel, acting as filters or gates. This is a convenient evolutionary solution because it does not require imposing conditions on the whole sequence.

Pohorille, Andrew↗

NASA NTRS2004

X-ray Microscopic Characterization of Protein Crystals

The microscopic mapping of the variation in degree of perfection and in type of defects in entire protein crystals by x-rays may well be a prerequisite for better understanding causes of lattice imperfections, the growth history, and properties of protein crystals. However, x-ray microscopic characterization of bulk protein crystals, in the as-grown state, is frequently more challenging than that of small molecular crystals due to the experimental difficulties arising largely from the unique features possessed by protein crystals. In this presentation, we will illustrate ssme recent activities in employing coherence-based phase contrast x-ray imaging and high-angular-resolution diffraction techniques for mapping microdefects and the degree of perfection of protein crystals, and demonstrate a correlation between crystal perfection, diffraction phenomena., and crystallization conditions. The observed features and phenomena will be discussed in context to gain insight into the nature of defects, nucleation and growth, and the properties of protein crystals.

Hu, Z. W.↗

NASA NTRS2002

Alterations in protein metabolism during space flight and inactivity

Space flight and the accompanying diminished muscular activity lead to a loss of body nitrogen and muscle function. These losses may affect crew capabilities and health in long-duration missions. Space flight alters protein metabolism such that the body is unable to maintain protein synthetic rates. A concomitant hypocaloric intake and altered anabolic/catabolic hormonal profiles may contribute to or exacerbate this problem. The inactivity associated with bedrest also reduces muscle and whole-body protein synthesis. For this reason, bedrest provides a good model for the investigation of potential exercise and nutritional countermeasures to restore muscle protein synthesis. We have demonstrated that minimal resistance exercise preserves muscle protein synthesis throughout bedrest. In addition, ongoing work indicates that an essential amino acid and carbohydrate supplement may ameliorate the loss of lean body mass and muscle strength associated with 28 d of bedrest. The investigation of inactivity-induced alterations in protein metabolism, during space flight or prolonged bedrest, is applicable to clinical populations and, in a more general sense, to the problems associated with the decreased activity that occur with aging.

Non-NASA Center↗

NASA NTRS1997

Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

Non-NASA Center↗