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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Hydration‐Induced β‐Sheet Crosslinking of α‐Helical‐Rich Spider Prey‐Wrapping Silk

Abstract Due to its moderate strength (≈700 MPa) and impressive extensibility before breaking (≈60–80%), orb‐weaving spider aciniform (AC) prey‐wrapping silks are actually the toughest of the spider silks but are remarkably understudied. The previous results indicate that native AC silk fibers are an α‐helix rich coiled‐coil/β‐sheet hybrid nanofiber, and that conversion of disordered or helical domains to β‐sheet aggregates is surprisingly minimal and overall β‐sheet content is low (≈15%). In this work, it is demonstrated through scanning electron microscopy that native AC silk fibers undergo matted cross‐linking upon exposure to moisture that increases silk stiffness. The unique molecular mechanism of water‐induced cross‐linking is revealed with solid‐state NMR (SSNMR) methods; water‐induced morphological changes are correlated with an increase in AC silk protein β‐sheet content, and additionally a minor unfolding of coiled‐coil regions is observed. Continued and increased β‐sheet cross‐linking is observed upon application of mechanical shear. The size of these β‐sheet domains to be 4–6 nm using Wide‐Line Separation SSNMR is determined. The observation that merely water treatment can be used to convert a protein‐based material from a flexible/extensible α‐helix‐rich fiber to a rigid crossed‐linked β‐sheet mat is a novel observation that should provide new avenues in bioinspired materials design.

Stengel, Dillan↗
DOE OSTI2021

Genomic and Phenotypic Characterization of Chloracidobacterium Isolates Provides Evidence for Multiple Species

Chloracidobacterium is the first and until now the sole genus in the phylum Acidobacteriota (formerly Acidobacteria ) whose members perform chlorophyll-dependent phototrophy (i.e., chlorophototrophy). An axenic isolate of Chloracidobacterium thermophilum (strain B T ) was previously obtained by using the inferred genome sequence from an enrichment culture and diel metatranscriptomic profiling analyses in situ to direct adjustments to the growth medium and incubation conditions, and thereby a defined growth medium for Chloracidobacterium thermophilum was developed. These advances allowed eight additional strains of Chloracidobacterium spp. to be isolated from microbial mat samples collected from Mushroom Spring, Yellowstone National Park, United States, at temperatures of 41, 52, and 60°C; an axenic strain was also isolated from Rupite hot spring in Bulgaria. All isolates are obligately photoheterotrophic, microaerophilic, non-motile, thermophilic, rod-shaped bacteria. Chloracidobacterium spp. synthesize multiple types of (bacterio-)chlorophylls and have type-1 reaction centers like those of green sulfur bacteria. Light harvesting is accomplished by the bacteriochlorophyll a -binding, Fenna-Matthews-Olson protein and chlorosomes containing bacteriochlorophyll c . Their genomes are approximately 3.7 Mbp in size and comprise two circular chromosomes with sizes of approximately 2.7 Mbp and 1.0 Mbp. Comparative genomic studies and phenotypic properties indicate that the nine isolates represent three species within the genus Chloracidobacterium . In addition to C. thermophilum , the microbial mats at Mushroom Spring contain a second species, tentatively named Chloracidobacterium aggregatum , which grows as aggregates in liquid cultures. The Bulgarian isolate, tentatively named Chloracidobacterium validum , will be proposed as the type species of the genus, Chloracidobacterium. Additionally, Chloracidobacterium will be proposed as the type genus of a new family, Chloracidobacteriaceae , within the order Blastocatellales , the class Blastocatellia , and the phylum Acidobacteriota.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Assembly of ordered DNA-curli fibril complexes during Salmonella biofilm formation correlates with strengths of the type I interferon and autoimmune responses

Deposition of human amyloids is associated with complex human diseases such as Alzheimer’s and Parkinson’s. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro . The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to autoimmunity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Metal Ion Binding to the Amyloid β Monomer Studied by Native Top-Down FTICR Mass Spectrometry

Native top-down mass spectrometry is a fast, robust biophysical technique that can provide molecular-scale information on the interaction between proteins or peptides and ligands, including metal cations. Here we have analyzed complexes of the full-length amyloid β (1-42) monomer with a range of (patho)physiologically relevant metal cations using native Fourier transform ion cyclotron resonance mass spectrometry and three different fragmentation methods—collision-induced dissociation, electron capture dissociation, and infrared multiphoton dissociation—all yielding consistent results. Amyloid β is of particular interest as its oligomerization and aggregation are major events in the etiology of Alzheimer’s disease, and it is known that interactions between the peptide and bioavailable metal cations have the potential to significantly damage neurons. Those metals which exhibited the strongest binding to the peptide (Cu{sup 2+}, Co{sup 2+}, Ni{sup 2+}) all shared a very similar binding region containing two of the histidine residues near the N-terminus (His6, His13). Notably, Fe{sup 3+} bound to the peptide only when stabilized toward hydrolysis, aggregation, and precipitation by a chelating ligand, binding in the region between Ser8 and Gly25. We also identified two additional binding regions near the flexible, hydrophobic C-terminus, where other metals (Mg{sup 2+}, Ca{sup 2+}, Mn{sup 2+}, Na{sup +}, and K{sup +}) bound more weakly—one centered on Leu34, and one on Gly38. Unexpectedly, collisional activation of the complex formed between the peptide and [Co{sup III}(NH{sub 3}){sub 6}]{sup 3+} induced gas-phase reduction of the metal to Co{sup II}, allowing the peptide to fragment via radical-based dissociation pathways. This work demonstrates how native mass spectrometry can provide new insights into the interactions between amyloid β and metal cations. .

74 ATOMIC AND MOLECULAR PHYSICS↗
DOE OSTI2021

Sequence Analysis of 20,453 Severe Acute Respiratory Syndrome Coronavirus 2 Genomes from the Houston Metropolitan Area Identifies the Emergence and Widespread Distribution of Multiple Isolates of All Major Variants of Concern

Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there has been international concern about the emergence of virus variants with mutations that increase transmissibility, enhance escape from the human immune response, or otherwise alter biologically important phenotypes. In late 2020, several variants of concern emerged globally, including the UK variant (B.1.1.7), the South Africa variant (B.1.351), Brazil variants (P.1 and P.2), and two related California variants of interest (B.1.429 and B.1.427). These variants are believed to have enhanced transmissibility. For the South Africa and Brazil variants, there is evidence that mutations in spike protein permit it to escape from some vaccines and therapeutic monoclonal antibodies. On the basis of our extensive genome sequencing program involving 20,453 coronavirus disease 2019 patient samples collected from March 2020 to February 2021, we report identification of all six of these SARS-CoV-2 variants among Houston Methodist Hospital (Houston, TX) patients residing in the greater metropolitan area. Although these variants are currently at relatively low frequency (aggregate of 1.1%) in the population, they are geographically widespread. Houston is the first city in the United States in which active circulation of all six current variants of concern has been documented by genome sequencing. Finally, as vaccine deployment accelerates, increased genomic surveillance of SARS-CoV-2 is essential to understanding the presence, frequency, and medical impact of consequential variants and their patterns and trajectory of dissemination.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Structure of the Get3 targeting factor in complex with its membrane protein cargo

How to GET to the right membrane Membrane proteins with a hydrophobic transmembrane domain (TMD) play critical roles in virtually all aspects of cell physiology. After it has been synthesized in the cytosol, this TMD must be targeted to and inserted into the correct membrane. The GET pathway is one of two targeting pathways to the endoplasmic reticulum conserved across all eukaryotes. It is not clear how the central targeting factor, Get3, recognizes a TMD to shield it from aggregation until it is successfully inserted into the membrane. Now, Mateja et al. show that the functional targeting complex comprises a Get3 dimer bound to a single TMD. The helical hydrophobic TMD binds deep within a large hydrophobic groove in the Get3 dimer. This groove closes slightly upon TMD binding, forming a dynamic “lid” over the mouth of the groove. Science , this issue p. 1152

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Peak selection matters in Principal Component Analysis: A case study of syntrophic microbes

In situ liquid time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful technique to address surface characterization of living biofilms in hydrated conditions. Principal component analysis (PCA) is an important tool to process a large amount of data and interpret the information from ToF-SIMS spectra. However, ToF-SIMS data analysis is still a great challenge in complicated bacterial biofilms, because many interference peaks from the medium may result in inaccurate interpretation of ToF-SIMS spectral analysis. In this study, two syntrophic Geobacter populations were investigated using in situ liquid ToF-SIMS to reveal the biofilm surface changes between them as due to direct interspecies electron transfer (DIET). Peak overlay is an effective strategy to eliminate the matrix effects in handling complex ToF-SIMS data and was used to accomplish peak selection in this work. Both all peak and selected peak spectral PCA showed a distinctive separation among different samples. However, medium samples occupied high scores values in all peak PCA compared with the selected peak PCA, suggesting a strong interference from the medium as the matrix effect. Amino acids and lipids contributed significantly in separating all samples in selected peak PCA, which indicated that surface membrane components such as pili and proteins are important in the co-cultured Geobacter community compared to controls such as the single population planktonic cells and biofilms, respectively. Additionally, the spectral PCA results of high intensity and high resolution data obtained from liquid ToF-SIMS were compared. Selected peak PCA obtained nice separation among samples in both data sets; however, the high resolution data showed better separation between co-culture planktonics and co-culture aggregates, confirming that the higher mass accuracy is useful in analysis of microbial samples. Furthermore, amino acid and water cluster as an important media for microbial metabolite were selected for a more focused selected peak PCA comparison. Consistent scores and loadings plots were achieved. In conclusion, we show that peak selection is critical for acquiring effective microbial information and interpretation of syntrophic Geobacter using spectral data from in situ liquid ToF-SIMS.

spectral overlay, peak selection, matrix effect, P↗
DOE OSTI2012

Solving structure with sparse, randomly-oriented x-ray data

Single-particle imaging experiments of biomolecules at x-ray free-electron lasers (XFELs) require processing hundreds of thousands of images that contain very few x-rays. Each low-fluence image of the diffraction pattern is produced by a single, randomly oriented particle, such as a protein. We demonstrate the feasibility of recovering structural information at these extremes using low-fluence images of a randomly oriented 2D x-ray mask. Successful reconstruction is obtained with images averaging only 2.5 photons per frame, where it seems doubtful there could be information about the state of rotation, let alone the image contrast. This is accomplished with an expectation maximization algorithm that processes the low-fluence data in aggregate, and without any prior knowledge of the object or its orientation. The versatility of the method promises, more generally, to redefine what measurement scenarios can provide useful signal.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2014

The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution

Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in promoting nucleation, controlling growth, and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, the quaternary structure of LRAP is not as well studied. Here, analytical ultracentrifugation sedimentation velocity (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP over a range of pH values, ionic strengths, and concentrations. SV has advantages over other techniques in accurately quantifying protein speciation in polydisperse solutions. We found that the monomer was the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7 to 4.1, pH 4.5 to 8, 50 mmol/L( mM) to 200 mM NaCl, 0.065 to 2 mg/mL). The monomer was also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregated in a narrow pH range near the isoelectric point (pH 4.1). We conclude that LRAP does not form nanospheres under physiological solution conditions. Both SV and SANS showed that the LRAP monomer has a radius of ~2.0 nm and adopts an extended structure which solution NMR studies show is intrinsically disordered. This work provides new insights into the tertiary and quaternary structure of LRAP and further evidence that the monomeric species is an important functional form of amelogenins

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Self-assembly of pi-conjugated peptides in aqueous environments leading to energy-transporting bioelectronic nanostructures

The realization of new supramolecular pi-conjugated organic structures inspired and driven by peptide-based self-assembly will offer a new approach to interface with the biotic environment in a way that will help to meet many DOE-recognized grand challenges. Previously, we developed pi-conjugated peptides that undergo supramolecular self-assembly into one-dimensional (1-D) organic electronic nanomaterials under benign aqueous conditions. The intermolecular interactions among the pi-conjugated organic segments within these nanomaterials lead to defined perturbations of their optoelectronic properties and yield nanoscale conduits that support energy transport within individual nanostructures and throughout bulk macroscopic collections of nanomaterials. Our objectives for future research are to construct and study biomimetic electronic materials for energy-related technology optimized for harsher non-biological environments where peptide-driven self-assembly enhances pi-stacking within nanostructured biomaterials, as detailed in the following specific tasks: (1) synthesis and detailed optoelectronic characterization of new pi-electron units to embed within homogeneous self assembling peptides, (2) molecular and data-driven modeling of the nanomaterial aggregates and their higher-order assemblies, and (3) development of new hierarchical assembly paradigms to organize multiple electronic subunits within the nanomaterials leading to heterogeneous electronic properties (i.e. gradients and localized electric fields). These intertwined research tasks will lead to the continued development and fundamental mechanistic understanding of a powerful bioinspired materials set capable of making connections between nanoscale electronic materials and macroscopic bulk interfaces, be they those of a cell, a protein or a device.

36 MATERIALS SCIENCE↗
DOE OSTI2011

Vasculatures in Tumors Growing From Preirradiated Tissues: Formed by Vasculogenesis and Resistant to Radiation and Antiangiogenic Therapy

Purpose: To investigate vasculatures and microenvironment in tumors growing from preirradiated tissues (pre-IR tumors) and study the vascular responses of pre-IR tumors to radiation and antiangiogenic therapy. Methods and Materials: Transgenic adenocarcinoma of the mouse prostate C1 tumors were implanted into unirradiated or preirradiated tissues and examined for vascularity, hypoxia, and tumor-associated macrophage (TAM) infiltrates by immunohistochemistry. The origin of tumor endothelial cells was studied by green fluorescent protein-tagged bone marrow (GFP-BM) transplantation. The response of tumor endothelial cells to radiation and antiangiogenic agent was evaluated by apoptotic assay. Results: The pre-IR tumors had obvious tumor bed effects (TBE), with slower growth rate, lower microvascular density (MVD), and more necrotic and hypoxic fraction compared with control tumors. The vessels were dilated, tightly adhered with pericytes, and incorporated with transplanted GFP-BM cells. In addition, hypoxic regions became aggregated with TAM. As pre-IR tumors developed, the TBE was overcome at the tumor edge where the MVD increased, TAM did not aggregate, and the GFP-BM cells did not incorporate into the vessels. The vessels at tumor edge were more sensitive to the following ionizing radiation and antiangiogenic agent than those in the central low MVD regions. Conclusions: This study demonstrates that vasculatures in regions with TBE are mainly formed by vasculogenesis and resistant to radiation and antiangiogenic therapy. Tumor bed effects could be overcome at the edge of larger tumors, but where vasculatures are formed by angiogenesis and sensitive to both treatments. Vasculatures formed by vasculogenesis should be the crucial target for the treatment of recurrent tumors after radiotherapy.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
NASA NTRS1981

Terrestrial evolution of polymerization of amino acids - Heat to ATP

Sets of amino acids containing sufficient trifunctional monomer are thermally polymerized at temperatures such as 65 deg; the amino acids order themselves. Various polymers have diverse catalytic activities. The polymers aggregate, in aqueous solution, to cell-like structures having those activities plus emergent properties, e.g. proliferatability. Polyamino acids containing sufficient lysine catalyze conversion of free amino acids, by ATP, to small peptides and a high molecular weight fraction. The lysine-rich proteinoid is active in solution, within suspensions of cell-like particles, or in other particles composed of lysine-rich proteinoid and homopolyribonucleotide. Selectivities are observed. An archaic polyamino acid prelude to coded protein synthesis is indicated.

Fox, S. W.↗
NASA NTRS1999

Biotechnology Science Experiments on Mir

This paper describes the microgravity biotechnology experiments carried out on the Shuttle/Mir program. Four experiments investigated the growth of protein crystals, and three investigated cellular growth. Many hundreds of protein samples were processed using four different techniques. The objective of these experiments was to determine optimum conditions for the growth of very high quality single crystals to be used for structure determination. The Biotechnology System (BTS) was used to process the three cell growth investigations. The samples processed by these experiments were: bovine chondrocytes, human renal epithelial cells, and human breast cancer cells and endothelial cells. The objective was to determine the unique properties of cell aggregates produced in the microgravity environment.

Kroes, Roger L.↗
NASA NTRS1998

Determining the Molecular Growth Mechanisms of Protein Crystal faces by Atomic Force Microscopy

A high resolution atomic force microscopy (AFM) study had shown that the molecular packing on the tetragonal lysozyme (110) face corresponded to only one of two possible packing arrangements, suggesting that growth layers on this face were of bimolecular height (Li et al., 1998). Theoretical analyses of the packing had also indicated that growth of this face should proceed by the addition of growth units of at least tetramer size corresponding to the 43 helices in the crystal. In this study an AFM linescan technique was devised to measure the dimensions of individual growth units on protein crystal faces. The growth process of tetragonal lysozyme crystals was slowed down by employing very low supersaturations. As a result images of individual growth events on the (110) face were observed, shown by jump discontinuities in the growth step in the linescan images. The growth unit dimension in the scanned direction was obtained by suitably averaging these images. A large number of scans in two directions on the (110) face were performed and the distribution of lysozyme aggregate sizes were obtained. A variety of growth units, all of which were 43 helical lysozyme aggregates, were shown to participate in the growth process with a 43 tetramer being the minimum observed size. This technique represents a new application for AFM allowing time resolved studies of molecular process to be carried out.

Li, Huayu↗
DOE OSTI2013

Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: Role of β-catenin/connexin43 association

Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of β-catenin, α-catenin and ZO-2, it decreased nuclear levels of β-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, β-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, β-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering β-catenin away from nucleus. - Highlights: • Cx43 over-expressing MCF-7 and MDA-MB-231 were grown in 2D and 3D cultures. • Proliferation and growth morphology were affected in a context dependent manner. • Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced. • Cx43-mediated gap junction complex assembly correlated with observed changes. • We propose that membranous Cx43 sequesters β-catenin away from the nucleus.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Autophagy induction in tobacco leaves infected by potato virus Y{sup O} and its putative roles

Autophagy plays a critical role in the innate immune response of plants to pathogen infection. In the present study, we examined autophagy induced by potato virus Y ordinary strain (PVY{sup O}) infection in tobacco (Nicotiana benthamiana). Enzyme-linked immunosorbent assays revealed that the number of virus particles in the plant peaked at 2 weeks post-inoculation and then gradually decreased. Additionally, the amount of virus increased significantly in the 3rd and 4th leaves distal to the inoculated leaf and decreased slightly in the 5th leaf. Within 2 weeks of PVY{sup O} inoculation, the tobacco leaves showed typical symptoms of Potyvirus inoculation, including mottling, yellowing, a mosaic pattern, and necrotic tissue changes at the inoculated site. Based on an ultrastructural analysis of the PVY{sup O}-infected tobacco leaves, virus aggregates appeared as longitudinal and transverse arrays and pinwheels, which are typical of Potyvirus inoculation. Moreover, PVY{sup O} infection caused changes in the number, size, and shape of chloroplasts, whereas the number of plastogranules increased markedly. Furthermore, double-membrane autophagosome-like vacuoles, including electron-dense materials, laminated structures, and cellular organelles, were found. The induction of autophagy after the PVY{sup O} infection of tobacco leaves was further confirmed by the expression of lipidated microtubule-associated protein 1 light chain 3 (LC3)-II, an autophagy marker and p62, an autophagy adaptor protein. The LC3-II levels increased daily over the 4-week period. Although virus inoculation was performed systemically on the basal leaves of the plants, LC3-II was expressed throughout the leaves and the expression was higher in leaves distal to the inoculated leaf. Moreover, PVY{sup O} infection caused the activation of stress-activated protein kinases/c-Jun N-terminal kinases. Therefore, PVY{sup O} infection-induced autophagy was positively correlated with the virus content, suggesting that autophagy induction following PVY{sup O} infection is involved in the anti-pathogen response of the host. -- Highlights: •The amount of PVY{sup O} revealed a peak at 2 weeks post-inoculation. •The amount of PVY{sup O} was higher in leaves distal to the inoculated leaf. •PVY{sup O} infection produced typical PVY infection symptoms. •Double-membranous vacuoles, including cellular organelles, were induced by PVY{sup O} infection. •PVY{sup O} infection induced LC3-II and JNK activation.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2000

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Hong, Y. S.↗