Biomedical subjects

Andreas Holzenburg

Publications and source records attributed to Andreas Holzenburg.

At least 19 recordsLinked to original sources

PubMed2007

Core-controlled polymorphism in virus-like particles.

This study concerns the self-assembly of virus-like particles (VLPs) composed of an icosahedral virus protein coat encapsulating a functionalized spherical nanoparticle core. The recent development of efficient methods for VLP self-assembly has opened the way to structural studies. Using electron microscopy with image reconstruction, the structures of several VLPs obtained from brome mosaic virus capsid proteins and gold nanoparticles were elucidated. Varying the gold core diameter provides control over the capsid structure. The number of subunits required for a complete capsid increases with the core diameter. The packaging efficiency is a function of the number of capsid protein subunits per gold nanoparticle. VLPs of varying diameters were found to resemble to three classes of viral particles found in cells (T=1, 2, and 3). As a consequence of their regularity, VLPs form three-dimensional crystals under the same conditions as the wild-type virus. The crystals represent a form of metallodielectric material that exhibits optical properties influenced by multipolar plasmonic coupling.

Crystallization↗

PubMed2006

RNA recognition and cleavage by the SARS coronavirus endoribonuclease.

The emerging disease SARS is caused by a novel coronavirus that encodes several unusual RNA-processing enzymes, including non-structural protein 15 (Nsp15), a hexameric endoribonuclease that preferentially cleaves at uridine residues. How Nsp15 recognizes and cleaves RNA is not well understood and is the subject of this study. Based on the analysis of RNA products separated by denaturing gel electrophoresis, Nsp15 has been reported to cleave both 5' and 3' of the uridine. We used several RNAs, including some with nucleotide analogs, and mass spectrometry to determine that Nsp15 cleaves only 3' of the recognition uridylate, with some cleavage 3' of cytidylate. A highly conserved RNA structure in the 3' non-translated region of the SARS virus was cleaved preferentially at one of the unpaired uridylate bases, demonstrating that both RNA structure and base-pairing can affect cleavage by Nsp15. Several modified RNAs that are not cleaved by Nsp15 can bind Nsp15 as competitive inhibitors. The RNA binding affinity of Nsp15 increased with the content of uridylate in substrate RNA and the co-factor Mn(2+). The hexameric form of Nsp15 was found to bind RNA in solution. A two-dimensional crystal of Nsp15 in complex with RNA showed that at least two RNA molecules could be bound per hexamer. Furthermore, an 8.3 A structure of Nsp15 was developed using cyroelectron microscopy, allowing us to generate a model of the Nsp15-RNA complex.

Binding, Competitive↗

PubMed2006

Structural and functional analyses of the human Toll-like receptor 3. Role of glycosylation.

Toll-like receptors (TLRs) play critical roles in bridging the innate and adaptive immune responses. The human TLR3 recognizes foreign-derived double-stranded RNA and endogenous necrotic cell RNA as ligands. Herein we characterized the contribution of glycosylation to TLR3 structure and function. Exogenous addition of purified extracellular domain of TLR3 (hTLR3 ECD) expressed in human embryonic kidney cells was found to inhibit TLR3-dependent signaling, thus providing a reagent for structural and functional characterization. Approximately 35% of the mass of the hTLR3 ECD was due to posttranslational modification, with N-linked glycosyl groups contributing substantially to the additional mass. Cells treated with tunicamycin, an inhibitor of glycosylation, prevented TLR3-induced NF-kappaB activation, confirming that N-linked glycosylation is required for bioactivity of this receptor. Further, mutations in two of these predicted glycosylation sites impaired TLR3 signaling without obviously affecting the expression of the protein. Single-particle structures reconstructed from electron microscopy images and two-dimensional crystallization revealed that hTLR3 ECD forms a horseshoe structure similar to the recently elucidated x-ray structure of the protein expressed in insect cells using baculovirus vectors (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585 and Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980). There are, however, notable differences between the human cell-derived and insect cell-derived structures, including features attributable to glycosylation.

Amino Acid Sequence↗

PubMed2006

Quantification and confocal imaging of protein specific molecularly imprinted polymers.

We have employed FITC--albumin as the protein template molecule in an aqueous phase molecular imprinted polymer (HydroMIP) strategy. For the first time, the use of a fluorescently labeled template is reported, with subsequent characterization of the smart material to show that the HydroMIP possesses a significant molecular memory in comparison to that of the nonimprinted control polymer (HydroNIP). The imaging of the FITC--albumin imprinted HydroMIP using confocal microscopy is described, with the in situ removal of the imprinted protein displayed in terms of observed changes in the fluorescence of the imprinted polymer, both before and after template elution (using a 10% SDS/10% AcOH (w/v) solution). We also report the imaging of a bovine hemoglobin (BHb) imprinted HydroMIP using two-photon confocal microscopy and describe the effects of template elution upon protein autofluorescence. The findings further contribute to the understanding of aqueous phase molecular imprinting protocols and document the use of fluorescence as a useful tool in template labeling/detection and novel imaging strategies.

Albumins↗

PubMed2005

Mutational analysis of the SARS virus Nsp15 endoribonuclease: identification of residues affecting hexamer formation.

The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-dependent endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration, while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low endoribonuclease activity, suggesting that oligomerization is required for endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites.

Amino Acids↗

PubMed2005

Asymmetric binding of membrane proteins to GroEL.

The interaction of GroEL with non-native soluble proteins has been studied intensively and structure-function relationships have been established in considerable detail. Recently, we found that GroEL is also able to bind membrane proteins in the absence of detergents and deliver them to liposomes in a biologically active state. Here, we report that three well-studied membrane proteins (bacteriorhodopsin, LacY, and the bacteriophage lambda holin) bind asymmetrically to tetradecameric GroEL. Each of the membrane proteins was visualized in one of the center cavities of GroEL using single particle analysis.

Bacteriorhodopsins↗

PubMed2005

Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions.

We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.

Amino Acid Sequence↗

PubMed2005

The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol.

Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR-pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two-dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.

Adaptation, Physiological↗

PubMed2004

Insights into the structure of human cytomegalovirus large terminase subunit pUL56.

Terminases are a class of proteins which catalyze the generation of unit-length genomes during DNA packaging. These essential proteins are conserved throughout the herpesviruses and many double-stranded DNA bacteriophages. We have determined the structure of the large terminase subunit pUL56 of human cytomegalovirus, a highly pathogenic virus, to 2.6 nm resolution. Image analysis of purified pUL56 suggests that the molecule exists as a dimer formed by the association of two ring-like structures positioned on top of each other and connected by a pronounced density on one side. The 3D reconstruction of pUL56 provides first structural insights into the active protein.

Adenosine Triphosphatases↗

PubMed2004

Functional bacteriorhodopsin is efficiently solubilized and delivered to membranes by the chaperonin GroEL.

Soluble complexes between the tetradecameric chaperonin GroEL and integral membrane proteins can be efficiently formed by detergent dialysis. For example, GroEL14 was found to bind a limit of two molecules of bacteriorhodopsin (BR). The GroEL-solubilized BR molecules were rapidly ejected from the chaperonin complexes on the addition of ATP or adenosine 5'-[beta,gamma-imido]triphosphate but not AMP, indicating that conformational changes induced by nucleotide binding eliminate a binding site for the hydrophobic transmembrane domains. BR retains its native conformation in the GroEL complexes, as judged by the spectral characteristics of the bound retinal. Moreover, the chaperonin-solubilized BR could be transferred efficiently to liposomes and used to effect a light-driven proton gradient, indicating that both native conformation and vectorial insertion were accomplished. These results suggest new approaches to the study of purified integral membrane proteins in their natural membrane environment and raise the prospect that GroEL may have a role in the integration of proteins into the cytoplasmic membrane in vivo.

Bacteriorhodopsins↗

PubMed2004

NMR structure of the KaiC-interacting C-terminal domain of KaiA, a circadian clock protein: implications for KaiA-KaiC interaction.

KaiA is a two-domain circadian clock protein in cyanobacteria, acting as the positive element in a feedback loop that sustains the oscillation. The structure of the N-terminal domain of KaiA is that of a pseudo-receiver, similar to those of bacterial response regulators, which likely interacts with components of the clock-resetting pathway. The C-terminal domain of KaiA is highly conserved among cyanobacteria and enhances the autokinase activity of KaiC. Here we present the NMR structure of the C-terminal domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. This domain adopts a novel all alpha-helical homodimeric structure. Several mutations known to affect the period of the circadian oscillator are shown to be located at an exposed groove near the dimer interface. This NMR structure and a 21-A-resolution electron microscopy structure of the hexameric KaiC particle allow us to postulate a mode of KaiA-KaiC interaction, in which KaiA binds a linker region connecting two globular KaiC domains.

Bacterial Proteins↗

PubMed2004

Solubilization and delivery by GroEL of megadalton complexes of the lambda holin.

GroEL can solubilize membrane proteins by binding them in its hydrophobic cavity when detergent is removed by dialysis. The best-studied example is bacteriorhodopsin, which can bind in the GroEL chaperonin at two molecules per tetradecamer. Applying this approach to the holin and antiholin proteins of phage lambda, we find that both proteins are solubilized by GroEL, in an ATP-sensitive mode, but to vastly different extents. The antiholin product, S107, saturates the chaperonin at six molecules per tetradecameric complex, whereas the holin, S105, which is missing the two N-terminal residues of S107, forms a hyper-solubilization complex with up to 350 holin molecules per GroEL, or approximately 4 MDa of protein per 0.8 MDa tetradecamer. Gel filtration chromatography and immunoprecipitation experiments confirmed the existence of complexes of the predicted masses for both S105 and S107 solubilization. For S105, negatively stained electron microscopic images show structures consistent with protein shells of the holin assembled around the chaperonin tetradecamer. Importantly, S105 can be delivered rapidly and efficiently to artificial liposomes from these complexes. In these delivery experiments, the holin exhibits efficient membrane-permeabilizing activity. The S107 antiholin can block formation of the hypersolubilization complexes, suggesting that their formation is related to an oligomerization step intrinsic to holin function.

Adenosine Triphosphate↗

PubMed2003

Crystal structure of Mycobacterium tuberculosis SecA, a preprotein translocating ATPase.

In bacteria, the majority of exported proteins are translocated by the Sec system, which recognizes the signal sequence of a preprotein and uses ATP and the proton motive force to mediate protein translocation across the cytoplasmic membrane. SecA is an essential protein component of this system, containing the molecular motor that facilitates translocation. Here we report the three-dimensional structure of the SecA protein of Mycobacterium tuberculosis. Each subunit of the homodimer contains a "motor" domain and a translocation domain. The structure predicts that SecA can interact with the SecYEG pore and function as a molecular ratchet that uses ATP hydrolysis for physical movement of the preprotein. Knowledge of this structure provides a framework for further elucidation of the translocation process.

Adenosine Diphosphate↗

PubMed2003

Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin.

The host range of swinepox virus (SPV) is restricted to swine, although SPV has been shown to infect mammalian, non-swine cells, without recovery of infectious virus. SPV is a reasonable candidate for development as a non-productively replicating viral vector for use in non-swine, mammalian species, such as the cat. A novel SPV gene deletion (SPV 043) was created and found to be non-attenuating. This deletion was utilized to generate a stable recombinant virus expressing the Gag-Pro and Env proteins of feline leukemia virus (FeLV). Expression and replication of this vector was studied in embryonic swine kidney cells (ESK-4), and two feline cell lines, Crandell feline kidney cells (CRFK) and feline skin fibroblasts (FSF). Our results showed that feline cells were susceptible to infection by SPV and supported expression of foreign genes driven by synthetic poxvirus promoters, however, SPV viral DNA was not replicated in feline cells and infectious virus was not recovered. In addition, FeLV Gag virus-like particles were produced from both ESK-4 and CRFK cells and foreign antigens were incorporated into infectious SPV intracellular mature virions (IMV). These results suggest that SPV may have potential as a safe vaccine delivery vector for cats.

Animals↗

PubMed2003

Viral internal ribosome entry site structures segregate into two distinct morphologies.

An increasing number of viruses have been shown to initiate protein synthesis by a cap-independent mechanism involving internal ribosome entry sites (IRESs). Predictions of the folding patterns of these RNA motifs have been based primarily on sequence and biochemical analyses. Biophysical confirmation of the models has been achieved only for the IRES of hepatitis C virus (HCV), which adopts an open structure consisting of two major stems. We have conducted an extensive comparison of flavivirus and picornavirus IRES elements by negative stain transmission electron microscopy. All of the flavivirus IRESs we examined (those of GB virus-B, GB virus-C, and classical swine fever virus) fold to give a structure similar to that of the HCV IRES, as does an IRES recently found on mRNA encoded by human herpesvirus 8. The larger picornavirus IRESs (those of foot-and-mouth disease virus, rhinovirus, encephalomyocarditis virus, and hepatitis A virus) are morphologically similar, comprising a backbone with two protruding stems, and distinct from the flavivirus IRESs.

Animals↗

PubMed2002

Light-induced trimer to monomer transition in the main light-harvesting antenna complex of plants: thermo-optic mechanism.

The main chlorophyll a/b light-harvesting complex of photosystem II, LHCIIb, has earlier been shown to be capable of undergoing light-induced reversible structural changes and chlorophyll a fluorescence quenching in a way resembling those observed in granal thylakoids when exposed to excess light [Barzda, V., et al. (1996) Biochemistry 35, 8981-8985]. The nature and mechanism of this unexpected structural flexibility has not been elucidated. In this work, by using density gradient centrifugation and nondenaturing green gel electrophoresis, as well as absorbance and circular dichroic spectroscopy, we show that light induces a significant degree of monomerization, which is in contrast with the preferentially trimeric organization of the isolated complexes in the dark. Monomerization is accompanied by a reversible release of Mg ions, most likely from the outer loop of the complexes. These data, as well as the built-in thermal and light instability of the trimeric organization, are explained in terms of a simple theoretical model of thermo-optic mechanism, effect of fast thermal transients (local T-jumps) due to dissipated photon energies in the vicinity of the cation binding sites, which lead to thermally assisted elementary structural transitions. Disruption of trimers to monomers by excess light is not confined to isolated trimers and lamellar aggregates of LHCII but occurs in photosystem II-enriched grana membranes, intact thylakoid membranes, and whole plants. As indicated by differences in the quenching capability of trimers and monomers, the appearance of monomers could facilitate the nonphotochemical quenching of the singlet excited state of chlorophyll a. The light-induced formation of monomers may also be important in regulated proteolytic degradation of the complexes. Structural changes driven by thermo-optic mechanisms may therefore provide plants with a novel mechanism for regulation of light harvesting in excess light.

Arabidopsis Proteins↗

PubMed2002

The location of plastocyanin in vascular plant photosystem I.

We have studied the binding sites of the electron donor and acceptor proteins of vascular plant photosystem I by electron microscopy/crystallography. Previously, we identified the binding site for the electron acceptor (ferredoxin). In this paper we complete these studies with the characterization of the electron donor (plastocyanin) binding site. After cross-linking, plastocyanin is detected using Fourier difference analysis of two dimensionally ordered arrays of photosystem I located at the periphery of chloroplast grana. Plastocyanin binds in a small cavity on the lumenal surface of photosystem I, close to the center and with a slight bias toward the PsaL subunit of the complex. The recent release of the full coordinates for the cyanobacterial photosystem I reaction center has allowed a detailed comparison between the structures of the eukaryotic and prokaryotic systems. This reveals a very close homology, which is particularly striking for the lumenal side of photosystem I.

Binding Sites↗

PubMed2002

The terminase subunits pUL56 and pUL89 of human cytomegalovirus are DNA-metabolizing proteins with toroidal structure.

Herpesvirus DNA packaging involves binding and cleavage of DNA containing the specific DNA-packaging motifs. Here we report a first characterization of the terminase subunits pUL56 and pUL89 of human cytomegalovirus (HCMV). Both gene products were shown to have comparable nuclease activities in vitro. Under limiting protein concentrations the nuclease activity is enhanced by interaction of pUL56 and pUL89. High amounts of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole partially inhibited the pUL89-associated nuclease activity. It was demonstrated that pUL56 is able to bind to nucleocapsids in vivo. Electron microscopy (EM) and image analysis of purified pUL56 revealed that the molecules occurred as a distinct ring-shaped structure with a pronounced cleft. EM analysis of purified pUL89 demonstrated that this protein is also a toroidal DNA-metabolizing protein. Upon interaction of pUL56 with linearized DNA, the DNA remains uncut while the cutting event itself is mediated by pUL89. Using biochemical assays in conjunction with EM pUL56 was shown to (i) bind to DNA and (ii) associate with the capsid. In contrast to this, EM analysis implied that pUL89 is required to effect DNA cleavage. The data provide the first insights into the terminase-dependent viral DNA-packaging mechanism of HCMV.

Animals↗