Publications by authors named "Giuliano Bellapadrona"

12 Publications

  • Page 1 of 1

Detection of isolated protein-bound metal ions by single-particle cryo-STEM.

Proc Natl Acad Sci U S A 2017 10 2;114(42):11139-11144. Epub 2017 Oct 2.

Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 7610001, Israel;

Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.
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http://dx.doi.org/10.1073/pnas.1708609114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651765PMC
October 2017

Design of a Redox-Sensitive Supramolecular Protein Assembly System Operating in Live Cells.

Nano Lett 2016 Oct 6;16(10):6231-6235. Epub 2016 Sep 6.

Department of Materials and Interfaces, Weizmann Institute of Science , Rehovot 76100, Israel.

A fusion construct between Citrine (a YFP variant) and human ferritin (H-chain) was recently shown to form supramolecular assemblies of micrometer size when expressed in mammalian cells. The assembly process is driven by weak hydrophobic interactions leading to dimerization of YFP. Protein assembly could be suppressed at the gene level by mutation in the primary sequence of the construct. In this work, we describe the engineering of a self-assembly interface sensitive to redox state in the cell. Key hydrophobic residues of YFP were mutated systematically to cysteines. Supramolecular assembly of the Citrine-ferritin construct was in some cases preserved by formation of disulfide bonds in place of hydrophobic interactions. In others cases, assembly was abolished, resulting in a diffuse distribution of the expressed protein. A specific variant that remained diffuse under normally reducing intracellular conditions was found to self-assemble rapidly upon exposure to a thiol-specific oxidizing reagent.
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http://dx.doi.org/10.1021/acs.nanolett.6b02480DOI Listing
October 2016

Supramolecular Assembly and Coalescence of Ferritin Cages Driven by Designed Protein-Protein Interactions.

Biomacromolecules 2015 Jul 9;16(7):2006-11. Epub 2015 Jun 9.

†Department of Materials and Interfaces, Weizmann Institute of Science, 76100 Rehovot, Israel.

A genetically encoded system for expression of supramolecular protein assemblies (SMPAs) based on a fusion construct between ferritin and citrine (YFP) was transferred from a mammalian to a bacterial host. The assembly process is revealed to be independent of the expression host, while dimensions and level of order of the assembled structures were influenced by the host organism. An additional level of interactions, namely, coalescence between the preformed SMPAs, was observed during the purification process. SAXS investigation revealed that upon coalescence, the local order of the individual SMPAs was preserved. Finally, the chaotropic agent urea effectively disrupted both the macroscopic coalescence and the interactions at the nanoscale until the level of the single ferritin cage.
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http://dx.doi.org/10.1021/acs.biomac.5b00435DOI Listing
July 2015

Probing protein conformation in cells by EPR distance measurements using Gd3+ spin labeling.

J Am Chem Soc 2014 Sep 11;136(38):13458-65. Epub 2014 Sep 11.

Department of Chemical Physics and ‡Department of Materials and Interfaces, Weizmann Institute of Science , Rehovot, Israel 7610001.

Protein structure investigations are usually carried out in vitro under conditions far from their native environment in the cell. Differences between in-cell and in vitro structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electron-electron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to in-cell measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for in-cell DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd(3+) chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells.
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http://dx.doi.org/10.1021/ja5079392DOI Listing
September 2014

Supramolecular protein assemblies in the nucleus of human cells.

Angew Chem Int Ed Engl 2014 Feb 22;53(6):1534-7. Epub 2014 Jan 22.

Dept of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100 (Israel).

Genetically encoded supramolecular protein assemblies (SMPAs) are induced to form in living cells by combination of distinct self-assembly properties. A single fusion construct contains genes encoding the heavy chain (H) of human ferritin and the citrine fluorescent protein, the latter exposing a weak dimerization interface, as well as a nuclear localization signal. Upon expression in HeLa cells, in vivo confocal fluorescence and differential interference contrast imaging revealed extended SMPA structures exclusively in the nuclei. Assemblies were typically round and took alveolar, shell-like, or hybrid structure. Transmission electron microscopy revealed a crystalline packing. Site-specific mutagenesis of the citrine dimerization interface clarified the mechanism of SMPA formation. The constituent proteins retained their activity in iron binding and fluorescence emission, thus suggesting a general strategy for formation of synthetic cellular bodies with specific biochemical function.
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http://dx.doi.org/10.1002/anie.201309163DOI Listing
February 2014

Optimization of localized surface plasmon resonance transducers for studying carbohydrate-protein interactions.

Anal Chem 2012 Jan 7;84(1):232-40. Epub 2011 Dec 7.

Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, 76100, Israel.

Noble metal nanostructures supporting localized surface plasmons (SPs) have been widely applied to chemical and biological sensing. Changes in the refractive index near the nanostructures affect the SP extinction band, making localized surface plasmon resonance (LSPR) spectroscopy a convenient tool for studying biological interactions. Carbohydrate-protein interactions are of major importance in living organisms; their study is crucial for understanding of basic biological processes and for the construction of biosensors for diagnostics and drug development. Here LSPR transducers based on gold island films prepared by evaporation on glass and annealing were optimized for monitoring the specific interaction between Concanavalin A (Con A) and D-(+)-mannose. The sugar was modified with a PEG-thiol linker and immobilized on the Au islands. Sensing assays were performed under stationary and flow conditions, the latter providing kinetic parameters for protein binding and dissociation. Ellipsometry and Fourier transform-infrared (FT-IR) data, as well as scanning electron microscopy (SEM) imaging of fixated and stained samples, furnished independent evidence for the protein-sugar recognition. Enhanced response and visual detection of protein binding was demonstrated using Au nanoparticles stabilized with the linker-modified mannose molecules. Mannose-coated transducers display an excellent selectivity toward Con A in the presence of a large excess of bovine serum albumin (BSA).
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http://dx.doi.org/10.1021/ac202363tDOI Listing
January 2012

A quantitative, real-time assessment of binding of peptides and proteins to gold surfaces.

Chemistry 2011 Jan 1;17(4):1327-36. Epub 2010 Dec 1.

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

Interactions of peptides and proteins with inorganic surfaces are important to both natural and artificial systems; however, a detailed understanding of such interactions is lacking. In this study, we applied new approaches to quantitatively measure the binding of amino acids and proteins to gold surfaces. Real-time surface plasmon resonance (SPR) measurements showed that TEM1-β-lactamase inhibitor protein (BLIP) interacts only weakly with Au nanoparticles (NPs). However, fusion of three histidine residues to BLIP (3H-BLIP) resulted in a significant increase in the binding to the Au NPs, which further increased when the histidine tail was extended to six histidines (6H-BLIP). Further increasing the number of His residues had no effect on the binding. A parallel study using continuous (111)-textured Au surfaces and single-crystalline, (111)-oriented, Au islands by ellipsometry, FTIR, and localized surface plasmon resonance (LSPR) spectroscopy further confirmed the results, validating the broad applicability of Au NPs as model surfaces. Evaluating the binding of all other natural amino acid homotripeptides fused to BLIP (except Cys and Pro) showed that aromatic and positively-charged residues bind preferentially to Au with respect to small aliphatic and negatively charged residues, and that the rate of association is related to the potency of binding. The binding of all fusions was irreversible. These findings were substantiated by SPR measurements of synthesized, free, soluble tripeptides using Au-NP-modified SPR chips. Here, however, the binding was reversible allowing for determination of binding affinities that correlate with the binding potencies of the related BLIP fusions. Competition assays performed between 3H-BLIP and the histidine tripeptide (3 His) suggest that Au binding residues promote the adsorption of proteins on the surface, and by this facilitate the irreversible interaction of the polypeptide chain with Au. The binding of amino acids to Au was simulated by using a continuum solvent model, showing agreement with the experimental values. These results, together with the observed binding potencies and kinetics of the BLIP fusions and free peptides, suggest a binding mechanism that is markedly different from biological protein-protein interactions.
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http://dx.doi.org/10.1002/chem.201001781DOI Listing
January 2011

Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell.

Free Radic Biol Med 2010 Jan 3;48(2):292-7. Epub 2009 Nov 3.

CNR Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi-Fanelli, Sapienza University of Rome, 00185 Rome, Italy.

Dps (DNA-binding proteins from starved cells) proteins belong to a widespread bacterial family of proteins expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton-mediated oxidative stress, as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radical production. This work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped-flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role for the Dps protein cage.
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http://dx.doi.org/10.1016/j.freeradbiomed.2009.10.053DOI Listing
January 2010

Synthesis of iron oxide nanoparticles in Listeria innocua Dps (DNA-binding protein from starved cells): a study with the wild-type protein and a catalytic centre mutant.

Chemistry 2010 Jan;16(2):709-17

C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi-Fanelli, Sapienza, University of Rome, 00185 Rome, Italy.

A comparative analysis of the magnetic properties of iron oxide nanoparticles grown in the cavity of the DNA-binding protein from starved cells of the bacterium Listeria innocua, LiDps, and of its triple-mutant lacking the catalytic ferroxidase centre, LiDps-tm, is presented. TEM images and static and dynamic magnetic and electron magnetic resonance (EMR) measurements reveal that, under the applied preparation conditions, namely alkaline pH, high temperature (65 degrees C), exclusion of oxygen, and the presence of hydrogen peroxide, maghemite and/or magnetite nanoparticles with an average diameter of about 3 nm are mineralised inside the cavities of both LiDps and LiDps-tm. The magnetic nanoparticles (MNPs) thus formed show similar magnetic properties, with superparamagnetic behaviour above 4.5 K and a large magnetic anisotropy. Interestingly, in the EMR spectra an absorption at half-field is observed, which can be considered as a manifestation of the quantum behaviour of the MNPs. These results indicate that Dps proteins can be advantageously used for the production of nanomagnets at the interface between molecular clusters and traditional MNPs and that the presence of the ferroxidase centre, though increasing the efficiency of nanoparticle formation, does not affect the nature and fine structure of the MNPs. Importantly, the self-organisation of MNP-containing Dps on HRTEM grids suggests that Dps-enclosed MNPs can be deposited on surfaces in an ordered fashion.
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http://dx.doi.org/10.1002/chem.200901138DOI Listing
January 2010

Iron translocation into and out of Listeria innocua Dps and size distribution of the protein-enclosed nanomineral are modulated by the electrostatic gradient at the 3-fold "ferritin-like" pores.

J Biol Chem 2009 Jul 20;284(28):19101-9. Epub 2009 May 20.

CNR Institute of Molecular Biology and Pathology, Department of Biochemical Sciences, University of Rome, 00185 Rome, Italy.

Elucidating pore function at the 3-fold channels of 12-subunit, microbial Dps proteins is important in understanding their role in the management of iron/hydrogen peroxide. The Dps pores are called "ferritin-like" because of the structural resemblance to the 3-fold channels of 24-subunit ferritins used for iron entry and exit to and from the protein cage. In ferritins, negatively charged residues lining the pores generate a negative electrostatic gradient that guides iron ions toward the ferroxidase centers for catalysis with oxidant and destined for the mineralization cavity. To establish whether the set of three aspartate residues that line the pores in Listeria innocua Dps act in a similar fashion, D121N, D126N, D130N, and D121N/D126N/D130N proteins were produced; kinetics of iron uptake/release and the size distribution of the iron mineral in the protein cavity were compared. The results, discussed in the framework of crystal growth in a confined space, indicate that iron uses the hydrophilic 3-fold pores to traverse the protein shell. For the first time, the strength of the electrostatic potential is observed to modulate kinetic cooperativity in the iron uptake/release processes and accordingly the size distribution of the microcrystalline iron minerals in the Dps protein population.
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http://dx.doi.org/10.1074/jbc.M109.014670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2707210PMC
July 2009

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi.

Arch Biochem Biophys 2008 Oct 28;478(1):69-74. Epub 2008 Jun 28.

Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Sapienza, Università di Roma, P.le A. Moro, 5, 00185 Roma, Italy.

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.
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http://dx.doi.org/10.1016/j.abb.2008.06.022DOI Listing
October 2008

The mutations Lys 114 --> Gln and Asp 126 --> Asn disrupt an intersubunit salt bridge and convert Listeria innocua Dps into its natural mutant Listeria monocytogenes Dps. Effects on protein stability at Low pH.

Proteins 2007 Mar;66(4):975-83

Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università La Sapienza, Rome, Italy.

The stability of the dodecameric Listeria monocytogenes Dps has been compared with that of the Listeria innocua protein. The two proteins differ only in two amino acid residues that form an intersubunit salt-bridge in L. innocua Dps. This salt-bridge is replaced by a hydrogen bonding network in L. monocytogenes Dps as revealed by the X-ray crystal structure. The resistance to low pH and high temperature was assayed for both Dps proteins under equilibrium conditions and kinetically. Despite the identical equilibrium behavior, significant differences in the kinetic stability and activation energy of the unfolding process are apparent at pH 1.5. The higher stability of L. monocytogenes Dps has been accounted for in terms of the persistence of the hydrogen bonding network at this low pH value. In contrast, the salt-bridge between Lys 114 and Asp 126 characteristic of L. innocua Dps is most likely abolished due to protonation of Asp 126.
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http://dx.doi.org/10.1002/prot.21305DOI Listing
March 2007