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ABSTRACT: Resolution of the intrinsic emission properties of a protein by different fluorescence spectroscopy techniques is an invaluable tool to detect and characterize its structural architecture and conformational changes under different experimental conditions. Indeed, the multidimensional character of fluorescence can provide information on local chemical features, on solvent diffusional processes, and on rotational movements of peptide chains or whole proteins. Here, we describe the details of quenching fluorescence experiments and how to correlate the results to the peculiar structural information on the organization of intrinsically disordered proteins (IDPs).
Methods in molecular biology (Clifton, N.J.) 01/2012; 895:435-40.
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ABSTRACT: Intrinsically disordered proteins (IDPs) differ from ordered proteins at several levels: structural, functional, and conformational. Amino acid biases also drive atypical responses of IDPs to changes in their environment. Among several specific features, the conformational behavior of IDPs is characterized by the low cooperativity (or the complete lack thereof) of the denaturant-induced unfolding. In fact, the denaturant-induced unfolding of native molten globules can be described by shallow sigmoidal curves, whereas urea- or guanidinium hydrochloride-induced unfolding of native pre-molten globules or native coils is a noncooperative process and typically is seen as monotonous feature-less changes in the studied parameters. This chapter describes some of the most characteristic features of the IDP conformational behavior.
Methods in molecular biology (Clifton, N.J.) 01/2012; 896:197-213.
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ABSTRACT: In the past, enzymatic activity has always been expected to be dependent on overall protein rigidity, necessary for substrate recognition and optimal orientation. However, increasing evidence is now accumulating, revealing that some proteins characterized by intrinsic disorder are actually able to perform catalysis. Among them, the only known natural intrinsically disordered enzyme is UreG, a GTPase that, in plants and bacteria, is involved in the protein interaction network leading to Ni(2+) ions delivery into the active site of urease. In this paper, we report a detailed analysis of the unfolding behaviour of UreG from Bacillus pasteurii (BpUreG), following its thermal and chemical denaturation with a combination of fluorescence spectroscopy, calorimetry, CD and NMR. The results demonstrate that BpUreG exists as an ensemble of inter-converting conformations, whose degrees of secondary structure depend on temperature and denaturant concentration. In particular, three major types of conformational ensembles with different degrees of residual structure were identified, with major structural characteristics resembling those of a molten globule (low temperature, absence of denaturant), pre-molten globule (high temperature, absence or presence of denaturant) and random coil (low temperature, presence of denaturant). Transitions among these ensembles of conformational states occur non-cooperatively although reversibly, with a gradual loss or acquisition of residual structure depending on the conditions. A possible role of disorder in the biological function of UreG is envisaged and discussed.
Molecular BioSystems 09/2011; 8(1):220-8. · 3.53 Impact Factor
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Laura Fiammenghi,
Valentina Ancarani,
Tilman Rosales,
Jay R Knutson,
Massimiliano Petrini,
Anna Maria Granato,
Elena Pancisi,
Laura Ridolfi,
Ruggero Ridolfi,
Angela Riccobon, Paolo Neyroz
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ABSTRACT: Antigen processing by dendritic cells (DC) exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials) are similarly processed by these cells has not yet been resolved.
In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II) in mature dendritic cells (mDC) from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by Förster Resonance Energy Transfer (FRET) measurements, which effectively extends the application of fluorescence microscopy to the molecular level (<100A). Tumor lysates were labelled with Alexa-488, as the donor, and mDC MHC II HLA-DR molecules were labelled with Alexa-546-conjugated IgG, as the acceptor.
We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr.
The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates.
Journal of Translational Medicine 01/2010; 8:52. · 3.41 Impact Factor
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ABSTRACT: NikR is a prokaryotic transcription factor that regulates the expression of Ni2+ enzymes and other proteins involved in Ni2+ trafficking. In the human pathogen Helicobacter pylori, NikR controls transcription of the Ni2+ enzyme urease, which allows survival of the bacterium in the acidic gastric niche. The in vitro affinity of NikR from H. pylori (HpNikR) for different metal ions and the metal-ion-dependent capability of HpNikR to bind PureA, the promoter of the urease operon, were the object of this study. Electrophoretic mobility shift and DNase I footprinting assays indicated that Ni2+ is necessary and sufficient to promote HpNikR binding to PureA, while the effect of other metal ions in identical conditions is significantly lower (Zn2+ and Co2+) or absent (Ca2+ and Mg2+). Isothermal titration calorimetry (ITC) demonstrated the absence of specific Ca2+ and Mg2+ binding to the protein. ITC also established the binding of Zn2+ and Co2+ to two sets of high-affinity sites on HpNikR, differing in stoichiometry (n1=2, n2=4) and dissociation constant (Kd1=6 nM, Kd2=90 nM for Zn2+; Kd1=0.3 microM, Kd2=2.7 microM for Co2+). Additional low-affinity binding sites were observed for Zn2+ (n=8, Kd=1.6 microM). Mobility shift assays and ITC proved that binding of stoichiometric Ni2+ (but not Zn2+ or Co2+) to the high-affinity sites (but not to the low-affinity sites) selectively activates HpNikR to bind its target operator with 1:1 stoichiometry and Kd=56 nM. A protein conformational rearrangement is selectively induced by Ni2+ and not by Zn2+, as indicated by fluorescence spectroscopy and microcalorimetry. Accordingly, competition experiments showed that stoichiometric Ni2+ outperforms Zn2+, as well as Co2+, in functionally activating HpNikR toward high affinity binding to PureA. A general scheme for the nickel-selective HpNikR-DNA interaction is proposed.
Journal of Molecular Biology 10/2008; 383(5):1129-43. · 4.00 Impact Factor
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ABSTRACT: The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 microM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 microM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism.
Proteins Structure Function and Bioinformatics 10/2008; 74(1):222-39. · 3.39 Impact Factor
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ABSTRACT: UreG is an essential protein for the in vivo activation of urease. In a previous study, UreG from Bacillus pasteurii was shown to behave as an intrinsically unstructured dimeric protein. Here, intrinsic and extrinsic fluorescence experiments were performed, in the absence and presence of denaturant, to provide information about the form (fully folded, molten globule, premolten globule, or random coil) that the native state of BpUreG assumes in solution. The features of the emission band of the unique tryptophan residue (W192) located on the C-terminal helix, as well as the rate of bimolecular quenching by potassium iodide, indicated that, in the native state, W192 is protected from the aqueous polar solvent, while upon addition of denaturant, a conformational change occurs that causes solvent exposure of the indole side chain. This structural change, mainly affecting the C-terminal helix, is associated with the release of static quenching, as shown by resolution of the decay-associated spectra. The exposure of protein hydrophobic sites, monitored using the fluorescent probe bis-ANS, indicated that the native dimeric state of BpUreG is disordered even though it maintains a significant amount of tertiary structure. ANS fluorescence also indicated that, upon addition of a small amount of GuHCl, a transition to a molten globule state occurs, followed by formation of a pre-molten globule state at a higher denaturant concentration. The latter form is resistant to full unfolding, as also revealed by far-UV circular dichroism spectroscopy. The hydrodynamic parameters obtained by time-resolved fluorescence anisotropy at maximal denaturant concentrations (3 M GuHCl) confirmed the existence of a disordered but stable dimeric protein core. The nature of the forces holding together the two monomers of BpUreG was investigated. Determination of free thiols in native or denaturant conditions, as well as light scattering experiments in the absence and presence of dithiothreitol as a reducing agent, under native or denaturing conditions, indicates that a disulfide bond, involving the unique conserved cysteine C68, is present under native conditions and maintained upon addition of denaturant. This covalent bond is therefore important for the stabilization of the dimer under native conditions. The intrinsically disordered structure of UreG is discussed with respect to the role of this protein as a chaperone in the urease assembly system.
Biochemistry 08/2006; 45(29):8918-30. · 3.42 Impact Factor
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ABSTRACT: The U4/U6*U5 tri-snRNP complex is the catalytic core of the pre-mRNA splicing machinery. The thioredoxin-like protein hDim1 (U5-15 kDa) constitutes an essential component of the U5 particle, and its functions have been reported to be highly conserved throughout evolution. Recently, the Dim1-like protein (DLP) family has been extended to other proteins harboring similar sequence motifs. Here we report the biochemical characterization and crystallographic structure of a 149 amino acid protein, hDim2, which shares 38% sequence identity with hDim1. The crystallographic structure of hDim2 solved at 2.5 A reveals a classical thioredoxin-fold structure. However, despite the similarity in the thioredoxin fold, hDim2 differs from hDim1 in many significant features. The structure of hDim2 contains an extra alpha helix (alpha3) and a beta strand (beta5), which stabilize the protein, suggesting that they may be involved in interactions with hDim2-specific partners. The stability and thermodynamic parameters of hDim2 were evaluated by combining circular dichroism and fluorescence spectroscopy together with chromatographic and cross-linking approaches. We have demonstrated that, in contrast to hDim1, hDim2 forms stable homodimers. The dimer interface is essentially stabilized by electrostatic interactions and involves tyrosine residues located in the alpha3 helix. Structural analysis reveals that hDim2 lacks some of the essential structural motifs and residues that are required for the biological activity and interactive properties of hDim1. Therefore, on the basis of structural investigations we suggest that, in higher eukaryotes, although both hDim1 and hDim2 are involved in pre-mRNA splicing, the two proteins are likely to participate in different multisubunit complexes and biological processes.
Biochemistry 10/2005; 44(36):11997-2008. · 3.42 Impact Factor
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ABSTRACT: The synthesis and physical-chemical characterization of the metal-ligand complex [Os(bpy)2(CO)(enIA)][OTf]2 (where enIA = ethylenediamine iodoacetamide) with a sulfhydryl-specific functional group is described. The UV and visible absorption and luminescence emission, including lifetime and steady-state anisotropy, are reported for the free probe and the probe covalently linked to two test proteins. The spectroscopic properties of the probe are unaffected by chemical modification and subsequent covalent linkage to the proteins. The luminescence lifetime in aqueous buffer is approximately 200 ns and the limiting anisotropy is greater than 0.125, suggesting a potentially useful probe for biophysical investigations.
Inorganic Chemistry 06/2005; 44(11):3875-9. · 4.60 Impact Factor
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ABSTRACT: Different strategies are presented to conjugate a fluorescein moiety to 9- and 10-hydroxystearic acids (HSAs). 5-Amino-fluorescein (5-AF) was used as a starting reagent. When reacted with acyl-chloride-modified HSAs, 5-AF gave rise to stable amide derivatives with a 75% reaction yield. These products exhibited the typical steady-state and time-resolved fluorescence properties of the fluorescein chromophore with absorption at 494 nm and emission at 519 nm. Flow cytometry studies confirmed the distinct proapoptotic effect of underivatized 9-HSA on Jurkat cells and revealed a comparable ability of its amide derivative. Confocal microscopy imaging studies showed that green fluorescence could stain intracellular membranous structures. Moreover, dual-dye labeling with Mito Tracker Red, followed by colocalization analysis, revealed that HSA can move to the mitochondria. Thus, fluorescent derivatives of HSA can be used to monitor the localization of these biologically active molecules in living cells and can provide a useful tool for linking biochemical investigation with optical visualization methods. In contrast, when unmodified HSAs were used, the reaction gave monoesterified and diesterified fluorescein derivatives. These products exhibited unusual steady-state and time-resolved fluorescence properties with the excitation wavelength at 342 nm and the emission wavelength at 432 nm. It is shown that the synthesized HSA amides of fluorescein provide all of the typical photophysical and instrumental advantages of this popular dye, whereas the unusual luminescence and excitation properties of the monoester and diester of the 5-aminofluorescein would make these dyes interesting to explore as potential candidates for two photon excitation applications.
Analytical Biochemistry 01/2005; 335(2):196-209. · 3.00 Impact Factor
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ABSTRACT: Peripheral blood CD14+ monocytes from multiple myeloma (MM) patients can be induced to differentiate into fully functional, mature, CD83+ dendritic cells (DCs) which are highly efficient in priming autologous T lymphocytes in response to the patient-specific tumor idiotype (Id). We have recently scaled up our manufacturing protocol for application in a phase I-II clinical trial of anti-Id vaccination with DCs in MM patients. Elegible patients received a series of by-monthly immunizations consisting of three subcutaneous and two intravenous injections of Id-keyhole limpet hemocyanin (KLH)-pulsed DCs (5 x -, 10 x -, 50 x 10(6) cells and 10 x -, 50 x 10(6) cells, respectively). To generate DCs, monocytes were labeled with clinical grade anti-CD14 conjugates and positively selected by immunomagnetic separation. Cells were then cultured, according to Good Manufacturing Practice guidelines, in FCS-free medium in cell culture bags, and differentiated to DCs with GM-CSF plus IL-4 followed by TNF-alpha or, more recently, by a cocktail of IL-1beta, IL-6, TNF-alpha and prostaglandin-E2. Before maturation, Mo-DCs were pulsed with the autologous Id as whole protein or Id (VDJ)-derived HLA class I restricted peptides. Ten MM patients, who had been treated with two courses of high-dose chemotherapy with peripheral blood stem cell support, entered into the clinical study. CD14+ monocytes were enriched from 16.1+/-5.7% to 95.5+/-3.2% (recovery 67.9+/-15%, viability > 97%). After cell culture, phenotypic analysis showed that 89.6+/-6.6% of the cells were mature DCs. We obtained 2.89+/-1 x 10(8) DCs/leukapheresis which represented 24.5+/-9% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (31+/-10.9 of initial CD14+ cells) of DCs than TNF-alpha alone, secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic and autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of pre-loaded DCs. The recovery of thawed, viable DCs, was 78+/-10%. Thus, positive selection of CD14+ monocytes allows the generation of a uniform population of mature pre-loaded DCs which can be cryopreserved with no effects on phenotype and function and are suitable for clinical trials. Based on these results, a DCs-based phase II trial of anti-Id vaccination with VDJ-derived HLA class I-restricted peptides and KLH is underway for lymphoma patients.
Leukemia and Lymphoma 08/2004; 45(7):1419-28. · 2.58 Impact Factor
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ABSTRACT: p13suc1 acts in the fission yeast cell division cycle as a component of p34cdc2. In the present work, structural information contained in the intrinsic fluorescence of p13suc1 has been extracted by steady-state and time-resolved fluorescence techniques. In its native form, the steady-state emission
spectrum of p13suc1 is centered at 336 nm. Upon denaturation by guanidine HCl (4.0 M), the emission spectrum is shifted to 355-360 nm and the fluorescence intensity decreases 70%. The same changes are not obtained
with p13suc1 at 56°C or after incubation at 100°C, and the protein appears to be substantially temperature-stable. The fluorescence decay
of p13suc1 is best described by three discrete lifetimes of 0.6 ns (τ1), 2.9 ns (τ2), and 6.1 ns (τ3), with amplitudes that are dependent on the native or unfolded state of the protein. Under native conditions, the two predominant
decay-associated spectra, DAS-τ2 (λmax = 332 nm) and DAS-τ3 (λmax = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities
(kq(τ2)≫kq(τ3)) are resolved for each lifetime. An interpretation in terms of specific tryptophan residue (or protein conformer)-lifetime
assignments is presented. The decay of the fluorescence anisotropy of native p13suc1 is best described by a double exponential decay. The longer correlation time recovered (9 ns ≤ Φ2 ≤ 15ns) can be associated with the rotational motion of the protein as a whole and a Stokes radius of 21.2 Å has been calculated
for p13suc1. Anisotropy measurements obtained as a function of temperature indicate that, in solution, the protein exists exclusively
as a prolate monomer. In 1 mM zinc, changes of the anisotropy decay parameters are compatible with subunits oligomerization.
Journal of Biological Chemistry 10/1996; 271(44):27249-27258. · 4.77 Impact Factor
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ABSTRACT: The fluorescence properties of the phospholipid derivative,N-[1-(2-naphthol)]-phosphatidylethanolamine (NAPH-PE), have been studied by steady-state and time-resolved fluorescence techniques. The new probe is a naphthol adduct of phosphatidylethanolamine. The emission spectrum of the fluorescent phospholipid depends on the pH and on the proton acceptor concentration as expected for a typical two-state excited-state proton transfer reaction. In ethanol solutions at an apparent pH of 6.7 and in the presence of acetate anion (0.14M), a biexponential decay is obtained from global analysis of the data. The lifetimes,
1=3.9 ns and
2=6.2 ns. are constant across the spectral region 350–460 nm. The decay-associated spectra and the species-associated spectra reproduce well the profiles reported for a two-state excited-state proton transfer reaction. The fluorescent phospholipid has been incorporated into dimyristoyllecithin and dipalmitoyllecithin vesicles. Although lower proton transfer is found, the reaction appears to be dependent on the gel-to-liquid-crystalline phase transition of the lipid membrane. In addition, the steady-state anisotropy of NAPH-PE measured as a function of temperature trace the phase transition of the two vesicle systems. Thus, it is shown that the physical state of the bilayer affects a reaction which takes place at the membrane surface. In the presence of acetate ions (0.3M), global analysis, performed in terms of fluorescence decay parameters, recovers preexponential coefficients that are consistent with an excited-state proton transfer reaction. The short lifetime drops from 3.9 to 0.44 ns without significant changes of the longer-lifetime component.
Journal of Fluorescence 08/1996; 6(3):127-138. · 2.11 Impact Factor
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ABSTRACT: This report describes a method to conjugate lucifer yellow to the external surface of liposomes. The heterobifunctional cross-linking reagentN-succinimidyl 3-(2-pyridyldithio)propionate has been used to activate DMPE molecules. The DMPE-dithiopyridine product has been mixed with DMPC to prepare liposome vesicles. These have been reduced by DTT and finally reacted with lucifer yellow-iodoacetamide to produce the fluorescence-labeled vesicles. The quenching of their fluorescence intensity by Kl is consistent with fully exposed fluorophores. The decay of the fluorescence intensity of the lipid-bound lucifer yellow is biexponential (1=7.9 ns; 2=1.1 ns), with a relative yield of 0.16. When the fluorescent liposomes are mixed with cells, the lucifer yellow-DMPE derivative is transferred. Boar spermatozoa and peripheral human blood lymphocytes have been used as cellular models. The extent of incorporation is dependent on the incubation time and temperature. At 36C, lucifer yellow fluorescence appears in the spermatozoa cells after 10 min of incubation and reaches its maximum at about 60 min. The fluorescent phospholipid derivative seems to incorporate specifically into membrane structures. The highest labeling ratio is observed with integer, scarcely motile, spermatozoa. A poorer labeling yield (15%) is found with lymphocytes. Interestingly, photobleaching due to epiillumination of the labeled cells is apparently negligible and cells are clearly visible after irradiation times ranging from several minutes to few hours.
Journal of Fluorescence 02/1993; 3(1):33-43. · 2.11 Impact Factor
