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Mario Piccioli

Publications and source records attributed to Mario Piccioli.

16 recordsLinked to original sources
PubMed2006

13C direct detected NMR increases the detectability of residual dipolar couplings.

13C direct detection is becoming an increasingly efficient approach to identify signals of residues that escape detection in 1H detected experiments. Pulse sequences have been developed to obtain 1H partially recoupled experiments for the measurement of the 1JHalphaCalpha and 1JHN couplings with the same resolution available in conventional 1H detected experiments. A consistent set of backbone rdc obtained without any 1H-based experiment has been obtained and shown to be effective for protein solution structure determination.

Calbindins↗
PubMed2006

13C direct detected COCO-TOCSY: a tool for sequence specific assignment and structure determination in protonless NMR experiments.

A novel experiment is proposed to provide inter-residue sequential correlations among carbonyl spins in (13)C detected, protonless NMR experiments. The COCO-TOCSY experiment connects, in proteins, two carbonyls separated from each other by three, four or even five bonds. The quantitative analysis provides structural information on backbone dihedral angles phi as well as on the side chain dihedral angles of Asx and Glx residues. This is the first dihedral angle constraint that can be obtained via a protonless approach. About 75% of backbone carbonyls in Calbindin D(9K), a 75 amino acid dicalcium protein, could be sequentially connected via a COCO-TOCSY spectrum. 49(3)J(C')(C') values were measured and related to backbone phi angles. Structural information can be extended to the side chain orientation of aminoacids containing carbonyl groups. Additionally, long range homonuclear coupling constants, (4)J(CC) and (5)J(CC), could be measured. This constitutes an unprecedented case for proteins of medium and small size.

Calbindins↗
PubMed2006

NMR structures of paramagnetic metalloproteins.

Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+ -binding proteins and for Ca2+ -binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions.

Computer Simulation↗
PubMed2006

Direct-detected 13C NMR to investigate the iron(III) hemophore HasA.

Hemophore HasA is a 19 kDa iron(III) hemoprotein that participates in the shuttling of heme to a specific membrane receptor. In HasA, heme iron has an original coordination environment with a His/Tyr pair as axial ligands. Recently developed two-dimensional protonless (13)C-detected experiments provide the sequence-specific assignment of all but three protein residues in the close proximity of the paramagnetic center, thus overcoming limitations due to the short relaxation times induced by the presence of the iron(III) center. Mono-dimensional (13)C and (15)N experiments tailored for the detection of paramagnetic signals allow the identification of resonances of the axial ligands. These experiments are used to characterize the conformational features and the electronic structure of the heme iron(III) environment. The good complementarity among (1)H-, (13)C-, and (15)N-detected experiments is highlighted. A thermal high-spin/low-spin equilibrium is observed and is related to a modulation of the strength of the coordination bond between the iron and the Tyr74 axial ligand. The key role of a neighboring residue, His82, for the stability of the axial coordination and its involvement in the heme delivery to the receptor is discussed.

Animals↗
PubMed2006

Assignment strategy for fast relaxing signals: complete aminoacid identification in thulium substituted calbindin D 9K.

Paramagnetic proteins generally contain regions with diverse relaxation properties. Nuclei in regions far from the metal center may behave like those in diamagnetic proteins, but those closer to the metal experience rapid relaxation with accompanying line broadening. We have used a set of NMR experiments optimized to capture data from these various concentric regions in assigning the signals from a paramagnetic Calbindin D 9K derivative in which one of the two calcium ions has been replaced by thulium(III). Normal double- and triple-resonance experiments with 1H detection were used in collecting data from nuclei in the diamagnetic-like region; these approaches identified signals from fewer than 50% of the amino acid residues (those with d > 17.5 A from thulium(III)). Paramagnetism-optimized two-dimensional NMR experiments with 1H detection were used in collecting data from nuclei in the next nearer region (d > 15 A). Standard (d > 14 A) and optimized (d > 9 A) 13C direct-detection experiments were used to capture data from nuclei in the next layer. Finally nuclei closest to the metal were detected by one-dimensional 13C (d > 5 A) and one-dimensional 15N data collection (d > 4.2 A). NMR signals were assigned on the basis of through-bond correlations and, for signals closest to the metal, pseudocontact shifts. The latter were determined from chemical shift differences between assigned signals in thulium(III) and lanthanum(III) derivatives of Calbindin D 9K and they were interpreted on the basis of a structural model for the lanthanide-substituted protein. This approach yielded assignments of at least one resonance per amino acid residue, including those in the thulium(III) coordination sphere.

Amino Acids↗
PubMed2005

Asymmetry in 13C-13C COSY spectra provides information on ligand geometry in paramagnetic proteins.

The relative intensity of Calpha-C' cross-peaks in homonuclear 13C COSY spectra depends on the relaxation properties of Calpha and C' spins, which, in the proximity of a paramagnetic center, are related to the metal-to-carbon distance. Their quantitative analysis has lead, for the cerium-substituted dicalcium protein, calbindin D9k, to the straightforward identification of peaks arising from metal-coordinating groups. The monodentate or bidentate metal binding mode of carboxylates was identified directly via NMR.

Algorithms↗
PubMed2005

13C direct detected experiments: optimization for paramagnetic signals.

To optimize 13C direct detected experiments for the observation of signals close to a paramagnetic center, we have assessed the sensitivity of different sequences based on CO-Cali coherence transfer. Features of CACO experiments were tested for Calbindin D9k, in which one of the two native Ca2+ ions is replaced by the paramagnetic Ce3+ ion. We have studied the comparison of single vs multiple quantum coherence transfer evolution as well as the influence of in-phase vs anti-phase detection of 13CO signals and finally the comparison of a coherence transfer step based on a CyO in plane with respect to a Cy ali in plane. The acquisition of the anti-phase component of the signal, accomplished by the removal of the last refocusing steps, allowed the identification of some signals unobserved with other pathways. The structural dependency of paramagnetism-induced nuclear relaxation is such that the identification of the most suitable coherence transfer pathway is not known "a priori" but it is driven by the relative proximity of Cali and CO to the paramagnetic center.

Animals↗
PubMed2003

New routes to the detection of relaxation allowed coherence transfer in paramagnetic molecules.

A new pulse sequence is proposed to measure cross correlation rates between 1H Curie spin relaxation and 1H-15N or 1H-13C dipole-dipole coupling (%@mt;sys@%Gamma%@sx@%H,HX%@be@%CS,DD%@sxx@%%@mx@% ) in paramagnetic systems. The new sequence has been conceived to obtain quantitative measurements of cross correlation rates in the close proximity to the paramagnetic center, preventing the loss of information due to fast transverse relaxation. The approach was tested on the dicalcium protein calbindin D9k in which Ca2+ has been substituted at site II with Ce3+. At variance with previously reported experiments, all peaks observed in HSQC experiments tailored to paramagnetic signals give quantitative estimates of %@mt;sys@%Gamma%@sx@%H%@ital@%,%@rsf@%HX%@be@%CS%@ital@%,%@rsf@%DD%@sxx@%%@mx@% . This is crucial to refine the immediate proximity of the metal ion.

Calbindins↗
PubMed2003

Monitoring the early steps of unfolding of dicalcium and mono-Ce3+-substituted forms of P43M calbindin D9k.

Early steps of unfolding of P43M Calbindin D(9k) have been evaluated by NMR spectroscopy on the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 M GdmHCl the protein core maintains its overall folding and structure, amide (15)N R(2) measurements and cross correlation rates between N-H dipole-dipole relaxation and (15)N CSA relaxation reveal a closer and stronger packing of the hydrophobic interactions in the protein as a response to the presence of denaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobic core is accomplished by the disappearance of the millisecond dynamics observed on the native form of Calbindin D(9k), while cross correlation rates provide evidence that the two-way hydrogen bond between Leu23 and Val61 is broken or substantially weakened. The substitution of the calcium ion in site II with the paramagnetic Ce(3+) ion allowed us to obtain a number of long-range nonconventional constraints, namely, pseudocontact shifts, which were used, together with the NOEs collected on the native state, to monitor subtle structural variations occurring in the non-native state of the protein. Although the average rmsd between the structures of native and non-native states is small (0.48 A), structural rearrangements could be reliably identified. Our results provide unprecedented information about the behavior of Calbindin D(9k) during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency of paramagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivity of an approach based only on NOE.

Amides↗
PubMed2003

Backbone dynamics of rusticyanin: the high hydrophobicity and rigidity of this blue copper protein is responsible for its thermodynamic properties.

Local dynamics and solute-solvent exchange properties of rusticyanin (Rc) from Thiobacillus ferrooxidans have been studied by applying heteronuclear ((1)H, (15)N) NMR spectroscopy. (15)N relaxation parameters have been determined for the reduced protein, and a model-free analysis has been applied. The high average value of the generalized order parameter, S(2) (0.93), indicates that Rc is very rigid. The analysis of cross correlation rates recorded in both the reduced and the oxidized forms conclusively proves that Rc possesses the same dynamic features in both oxidation states. The accessibility of backbone amide protons to the solvent at different time scales has also been studied by applying specific heteronuclear pulse sequences and by H(2)O/D(2)O exchange experiments. These experiments reveal that rusticyanin is extremely hydrophobic. The first N-35 amino acids, not present in the other BCPs, protect the beta-barrel core from its interaction with the solvent, and thus, this is one of the main factors contributing to the hydrophobicity. Both characteristics (high rigidity and hydrophobicity) are maintained in the metal ion surroundings.

Azurin↗
PubMed2003

A simple protocol to study blue copper proteins by NMR.

In the case of oxidized plastocyanin from Synechocystis sp. PCC6803, an NMR approach based on classical two and three dimensional experiments for sequential assignment leaves unobserved 14 out of 98 amino acids. A protocol which simply makes use of tailored versions of 2D HSQC and 3D CBCA(CO)NH and CBCANH leads to the identification of nine of the above 14 residues. The proposed protocol differs from previous approaches in that it does not involve the use of unconventional experiments designed specifically for paramagnetic systems, and does not exploit the occurrence of a corresponding diamagnetic species in chemical exchange with the blue copper form. This protocol is expected to extend the popularity of NMR in the structural studies of copper (II) proteins, allowing researchers to increase the amount of information available via NMR on the neighborhood of a paramagnetic center without requiring a specific expertise in the field. The resulting 3D spectra are standard spectra that can be handled by any standard software for protein NMR data analysis.

Carbon Isotopes↗
PubMed2002

A 15N NMR mobility study on the dicalcium P43M calbindin D9k and its mono-La3+-substituted form.

Calbindin D(9k) is a dicalcium binding protein consisting of two helix-loop-helix EF-hand motifs joined together by a flexible linker region where one metal ion can bind to each of the two loops. A proline residue at position 43 in the linker region displays cis-trans isomerism in the wild-type (WT) protein. Such isomerism appeared to be removed by substituting the proline with a glycine or a methionine in the P43G or P43M mutant. We have extended the available mobility studies on the P43M mutant through amide (15)N R(1), R(2), and R(1)(rho)() measurements. This has revealed unexpected conformational equilibria on the millisecond time scale involving residues 38, 42-44, and 46 in the linker region and residues 18 and 19 in calcium binding site I with similar energy barriers. These data are discussed in comparison with those available for the WT, as well as the apo-, mono-, and disubstituted P43G mutant. Quantification of water-amide proton exchange rates using saturation transfer and qualitative application of (15)N-(CLEANEX-PM)-FHSQC shows the values are in agreement with high mobility for the above-mentioned residues. Cross correlation between N-H dipole-dipole relaxation and (15)N CSA relaxation indicates that some of these mobility differences may extend to the sub-nanosecond time scale. Similar data were also obtained for the derivative where the calcium ion in the C-terminal loop was replaced with lanthanum. The results presented here show that, contrary to expectations, there are significant differences in dynamics between the dicalcium state of P43G and P43M and that these differences are not confined to the flexible linker region containing the point mutation. They also demonstrate that substitution of a lanthanide ion for calcium, which is a common procedure, does not significantly alter the mobility of the native protein.

Amides↗
PubMed2002

Tailored HCCH-TOCSY experiment for resonance assignment in the proximity of a paramagnetic center.

The presence of a paramagnetic center may disturb both coherent and incoherent communication between nuclear spins that are affected, to some extent, by the hyperfine interaction. This is a limiting factor to an extensive use of paramagnetic probes in NMR spectroscopy to enhance partial alignment and to exploit cross correlation effects and pseudocontact shifts. We propose here an HCCH-TOCSY experiment tailored to identify spin systems involving resonances that are partly or completely affected by hyperfine interaction. The efficiency of polarization transfer steps when fast relaxing nuclei are involved is discussed. The sequence is tested for the protein Calbindin D(9k), in which one of the two native Ca2+ ions is replaced by the paramagnetic Ce3+ ion as well as for the oxidized form of cytochrome b(562).

Calbindins↗
PubMed2002

Cross correlation rates between Curie spin and dipole-dipole relaxation in paramagnetic proteins: the case of cerium substituted calbindin D9k.

Cross correlation rates between Curie spin relaxation and H-N dipole-dipole coupling (gamma(HM,HN)CS,DD) have been determined for a calcium binding protein, Calbindin D9k, in which one of the two calcium ions is substituted with cerium(III). Gamma(HM,HN)CS,DD values depend on both the metal-to-proton distances and the M-H-N angles and can be used as an additional constraint in order to refine the solution structure of paramagnetic metalloproteins. For this purpose, we have implemented a new module (CCR-DYANA) in a version of the program DYANA (PARAMAGNETIC-DYANA), which can be used together with other paramagnetism-based constraints such as pseudocontact shifts, residual dipolar couplings and hyperfine based Karplus relationships. This integrated structure calculation protocol has the advantage that different paramagnetic-based constraints are treated by the same algorithm in a way that the efficiency of each class of constraints can be analyzed and compared.

Algorithms↗