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Paul R Vasos

Publications and source records attributed to Paul R Vasos.

11 recordsLinked to original sources
PubMed2007

Singlet-state exchange NMR spectroscopy for the study of very slow dynamic processes.

Singlet states with lifetimes that are longer than spin-lattice relaxation times TS > T1 offer unique opportunities for studying very slow dynamic processes in solution-state NMR. A set of novel experiments can achieve broadband excitation of singlet states in pairs of coupled spins. The most elaborate of these experiments, two-dimensional singlet-state exchange spectroscopy (SS-EXSY), is independent of the offsets of the two spins, their relative chemical shifts, and their scalar couplings. The new methods open the way to study very slow chemical exchange or translational diffusion using mixing times taum = Ts >> T1. The lifetimes TS of singlet states of pairs of protons in a partially deuterated saccharide are shown to be longer than the longitudinal proton relaxation times T1 in the same compound by a factor of ca. 37.

Furans↗
PubMed2006

Measurement of 15N relaxation in deuterated amide groups in proteins using direct nitrogen detection.

15N chemical shielding tensors contain useful structural information, and their knowledge is essential for accurate analysis of protein backbone dynamics. The anisotropic component (CSA) of 15N chemical shielding can be obtained from 15N relaxation measurements in solution. However, the predominant contribution to nitrogen relaxation from 15N-(1)H dipolar coupling in amide groups limits the sensitivity of these measurements to the actual CSA values. Here we present nitrogen-detected NMR experiments for measuring 15N relaxation in deuterated amide groups in proteins, where the dipolar contribution to 15N relaxation is significantly reduced by the deuteration. Under these conditions nitrogen spin relaxation becomes a sensitive probe for variations in 15N chemical shielding tensors. Using the nitrogen direct-detection experiments we measured the rates of longitudinal and transverse 15N relaxation for backbone amides in protein G in D(2)O at 11.7 T. The measured relaxation rates are validated by comparing the overall rotational diffusion tensor obtained from these data with that from the conventional 15N relaxation measurements in H(2)O. This analysis revealed a 17-24 degree angle between the NH-bond and the unique axis of the 15N chemical shielding tensor.

Amides↗
PubMed2005

Spin-state selection for increased confidence in cross-correlation rates measurements.

A new approach is described for measuring chemical shift anisotropy (CSA)/dipolar cross-correlated relaxation (CCR) rates based on the selection of the individual 15N doublet components prior to the relaxation period. The method uses the spin-state-selective element (S3E) of Sørensen and co-authors [Meissner et al. (1997) J. Mag. Reson., 128, 92-97]. The main advantage of the new method compared to other J-resolved experiments is that it does not create problems of additional signal overlap encountered in coupled spectra. At the same time, this approach allows a simpler control of magnetization pathways than the indirect methods. The method is demonstrated for the B3 domain of protein G.

Nuclear Magnetic Resonance, Biomolecular↗
PubMed2005

A selective experiment for the sequential protein backbone assignment from 3D heteronuclear spectra.

Two modifications of the triple-resonance CANCO sequence, designed for backbone assignment in proteins [Angew. Chem. Int. Ed. 43 (2004) 2257], are presented here. These two new sequences display the intra-residue Ca-CO correlation selectively, while in the original sequence both the inter- and the intra-residue correlations were present. In addition, one of the two variants benefits from an improved sensitivity. Both sequences are a useful complement to the CANCO sequence for facile sequence-specific protein assignment by protonless NMR.

Calcium-Binding Proteins↗
PubMed2004

Protein stability and mutations in the axial methionine loop of a minimal cytochrome c.

The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.

Amino Acids↗
PubMed2004

Cytochrome c and SDS: a molten globule protein with altered axial ligation.

Saccharomices cerevisiae (yeast iso-1) cytochrome c has been investigated in the presence of 100 mM SDS in order to simulate the interaction of cytochrome c with membrane. Under these circumstances, a high spin species with detached methionine axial ligand is observed through NMR, in analogy to findings on the horse heart protein. However, at variance with the latter system, for the yeast protein also a low spin species is detected, which appears to be present with a concentration of about 40% with respect to that of the high spin species. The R(1), R(2), [1H]-15N NOE of backbone amides which are not affected by paramagnetism are homogeneous and allow a simultaneous analysis of the data for the two species. The result is that the rotational correlation time is larger than in water and larger than expected on the basis of viscosity of the SDS-containing solution. This finding suggests interactions of cytochrome c with SDS. Furthermore, it appears that there is subnanosecond backbone mobility, which also accounts for the decreased intensity of NOE cross-peaks and may be associated with equilibria between helical and random coil structure. The dynamic behavior appears to be a common feature of the high spin and low spin species and is consistent with the presence of a molten globule state. The molten globule nature of the protein could account for the presence of the different axial coordination of the heme iron. Such findings are meaningful with respect to the physiology of cytochrome c as electron transfer protein and as promoter of apoptosis.

Animals↗
PubMed2003

Superoxide dismutase folding/unfolding pathway: role of the metal ions in modulating structural and dynamical features.

The unfolding and refolding of a monomeric mutant of copper-zinc superoxide dismutase was investigated by NMR spectroscopy in the copper-reduced form and by using guanidinium chloride as denaturing agent. It is found that the protein gives rise to a series of intermediates at low guanidinium concentration and to a globular unfolded state at a guanidinium concentration higher than 3.5 M, which closely resembles a random coil structure, but with a high degree of compactness. At 1.9 M guanidinium, the intermediate and unfolded forms are present in equimolar ratio. The intermediate states show changes in the 1H and 15N chemical shifts with respect to the native protein. The perturbations on the signals occur at different GdmCl concentrations for different regions of the protein. The residues affected first are located in the loops and in the beta3 strand, followed by changes in the sheet formed by beta4, beta5, beta7, beta8 strands. The transition into the unfolded structure implies the detachment of the metal ions from the native coordination sites, even if non-specific interactions with the metal ions remain. R(1), R(2), [1H]-15N NOE, and CLEANEX experiments provide information on the mobility at the various stages showing how protein rigidity is lost during unfolding. The whole process is reversible. The oxidized species behaves in a similar way. The apo protein shows formation of 50% of the unfolded species at a guanidinium concentration of 0.4 M, thus demonstrating the importance of metal ions with respect to the unfolding process and protein structure stability. Hints to understand the whole folding process are obtained and discussed.

Circular Dichroism↗
PubMed2003

A high-resolution NMR study of long-lived water molecules in both oxidation states of a minimal cytochrome c.

The interaction of water with oxidized and reduced cytochrome c from the Gram-positive bacterium Bacillus pasteurii (a 71-amino acid long monoheme cytochrome) is investigated through CLEANEX experiments and (15)N-edited ePHOGSY and Tr-ROESY experiments. It appears that a water molecule gives rise to dipolar cross-relaxation with the amide protons of Gly74 and Ile75, with a residence time longer than 0.4 ns, to account for a negative NOE. Such water molecule is present in both the oxidized and reduced species and in the X-ray structure. It appears to have a structural role. Other possible roles are discussed by comparison with the water molecules present in other c-type cytochromes. The amide proton of Cys35 is found to exchange rapidly with the solvent in the oxidized but not in the reduced protein, at variance with H/D exchange experiments, which probe a different time scale. The present data confirm that electron-transfer proteins evolved to minimize reorganization energy upon change of the oxidation state, even though the consequent variation of charge of the metal ion may induce some changes in the structure and/or dynamics of the protein.

Bacillus↗
PubMed2002

Solution structure of a monoheme ferrocytochrome c from Shewanella putrefaciens and structural analysis of sequence-similar proteins: functional implications.

Within the frame of the characterization of the structure and function of cytochromes c, an 81-amino acid cytochrome c was identified in the genome of Shewanella putrefaciens. Because of the scarce information about bacterial cytochromes of this type and the large variability in sequences and possibly function, we decided to proceed to its structural characterization. This protein was expressed in Escherichia coli and purified. The oxidized species is largely high spin, with a detached methionine, whereas the reduced species has the classical His/Met axial ligation to iron. The NMR solution structure of the reduced form was determined on a (15)N-labeled sample, for which 99% of all non-proline backbone (1)H and (15)N resonances have been assigned. One thousand three hundred two meaningful NOEs, out of 1775 NOEs, together with 66 dihedral angles provide a structure with rmsd values from the mean of 0.50 and 0.96 A for backbone and all heavy atoms, respectively. A search of gene banks allowed us to locate 10 different cytochromes c, the sequences of which are more than 30% identical to that of the S. putrefacienscytochrome. For two of them, the structures are known. The structures of the others have been modeled by using the available templates and internally validated. Structural similarities in terms of surface properties account for their biophysical features and provide hints about the function.

Amino Acid Sequence↗
PubMed2002

Browsing gene banks for Fe2S2 ferredoxins and structural modeling of 88 plant-type sequences: an analysis of fold and function.

One-hundred-and-seventy-nine sequences of Fe2S2 ferredoxins and ferredoxin precursors were identified in and retrieved from currently available protein and cDNA databases. On the basis of their cluster-binding patterns, these sequences were divided into three groups: those containing the CX4CX2CXnC pattern (plant-type ferredoxins), those with the CX5CX2CXnC pattern (adrenodoxins), and those with a different pattern. These three groups contain, respectively, 139, 36, and 4 sequences. After excluding ferredoxin precursors in the first group, two subgroups were identified, again based on their cluster-binding patterns: 88 sequences had the CX4CX2CX29C pattern, and 29 had the CX4CX2CXmC (m not equal 29) pattern. The structures of the 88 ferredoxins with the CX4CX2CX29C pattern were modeled based on the available experimental structures of nine proteins within this same group. The modeling procedure was tested by building structural models for the ferredoxins with known structures. The models resulted, on average, in being within 1 A of the backbone root-mean-square deviation from the corresponding experimental structures. In addition, these structural models were shown to be of high quality by using assessment procedures based on energetic and stereochemical parameters. Thus, these models formed a reliable structural database for this group of ferredoxins, which is meaningful within the framework of current structural genomics efforts. From the analysis of the structural database generated it was observed that the secondary structural elements and the overall three-dimensional structures are maintained throughout the superfamily. In particular, the residues in the hydrophobic core of the protein were found to be either absolutely conserved or conservatively substituted. In addition, certain solvent-accessible charged groups, as well as hydrophobic groups, were found to be conserved to the same degree as the core residues. The patterns of conservation of exposed residues identified the regions of the protein that are critical for its function in electron transfer. An extensive analysis of protein-protein interactions is now possible. Some conserved interactions between residues have been identified and related to structural and/or functional features. All this information could not be obtained from the analyses of the primary sequences alone. Finally, the analysis of the sequences of the related subgroup featuring the CX4CX2CXmC (m not equal 29) cluster-binding pattern in the light of the structural and functional insights provided by the inspection of the mentioned structural database affords some hints on the functional features of ferredoxins belonging to this subgroup.

Amino Acid Sequence↗