Biomedical subjects

Marcela Cataldi

Publications and source records attributed to Marcela Cataldi.

4 recordsLinked to original sources

PubMed2005

Resonance assignments and secondary structure analysis of E. coli thioredoxin by magic angle spinning solid-state NMR spectroscopy.

De novo site-specific 13C and 15N backbone and sidechain resonance assignments are presented for uniformly enriched E. coli thioredoxin, established using two-dimensional homo- and heteronuclear solid-state magic angle spinning NMR correlation spectroscopy. Backbone dihedral angles and secondary structure were derived from the statistical analysis of the secondary chemical shifts, and are in good agreement with solution values for the intact full-length thioredoxin, with the exception of a small number of residues located at the termini of the individual secondary structure elements. A large number of cross-peaks observed in the DARR spectra with long mixing times correspond to the pairs of carbon atoms separated by 4-6 angstroms, suggesting that DARR could be efficiently employed for observation of medium- and long-range correlations. The 108 amino acid residue E. coli thioredoxin is the largest uniformly enriched protein assigned to this degree of completeness by solid-state NMR spectroscopy to date. It is anticipated that with a combination of two-dimensional correlation experiments and high magnetic fields, resonance assignments and secondary structure can be generally derived for other noncrystalline proteins.

Carbon Isotopes↗

PubMed2005

pH dependence of amide chemical shifts in natively disordered polypeptides detects medium-range interactions with ionizable residues.

A growing number of natively disordered proteins undergo a folding/binding process that is essential for their biological function. An interesting question is whether these proteins have incompletely solvated regions that drive the folding/binding process. Although the presence of predominantly hydrophobic buried regions can be easily ascertained by high-sensitivity differential scanning calorimetry analysis, the identification of those residues implicated in the burial requires NMR analysis. We have selected a partially solvated natively disordered fragment of Escherichia coli, thioredoxin, C37 (38-108), for full NMR spectral assignment. The secondary chemical shifts, temperature coefficients, and relaxation rates (R(1) and R(2)) of this fragment indicate the presence of a flexible backbone without a stable hydrogen bond network near neutral pH. (1)H-(15)N heteronuclear single quantum coherence analysis of the pH dependence of amide chemical shifts in fragment C37 within pH 2.0 and 7.0 suggests the presence of interactions between nonionizable residues and the carboxylate groups of four Asp and four Glu residues. The pH midpoints (pH(m)) of the amides in the ionizable residues (Asp or Glu) and, consequently, the shifts in the pH(m) (DeltapH(m)) of these residues with respect to model tetrapeptides, are sequence-dependent; and the nonionizable residues that show pH dependence cluster around the ionizable ones. The same pH dependence has been observed in two fragments: M37 (38-73) and C73 (74-108), ruling out the participation of long-range interactions. Our studies indicate the presence of a 15-residue pH-dependent segment with the highest density of ionizable sites in the disordered ensembles of fragments C37 and M37. The observed correlations between ionizable and nonionizable residues in this segment suggest the organization of the backbone and side chains through local and medium-range interactions up to nine residues apart, in contrast to only a few interactions in fragment C73. These results agree qualitatively with the predominantly hydrophobic buried surface detected only in fragments C37 and M37 by highly sensitive differential scanning calorimetry analysis. This work offers a sensitive and rapid new tool to obtain clues about local and nonlocal interactions between ionizable and nonionizable residues in the growing family of natively disordered small proteins with full NMR assignments.

Amides↗

PubMed2004

Magic angle spinning solid-state NMR spectroscopy for structural studies of protein interfaces. resonance assignments of differentially enriched Escherichia coli thioredoxin reassembled by fragment complementation.

De novo site-specific backbone and side-chain resonance assignments are presented for U-15N(1-73)/U-13C,15N(74-108) reassembly of Escherichia coli thioredoxin by fragment complementation, determined using solid-state magic angle spinning NMR spectroscopy at 17.6 T. Backbone dihedral angles and secondary structure predicted from the statistical analysis of 13C and 15N chemical shifts are in general agreement with solution values for the intact full-length thioredoxin, confirming that the secondary structure is retained in the reassembled complex prepared as a poly(ethylene glycol) precipitate. The differential labeling of complementary thioredoxin fragments introduced in this work is expected to be beneficial for high-resolution structural studies of protein interfaces formed by protein assemblies by solid-state NMR spectroscopy.

Amino Acid Sequence↗

PubMed2004

Reversal of negative charges on the surface of Escherichia coli thioredoxin: pockets versus protrusions.

Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability. We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH). We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin. DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ). The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69. Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C. However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes. These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69. Our results led us to suggest that reversal of a negative charge at a pocket has a larger effect on stability than a similar reversal at a protrusion and that this difference arises largely from short-range interactions with polar groups within the pocket, rather than long-range interactions with solvent-exposed charged groups.

Alanine↗