Héctor A. Baldoni

Polyproline II Helix Conformation in a Proline-Rich Environment: A Theoretical Study

Interest centers here on whether a polyproline II helix can propagate through adjacent non-proline residues, and on shedding light on recent experimental observations suggesting the presence of significant PP(II) structure in a short alanine-based peptide with no proline in the sequence. For this purpose, we explored the formation of polyproline II helices in proline-rich peptides with the sequences Ac-(Pro)(3)-X-(Pro)(3)-Gly-Tyr-NH(2), with X = Pro (PPP), Ala (PAP), Gln (PQP), Gly (PGP), and Val (PVP), and Ac-(Pro)(3)-Ala-Ala-(Pro)(3)-Gly-Tyr-NH(2) (PAAP), by using a theoretical approach that includes a solvent effect as well as cis <--> trans isomerization of the peptide groups and puckering conformations of the pyrrolidine ring of the proline residues. Since (13)C chemical shifts have proven to be useful for identifying secondary-structure preferences in proteins and peptides, and because values of the dihedral angles (phi,psi) are the main determinants of their magnitudes, we have, therefore, computed the Boltzmann-averaged (13)C chemical shifts for the guest residues in the PXP peptide (X = Pro, Ala, Gln, Gly, and Val) with a combination of approaches, involving molecular mechanics, statistical mechanics, and quantum mechanics. In addition, an improved procedure was used to carry out the conformational searches and to compute the solvent polarization effects faster and more accurately than in previous work. The current theoretical work and additional experimental evidence show that, in short proline-rich peptides, alanine decreases the polyproline II helix content. In particular, the theoretical evidence accumulated in this work calls into question the proposal that alanine has a strong preference to adopt conformations in the polyproline II region of the Ramachandran map.

Unblocked statistical-coil tetrapeptides and pentapeptides in aqueous solution: A theoretical study

NMR studies of the molecular conformations of peptides and proteins rely on a comparison of the relevant spectral parameters with the corresponding values for so-called statistical-coilpolypeptides. For this reason, it is necessary to characterize the experimental ensemble of states populated by statistical-coilpeptides. Such a characterization, however, has proven to be both difficult and sensitive to changes in many environmental parameters such as solvent composition, temperature, pH, as well as the neighboring amino acids in the sequence. As a consequence, a series of significant discrepancies has been reported for some experimentally observed parameters, such as chemical shifts, or vicinal coupling constants, 3JNHα, whose values appear to be incompatible with a statistical-coilensemble. In this work, we report the results of a molecular mechanics study of a series of unblocked tetra- and pentapeptides under different pH conditions. These calculations were carried out with explicit consideration of both the coupling between the process of proton binding/release and conformation adopted by the molecule at a given pH and the contribution of the conformational entropy to the total free energy. Good agreement was found between the calculated and experimentally determined values of the vicinal coupling constant, 3JNHα, the α-proton chemical shift, and the 13Cαchemical shift. All the evidence accumulated in these theoretical calculations helps to rationalize some of the unsettled anomalies observed experimentally, and to provide an understanding of the effect of pH and amino acid sequence on the conformational preferences of statistical-coilpeptides.

Performance of density functional models to reproduce observed (13)C(alpha) chemical shifts of proteins in solution.

The purpose of this work is to test several density functional models (namely, OPBE, O3LYP, OPW91, BPW91, OB98, BPBE, B971, OLYP, PBE1PBE, and B3LYP) to determine their accuracy and speed for computing 13Cα chemical shifts in proteins. The test is applied to 10 NMR‐derived conformations of the 76‐residue α/β protein ubiquitin (protein data bank id 1D3Z). With each functional, the 13Cα shielding was computed for 760 amino acid residues by using a combination of approaches that includes, but is not limited to, treating each amino acid X in the sequence as a terminally blocked tripeptide with the sequence Ac‐GXG‐NMe in the conformation of the regularized experimental protein structure. As computation of the 13Cα chemical shifts, not their shielding, is the main goal of this work, a computation of the 13Cα shielding of the reference, namely, tetramethylsilane, is investigated here and an effective and a computed tetramethylsilane shielding value for each of the functionals is provided. Despite observed small differences among all functionals tested, the results indicate that four of them, namely, OPBE, OPW91, OB98, and OLYP, provide the most accurate functionals with which to reproduce observed 13Cα chemical shifts of proteins in solution, and are among the faster ones. This study also provides evidence for the applicability of these functionals to proteins of any size or class, and for the validation of our previous results and conclusions, obtained from calculations with the slower B3LYP functional.

A substituted sulfonamide and its Co (II), Cu (II), and Zn (II) complexes as potential antifungal agents

Abstract A sulfonamide 1-tosyl-1-H-benzo(d)imidazol-2-amine (TBZA) and three new complexes of Co(II), Cu(II), and Zn(II) have been synthesized. The compounds have been characterized by elemental analyses, FTIR, 1H, and 13C-NMR spectroscopy. The structure of the TBZA, and its Co(II) and Cu(II) complexes, was determined by X-ray diffraction methods. TBZA and its Co(II) complex crystallize in the triclinic P-1 space group, while the Cu(II) complex crystallizes in the monoclinic P21/c space group. Antifungal activity was screened against eight pathogenic yeasts: Candida albicans (DMic 972576), Candida krusei (DMic 951705), Candida glabrata (DMic 982882), Candida tropicalis (DMic 982884), Candida dubliniensis (DMic 93695), Candida guilliermondii (DMic 021150), Cryptococcus neoformans (ATCC 24067), and Cryptococcus gattii (ATCC MYA-4561). Results on the inhibition of various human (h) CAs, hCA I, II, IV, VII, IX, and XII, and pathogenic beta and gamma CAs are also reported.

Unblocked statistical-coil tetrapeptides in aqueous solution: Quantum-chemical computation of the carbon-13 NMR chemical shifts

We recently reported a theoretical characterization of representative ensembles of statistical-coil conformations for tetrapeptides with unblocked termini in aqueous solution, at pH 7. The results showed good agreement between the computed Boltzmann-averaged and experimentally-determined values for both the vicinal coupling constants 3JNHα and the α-proton chemical shifts. Here, we carry out a cluster analysis of the ensembles of conformations generated in that study, and use them to compute the Boltzmann-averaged values of the quantum-chemical 13C chemical shifts for different amino acids in the unblocked tetrapeptides GGXA (where X stands for Phe, Arg, His, Glu, Ile, Lys, Gln, Tyr, Leu, Thr, Ala, Gly and Val). The values of the 13C chemical shifts in these thirteen amino acids (for which experimental data are available) were computed by using Density Functional Theory with a 6−311+G(2d,p) basis set. Good agreement is found in terms of both the correlation coefficient (R) and standard deviations of the difference between the computed Bolztmann-averaged and the NMR-determined values for the 13C chemical shifts. These results suggest that it may be possible to build a reliable theoretically-derived database of chemical shifts for statistical-coil residues. The results of the current study contribute to our understanding of the relations between chemical shifts, dihedral angles and vicinal coupling constants, 3JNHα. In addition, they can shed light as to how the statistical-coilconformation is related to the conformational preference of more structured states, such as the α-helical conformation.

Fast and accurate computation of the 13C chemical shifts for an alanine-rich peptide.

The purpose of this work is, first, to present a fast and accurate technique to compute Boltzmann‐averaged values of the quantum‐chemical 13C chemical shifts for each amino acid in oligopeptides, demonstrated here by an application to the peptide Ac‐XXAAAAAAAOO‐NH2 (where X denotes diaminobutyric acid, A is alanine, and O is ornithine) [XAO] and, second, to discuss the capability of the 13Cα and 13Cβ chemical shifts to distinguish the PPII conformation from the α‐helix and statistical‐coil conformations. Use is made of a combination of approaches, summarized as follows: (1) derivation of an ensemble of conformations by using a molecular mechanics technique; (2) use of a clustering procedure to form families and build a reduced set of conformations consisting of the lowest‐energy conformations of each family, and (3) computation of the 13C chemical shifts for the lowest‐energy conformations of each family, using a quantum‐chemical approach that treats a selected residue, or group of residues, with a 6‐311+G(2d,p) locally‐dense basis set, while the remaining residues in the sequence are treated with a 3‐21G basis set. The whole procedure is quite accurate and speeds up the computation of the Boltzmann‐averaged values of the 13C‐chemical shifts by several orders of magnitude. The present application sheds some light on the conformational preference for alanine and non‐alanine residues to occupy the PPII helical region of the Ramachandran map. Proteins 2004.

New small-size peptides modulators of the exosite of BACE1 obtained from a structure-based design

We report here two new small-size peptides acting as modulators of the β-site APP cleaving enzyme 1 (BACE1) exosite. Ac-YPYFDPL-NH2 and Ac-YPYDIPL-NH2 displayed a moderate but significant inhibitory effect on BACE1. These peptides were obtained from a molecular modeling study. By combining MD simulations with ab initio and DFT calculations, a simple and generally applicable procedure to evaluate the binding energies of small-size peptides interacting with the exosite of the BACE1 is reported here. The structural aspects obtained for the different complexes were analyzed providing a clear picture about the binding interactions of these peptides. These interactions have been investigated within the framework of the density functional theory and the quantum theory of atoms in molecules using a reduced model. Although the approach used here was traditionally applied to the study of noncovalent interactions in small molecules complexes in gas phase, we show, through in this work, that this methodology is also a very powerful tool for the study of biomolecular complexes, providing a very detailed description of the binding event of peptides modulators at the exosite of BACE1.

Molecular design and synthesis of novel peptides from amphibians skin acting as inhibitors of cholinesterase enzymes

Cholinesterases are a family of enzymes that catalyze the hydrolysis of neurotransmitter acetylcholine. There are two types of cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which differ in their distribution in the body. Currently, cholinesterase inhibitors (ChEI) represent the treatment of choice for Alzheimers disease (AD). In this paper, we report the synthesis and inhibitory effect on both enzymes of four new peptides structurally related to P1‐Hp‐1971 (amphibian skin peptide found in our previous work. Sequence: TKPTLLGLPLGAGPAAGPGKR‐NH2). The bioassay data and cytotoxicity test show that some of the compounds possess a significant AChE and BChE inhibition and no toxic effect. The present work demonstrates that diminution of the size of the original peptide could potentially result in new compounds with significant cholinesterase inhibition activity, although it appears that there is an optimal size for the sequence. We also conducted an exhaustive molecular modeling study to better understand the mechanism of action of these compounds by combining docking techniques with molecular dynamics simulations on BChE. This is the first report about amphibian peptides and the second one of natural peptides with ChE inhibitory activity. Copyright

Structural and functional insights into the anti-BACE1 Fab fragment that recognizes the BACE1 exosite

A molecular modeling study giving structural, functional, and mutagenesis insights into the anti-BACE1 Fab fragment that recognizes the BACE1 exosite is reported. Our results allow extending experimental data resulting from X-ray diffraction experiments in order to examine unknown aspects for the Fab-BACE1 recognition and its binding mode. Thus, the study performed here allows extending the inherently static nature of crystallographic structures in order to gain a deeper understanding of the structural and dynamical basis at the atomic level. The characteristics and strength of the interatomic interactions involved in the immune complex formation are exhaustively analyzed. The results might explain how the anti-BACE1 Fab fragment and other BACE1 exosite binders are capable to produce an allosteric modulation of the BACE1 activity. Our site-directed mutagenesis study indicated that the functional anti-BACE1 paratope, residues Tyr32 (H1), Trp50 (H2), Arg98 (H3), Phe101 (H3), Trp104 (H3) and Tyr94 (L3), strongly dominates the binding energetics with the BACE1 exosite. The mutational studies described in this work might accelerate the development of new BACE1 exosite binders with interesting pharmacological activity.

Position dependence of the 13C chemical shifts of α-helical model peptides. Fingerprint of the 20 naturally occurring amino acids

The position dependence of the 13C chemical shifts was investigated at the density functional level for α‐helical model peptides represented by the sequence Ac‐(Ala)i‐X‐(Ala)j‐NH2, where X represents any of the 20 naturally occurring amino acids, with 0 ≤ i ≤ 8 and i + j = 8. Adoption of the locally dense basis approach for the quantum chemical calculations enabled us to reduce the length of the chemical‐shift calculations while maintaining good accuracy of the results. For the 20 naturally occurring amino acids in α‐helices, there is (1) significant variability of the computed 13C shielding as a function of both the guest residue (X) and the position along the sequence; for example, at the N terminus, the 13Cα and 13Cβ shieldings exhibit a uniform pattern of variation with respect to both the central or the C‐terminal positions; (2) good agreement between computed and observed 13Cα and 13Cβ chemical shifts in the interior of the helix, with correlation coefficients of 0.98 and 0.99, respectively; for 13Cα chemical shifts, computed in the middle of the helix, only five residues, namely Asn, Asp, Ser, Thr, and Leu, exhibit chemical shifts beyond the observed standard deviation; and (3) better agreement for four of these residues (Asn, Asp, Ser, and Thr) only for the computed values of the 13Cα chemical shifts at the N terminus. The results indicate that 13Cβ, but not 13Cβ, chemical shifts are sensitive enough to reflect the propensities of some amino acids for specific positions within an α‐helix, relative to the N and C termini of peptides and proteins.