302 research outputs found
Assessing the fractions of tautomeric forms of the imidazole ring of histidine in proteins as a function of pH
No full textA method is proposed to determine the fraction of the tautomeric forms of the imidazole ring of histidine in proteins as a function of pH, provided that the observed and chemical shifts and the protein structure, or the fraction of H(+) form, are known. This method is based on the use of quantum chemical methods to compute the (13)C NMR shieldings of all the imidazole ring carbons ((13)C(γ), , and ) for each of the two tautomers, N(δ1)-H and N(ε2)-H, and the protonated form, H(+), of histidine. This methodology enabled us (i) to determine the fraction of all the tautomeric forms of histidine for eight proteins for which the and chemical shifts had been determined in solution in the pH range of 3.2 to 7.5 and (ii) to estimate the fraction of tautomeric forms of eight histidine-containing dipeptide crystals for which the chemical shifts had been determined by solid-state (13)C NMR. Our results for proteins indicate that the protonated form is the most populated one, whereas the distribution of the tautomeric forms for the imidazole ring varies significantly among different histidines in the same protein, reflecting the importance of the environment of the histidines in determining the tautomeric forms. In addition, for ∼70% of the neutral histidine-containing dipeptides, the method leads to fairly good agreement between the calculated and the experimental tautomeric form. Coexistence of different tautomeric forms in the same crystal structure may explain the remaining 30% of disagreement.Fil: Vila, Jorge Alberto. Cornell University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis ; ArgentinaFil: Arnautova, Yelena A.. Cornell University; Estados UnidosFil: Vorobjev, Yury. Russian Academy of Science; RusiaFil: Scheraga, Harold A.. Cornell University; Estados Unido
A comprehensive analysis of the computed tautomer fractions of the imidazole ring of histidines in Loligo vulgaris
No full textA recently introduced electrostatic-based method to determine the pKa values of ionizable residues and fractions of ionized and tautomeric forms of histidine (His) and acid residues in proteins, at a given fixed pH, is applied here to the analysis of a His-rich protein, namely Loligo vulgaris (pdb id 1E1A), a 314-residue all-β protein. The average tautomeric fractions for the imidazole ring of each of the six histidines in the sequence were computed using an approach that includes, but is not limited to, molecular dynamic simulations coupled with calculations of the ionization states for all 94 ionizable residues of protein 1E1A in water at pH 6.5 and 300 K. The electrostatic-calculated tautomeric fractions of the imidazole ring of His were compared with predictions obtained from an existent NMR-based methodology. Our results indicate that: (i) the averaged electrostatic-based tautomeric predictions for the imidazole ring of all histidines of Loligo vulgaris are dominated by the Nε2-H rather than the Nδ1-H form, although such preferences from the NMR-based methodology are not so well defined; (ii) the computed average absolute difference between the electrostatic- and the NMR-based tautomeric predictions among all six histidines vary among 0% to 17%; (iii) for the His showing the largest fraction of the neutral form (81%), the absolute difference between the NMR- and electrostatic-based computed tautomeric predictions is only 3%; and (iv) the tautomeric predictions for the imidazole ring of His computed with the NMR-based methodology are stable within a certain, well-defined, range of variations of a tautomer-related parameter.Fil: Vorobjev, Yury N.. Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Science; Rusia. Novosibirsk State University; Rusia. Cornell University; Estados UnidosFil: Scheraga, Harold A.. Cornell University; Estados UnidosFil: Vila, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; Argentin
FAMBE-pH: a fast and accurate method to compute the total solvation free energies of proteins
No full textA fast and accurate method to compute the total solvation free energies of proteins as a function of pH is presented. The method makes use of a combination of approaches, some of which have already appeared in the literature; (i) the Poisson equation is solved with an optimized fast adaptive multigrid boundary element (FAMBE) method; (ii) the electrostatic free energies of the ionizable sites are calculated for their neutral and charged states by using a detailed model of atomic charges; (iii) a set of optimal atomic radii is used to define a precise dielectric surface interface; (iv) a multilevel adaptive tessellation of this dielectric surface interface is achieved by using multisized boundary elements; and (v) 1:1 salt effects are included. The equilibrium proton binding/release is calculated with the Tanford−Schellman integral if the proteins contain more than ∼20−25 ionizable groups; for a smaller number of ionizable groups, the ionization partition function is calculated directly. The FAMBE method is tested as a function of pH (FAMBE-pH) with three proteins, namely, bovine pancreatic trypsin inhibitor (BPTI), hen egg white lysozyme (HEWL), and bovine pancreatic ribonuclease A (RNaseA). The results are (a) the FAMBE-pH method reproduces the observed pKaʼs of the ionizable groups of these proteins within an average absolute value of 0.4 pK units and a maximum error of 1.2 pK units and (b) comparison of the calculated total pH-dependent solvation free energy for BPTI, between the exact calculation of the ionization partition function and the Tanford−Schellman integral method, shows agreement within 1.2 kcal/mol. These results indicate that calculation of total solvation free energies with the FAMBE-pH method can provide an accurate prediction of protein conformational stability at a given fixed pH and, if coupled with molecular mechanics or molecular dynamics methods, can also be used for more realistic studies of protein folding, unfolding, and dynamics, as a function of pH.Fil: Vorobjev, Yury N.. Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Science; RusiaFil: Vila, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; ArgentinaFil: Scheraga, Harold A.. Cornell University; Estados Unido
Identifying native‐like protein structures with scoring functions based on all‐atom ECEPP force fields, implicit solvent models and structure relaxation
No full textAvailability of energy functions which can discriminate native-like from non-native protein conformations is crucial for theoretical protein structure prediction and refinement of low-resolution protein models. This article reports the results of benchmark tests for scoring functions based on two all-atom ECEPP force fields, that is, ECEPP/3 and ECEPP05, and two implicit solvent models for a large set of protein decoys. The following three scoring functions are considered: (i) ECEPP05 plus a solvent-accessible surface area model with the parameters optimized with a set of protein decoys (ECEPP05/SA); (ii) ECEPP/3 plus the solvent-accessible surface area model of Ooi et al. (Proc Natl Acad Sci USA 1987;84:3086–3090) (ECEPP3/OONS); and (iii) ECEPP05 plus an implicit solvent model based on a solution of the Poisson equation with an optimized Fast Adaptive Multigrid Boundary Element (FAMBEpH) method (ECEPP05/FAMBEpH). Short Monte Carlo-with-Minimization (MCM) simulations, following local energy minimization, are used as a scoring method with ECEPP05/SA and ECEPP3/OONS potentials, whereas energy calculation is used with ECEPP05/FAMBEpH. The performance of each scoring function is evaluated by examining its ability to distinguish between native-like and non-native protein structures. The results of the tests show that the new ECEPP05/SA scoring function represents a significant improvement over the earlier ECEPP3/OONS version of the force field. Thus, it is able to rank native-like structures with Cα root-mean-square-deviations below 3.5 Å as lowest-energy conformations for 76% and within the top 10 for 87% of the proteins tested, compared with 69 and 80%, respectively, for ECEPP3/OONS. The use of the FAMBEpH solvation model, which provides a more accurate description of the protein-solvent interactions, improves the discriminative ability of the scoring function to 89%. All failed tests in which the native-like structures cannot be discriminated as those with low energy, are due to omission of protein–protein interactions. The results of this study represent a benchmark in force-field development, and may be useful for evaluation of the performance of different force fields. Proteins 2009.Fil: Arnautova, Yelena A.. Cornell University; Estados UnidosFil: Vorobjev, Yury N.. Russian Academy of Science; RusiaFil: Vila, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; ArgentinaFil: Scheraga, Haroldo A.. Cornell University; Estados Unido
Synergestic induction of NRF-2 gene by curcumin and sulforaphane and pharmacokinetics/metabolism of 13C/Dim in rats by UPLC/MS
No full textCurcumin (CUR) and Sulforaphane (SFN) have shown remarkable cancer chemopreventive effects in numerous studies and combinations of low doses of chemopreventive agents can reduce toxicity while augmenting efficacy. The first part of the thesis investigated the chemotherapeutic effects elicited by a combination of CUR and SFN on human hepatocarcinoma
cells. The combination treatment- mediated effects on phase II/antioxidant enzymatic induction and antioxidant response element (ARE) was investigated. It was proposed that the combination of CUR and SFN could synergistically enhance the induction of ARE and the nuclear E2-factor related factor 2 (Nrf2)-mediated enzymes. Low doses of CUR and SFN significantly induced the expression of Nrf2-mediated enzymes, HO-1 and UGT1A1, promoted nuclear translocation of
Nrf2– a key regulator of phase II detoxifying /antioxidant enzymes – and synergistically induced the ARE luciferase activity. Chemical inhibitors of mRNA and protein synthesis affected the combination therapy- mediated transcriptional regulation of both HO-1 and UGT1A1. Synergism of CUR and SFN was evident at low concentrations. Such synergism in ARE-luciferase activity may partly explain the significant induction in the expression of Nrf2- mediated expression of HO-1 and UGT1A1 and the nuclear translocation of Nrf2, suggesting that a combination of low doses may be a promising strategy for cancer chemoprevention. For the second part of this thesis, a liquid chromatographic method was validated for the simultaneous analysis and pharmacokinetic evaluation of Indole-3-Carbinol (I3C), Diindolylmethane (DIM), and several I3C metabolites. I3C and DIM are naturally derived phytochemicals with promising in-vitro and
in-vivo anticarcinogenic properties. Using reversed-phase ultra performance liquid
chromatography (UPLC) coupled with mass spectrophotometry (MS), a rapid, specific, and high
throughput method was developed and validated for the quantification and identification of I3C, DIM, and other I3C metabolites in plasma. Recovery, linearity, precision, accuracy, and stability analysis fulfilled the CDER guidelines criteria. The method was successfully applied to the determination of the pharmacokinetic parameters and elucidation of metabolites of I3C or DIM
after oral, intravenous, or intraperitoneal administration to Sprague- Dawley rats.M.S.Includes bibliographical referencesIncludes vitaby Yury Y. Gome
Outline of an experimental design aimed to detect protein A mirror image in solution
No full textThere is abundant theoretical evidence indicating that a mirror image of Protein A may occur during the protein folding process. However, as to whether such mirror image exists in solution is an unsolved issue. Here we provide outline of an experimental design aimed to detect the mirror image of Protein A in solution. The proposal is based on computational simulations indicating that the use of a mutant of protein A, namely Q10H, could be used to detect the mirror image conformation in solution. Our results indicate that the native conformation of the protein A should have a pKa, for the Q10H mutant, at ≈6.2, while the mirror-image conformation should have a pKa close to ≈7.3. Naturally, if all the population is in the native state for the Q10H mutant, the pKa should be ≈6.2, while, if all are in the mirror-image state, it would be ≈7.3, and, if it is a mixture, the pKa should be larger than 6.2, presumably in proportion to the mirror population. In addition, evidence is provided indicating the tautomeric distribution of H10 must also change between the native and mirror conformations. Although this may not be completely relevant for the purpose of determining whether the protein A mirror image exists in solution, it could provide valuable information to validate the pKa findings. We hope this proposal will foster experimental work on this problem either by direct application of our proposed experimental design or serving as inspiration and motivation for other experiments.Fil: Martín, Osvaldo Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; ArgentinaFil: Vorobjev, Yury. Institute Of Chemical Biology And Fundamental Medicine; RusiaFil: Scheraga, Harold A.. Cornell University; Estados UnidosFil: Vila, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; Argentina. Cornell University; Estados Unido
Coupling between conformation and proton binding in proteins
No full textInterest centers here on whether the use of a fixed charge distribution of a protein solute, or a treatment that considers proton-binding equilibria by solving the Poisson equation, is a better approach to discriminate native from non-native conformations of proteins. In this analysis of the charge distribution of 7 proteins, we estimate the solvation free energy contribution to the total free energy by exploring the 2ζ possible ionization states of the whole molecule, with ζ being the number of ionizable groups in the amino acid sequence, for every conformation in the ensembles of 7 proteins. As an additional consideration of the role of electrostatic interactions in determining the charge distribution of native folds, we carried out a comparison of alternative charge assignment models for the ionizable residues in a set of 21 native-like proteins. The results of this work indicate that (1) for 6 out of 7 proteins, estimation of solvent polarization based on the Generalized Born model with a fixed charge distribution provides the optimal trade-off between accuracy, with respect to the Poisson equation, and speed when compared to the accessible surface area model; for the seventh protein, consideration of all possible ionization states of the whole molecule appears to be crucial to discriminate the native from non-native conformations; (2) significant differences in the degree of ionization and hence the charge distribution for native folds are found between the different charge models examined; (3) the stability of the native state is determined by a delicate balance of all the energy components, and (4) conformational entropy, and hence the dynamics of folding, may play a crucial role for a successful ab initio protein folding prediction.Fil: Vila, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; Argentina. Cornell University; Estados UnidosFil: Ripoll, Daniel R.. Cornell University; Estados UnidosFil: Arnautova, Yelena A.. Cornell University; Estados UnidosFil: Vorobjev, Yury N.. Institute of Chemical Biology and Fundamental Medicine of Siberian Brunch of Russian Academy of Science; RusiaFil: Scheraga, Harold A.. Cornell University; Estados Unido
UDP-glucose glycoprotein glucosyltransferase (uggt-1) and UPR genes modulate C. elegans necrotic cell death:
No full textContrary to an initial understanding of necrosis as a chaotic, non-regulated type of cell death, recent studies suggest that necrotic cell death (NCD) is a consequence of a complex and lethal cascade of genetically-encoded events. Necrosis underlies the pathology of many neurodegenerative diseases, stroke, and traumatic injury. In the Driscoll Lab, NCD mechanisms have been addressed for several years taking advantage of unique genetic and molecular biology tools developed in the model organism Caenorhabditis elegans. The necrotic paradigm we study the most involves initiation of cell death by hyperactivated ion channels expressed in six touch-sensory neurons and requires elevation of intracellular Ca2+, which activates calpain and cathepsin proteases.
I exploited the unique features of our model system to uncover novel genetic factors influencing this process. To this end, I conducted a high-throughput forward genetic screen to identify mutations that block or delay necrotic cell death induced by MEC-4(d) channel hyperactivation, and genetically mapped novel mutations capable of blocking or slowing the death process. I exploited an automated mutational screening capacity that allows sorting of individual animals based on detection of fluorescent signals that, in our particular case, had been engineered to indicate neuronal viability. I focused on the cloning of two novel mutant loci and dissected molecular mechanisms responsible for death suppression. In addition, I studied the impact of a major subset of calcium homeostasis genes in a C. elegans model of Aβ toxicity.
My research adds a new component to the current understanding of NCD, suggesting that inability to cope with endoplasmic reticulum stress (presumably induced by calcium depletion inside the ER, which affects chaperone functionality) plays an important role in progression through necrosis. I discovered that mild activation of an intact unfolded protein response (UPR), e.g., as induced by downregulation of UDP-glucose:glycoprotein glucosyltransferase (UGGT, an ER-resident enzyme involved in high-fidelity protein folding quality control) or mild increments in ambient temperature, can partially suppress necrosis in our C. elegans model, reminiscent of beneficial preconditioning effects in mammals. Additionally I found that several UPR transducers contribute to such modulation of cell death in a “tug-of-war” fashion. Our refined model of molecular mechanisms contributing and modulating necrosis suggests new strategies that could eventually limit the devastating effects of necrosis in human injury and disease.Ph.D.Includes bibliographical references (p. 154-174)by Yury Orlando Nunez Lope
Advances in Implicit Models of Water Solvent to Compute Conformational Free Energy and Molecular Dynamics of Proteins at Constant pH
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