| 1. |
- Bettiga, Maurizio, 1978, et al.
(författare)
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Plasma membrane as a crucial player in acetic acid effect on yeast
- 2017
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Ingår i: IMYA12- 12th International Meeting on Yeast Apoptosis, Bari, Italy • 14-18 May 2017.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Weak organic acids such as formic, acetic or lactic acid are known inhibitors of microbial growth and fermentation. Acetic acid toxicity to yeast cells has been explained by different theories, involving specific signaling effects triggering an active cell death program, reduction of intracellular pH and acetate anion accumulation. Regardless of the fact whether the actual effect of acetate involves one of these mechanisms or a combination thereof, acetic acid inhibits yeast metabolism and affects yeast viability. This has a high impact on the feasibility of the new generation of fermentation processes, based on the naturally acetic acid-rich lignocellulosic substrates. It is therefore highly desirable to obtain a strain with increased capacity of coping with high acetic acid concentrations in the fermentation medium. Acetic acid is thought to be internalized by yeast cells in its undissociated form, by crossing the hydrophobic barrier of plasma membrane. Thus, in our work we focused on the investigation of membrane properties and how these influence the tolerance of yeast to acetic acid. First, we demonstrated with lipidomics analysis of membrane lipids that the yeast Zygosaccharomyces bailii, showing extraordinary tolerance to acetic acid, has a plasma membrane which is rich in sphingolipids. Next, we combined membrane molecular dynamics and in vivo measurements to confirm the specific role of sphingolipids in altering the permeability of plasma membrane to acetic acid. Finally, we investigated the effect of alcohols on the acetic acid permeation rate through the membrane. Our ultimate goal is to engineer the membrane composition of an industrial yeast strain towards reduced permeability, in order to obtain higher acetic acid tolerance.
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| 2. |
- Ciancetta, Antonella, et al.
(författare)
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A QM/MM study of the binding of RAPTA ligands to cathepsin B
- 2011
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Ingår i: Journal of Computer-Aided Molecular Design. - : Springer Science and Business Media LLC. - 1573-4951 .- 0920-654X. ; 25:8, s. 729-742
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Tidskriftsartikel (refereegranskat)abstract
- We have carried out quantum mechanical (QM) and QM/MM (combined QM and molecular mechanics) calculations, as well as molecular dynamics (MD) simulations to study the binding of a series of six RAPTA (Ru(II)-arene-1,3,5-triaza-7-phosphatricyclo-[3.3.1.1] decane) complexes with different arene substituents to cathepsin B. The recently developed QM/MM-PBSA approach (QM/MM combined with Poisson-Boltzmann solvent-accessible surface area solvation) has been used to estimate binding affinities. The QM calculations reproduce the antitumour activities of the complexes with a correlation coefficient (r(2)) of 0.35-0.86 after a conformational search. The QM/MM-PBSA method gave a better correlation (r(2) = 0.59) when the protein was fixed to the crystal structure, but more reasonable ligand structures and absolute binding energies were obtained if the protein was allowed to relax, indicating that the ligands are strained when the protein is kept fixed. In addition, the best correlation (r(2) = 0.80) was obtained when only the QM energies were used, which suggests that the MM and continuum solvation energies are not accurate enough to predict the binding of a charged metal complex to a charged protein. Taking into account the protein flexibility by means of MD simulations slightly improves the correlation (r(2) = 0.91), but the absolute energies are still too large and the results are sensitive to the details in the calculations, illustrating that it is hard to obtain stable predictions when full flexible protein is included in the calculations.
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| 3. |
- Diehl, Carl, et al.
(författare)
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Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3.
- 2009
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Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 45:1-2, s. 157-169
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Tidskriftsartikel (refereegranskat)abstract
- The conformational entropy of proteins can make significant contributions to the free energy of ligand binding. NMR spin relaxation enables site-specific investigation of conformational entropy, via order parameters that parameterize local reorientational fluctuations of rank-2 tensors. Here we have probed the conformational entropy of lactose binding to the carbohydrate recognition domain of galectin-3 (Gal3), a protein that plays an important role in cell growth, cell differentiation, cell cycle regulation, and apoptosis, making it a potential target for therapeutic intervention in inflammation and cancer. We used (15)N spin relaxation experiments and molecular dynamics simulations to monitor the backbone amides and secondary amines of the tryptophan and arginine side chains in the ligand-free and lactose-bound states of Gal3. Overall, we observe good agreement between the experimental and computed order parameters of the ligand-free and lactose-bound states. Thus, the (15)N spin relaxation data indicate that the molecular dynamics simulations provide reliable information on the conformational entropy of the binding process. The molecular dynamics simulations reveal a correlation between the simulated order parameters and residue-specific backbone entropy, re-emphasizing that order parameters provide useful estimates of local conformational entropy. The present results show that the protein backbone exhibits an increase in conformational entropy upon binding lactose, without any accompanying structural changes.
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| 4. |
- Diehl, Carl, et al.
(författare)
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Protein Flexibility and Conformational Entropy in Ligand Design Targeting the Carbohydrate Recognition Domain of Galectin-3.
- 2010
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 132, s. 14577-14589
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Tidskriftsartikel (refereegranskat)abstract
- Rational drug design is predicated on knowledge of the three-dimensional structure of the protein-ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. (15)N and (2)H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein-carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.
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| 5. |
- Genheden, Samuel, et al.
(författare)
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A comparison of different initialization protocols to obtain statistically independent molecular dynamics simulations.
- 2011
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Ingår i: Journal of Computational Chemistry. - : Wiley. - 1096-987X .- 0192-8651. ; 32:2, s. 187-195
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Tidskriftsartikel (refereegranskat)abstract
- We study how the results of molecular dynamics (MD) simulations are affected by various choices during the setup, e.g., the starting velocities, the solvation, the location of protons, the conformation of His, Asn, and Gln residues, the protonation and titration of His residues, and the treatment of alternative conformations. We estimate the binding affinity of ligands to four proteins calculated with the MM/GBSA method (molecular mechanics combined with a generalized Born and surface area solvation energy). For avidin and T4 lysozyme, all variations gave similar results within 2 kJ/mol. For factor Xa, differences in the solvation or in the selection of alternative conformations gave results that are significantly different from those of the other approaches by 4-6 kJ/mol, whereas for galectin-3, changes in the conformations, rotations, and protonation gave results that differed by 10 kJ/mol, but only if residues close to the binding site were modified. This shows that the results of MM/GBSA calculations are reasonably reproducible even if the MD simulations are set up with different software. Moreover, we show that the sampling of phase space can be enhanced by solvating the systems with different equilibrated water boxes, in addition to the common use of different starting velocities. If different conformations are available in the crystal structure, they should also be employed to enhance the sampling. Protonation, ionization, and conformations of Asn, Gln, and His may also be used to enhance sampling, but great effort should be spent to obtain as reliable predictions as possible close to the active site.
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| 6. |
- Genheden, Samuel, et al.
(författare)
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Accurate Predictions of Nonpolar Solvation Free Energies Require Explicit Consideration of Binding-Site Hydration
- 2011
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 133:33, s. 13081-13092
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Tidskriftsartikel (refereegranskat)abstract
- Continuum solvation methods are frequently used to increase the efficiency of computational methods to estimate free energies. In this paper, we have evaluated how well such methods estimate the nonpolar solvation free-energy change when a ligand binds to a protein. Three different continuum methods at various levels of approximation were considered, viz., the polarized continuum model (PCM), a method based on cavity and dispersion terms (CD), and a method based on a linear relation to the solvent-accessible surface area (SASA). Formally rigorous double-decoupling thermodynamic integration was used as a benchmark for the continuum methods. We have studied four protein-ligand complexes with binding sites of varying solvent exposure, namely the binding of phenol to ferritin, a biotin analogue to avidin, 2-aminobenzimidazole to trypsin, and a substituted galactoside to galectin-3. For ferritin and avidin, which have relatively hidden binding sites, rather accurate nonpolar solvation free energies could be obtained with the continuum methods if the binding site is prohibited to be filled by continuum water in the unbound state, even though the simulations and experiments show that the ligand replaces several water molecules upon binding. For the more solvent exposed binding sites of trypsin and galectin-3, no accurate continuum estimates could be obtained, even if the binding site was allowed or prohibited to be filled by continuum water. This shows that continuum methods fail to give accurate free energies on a wide range of systems with varying solvent exposure because they lack a microscopic picture of binding-site hydration as well as information about the entropy of water molecules that are in the binding site before the ligand binds. Consequently, binding affinity estimates based upon continuum solvation methods will give absolute binding energies that may differ by up to 200 kJ/mol depending on the method used. Moreover, even relative energies between ligands with the same scaffold may differ by up to 75 kJ/mol. We have tried to improve the continuum solvation methods by adding information about the solvent exposure of the binding site or the hydration of the binding site, and the results are promising at least for this small set of complexes.
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| 7. |
- Genheden, Samuel, et al.
(författare)
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All-atom/coarse-grained hybrid predictions of distribution coefficients in SAMPL5
- 2016
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Ingår i: Journal of Computer-Aided Molecular Design. - : Springer Science and Business Media LLC. - 0920-654X .- 1573-4951. ; 30:11, s. 969-976
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Tidskriftsartikel (refereegranskat)abstract
- We present blind predictions submitted to the SAMPL5 challenge on calculating distribution coefficients. The predictions were based on estimating the solvation free energies in water and cyclohexane of the 53 compounds in the challenge. These free energies were computed using alchemical free energy simulations based on a hybrid all-atom/coarse-grained model. The compounds were treated with the general Amber force field, whereas the solvent molecules were treated with the Elba coarse-grained model. Considering the simplicity of the solvent model and that we approximate the distribution coefficient with the partition coefficient of the neutral species, the predictions are of good accuracy. The correlation coefficient, R is 0.64, 82 % of the predictions have the correct sign and the mean absolute deviation is 1.8 log units. This is on a par with or better than the other simulation-based predictions in the challenge. We present an analysis of the deviations to experiments and compare the predictions to another submission that used all-atom solvent.
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| 8. |
- Genheden, Samuel, et al.
(författare)
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An MM/3D-RISM approach for ligand binding affinities.
- 2010
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Ingår i: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 114:25, s. 8505-8516
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Tidskriftsartikel (refereegranskat)abstract
- We have modified the popular MM/PBSA or MM/GBSA approaches (molecular mechanics for a biomolecule, combined with a Poisson-Boltzmann or generalized Born electrostatic and surface area nonelectrostatic solvation energy) by employing instead the statistical-mechanical, three-dimensional molecular theory of solvation (also known as 3D reference interaction site model, or 3D-RISM-KH) coupled with molecular mechanics or molecular dynamics ( Blinov , N. ; et al. Biophys. J. 2010 ; Luchko , T. ; et al. J. Chem. Theory Comput. 2010 ). Unlike the PBSA or GBSA semiempirical approaches, the 3D-RISM-KH theory yields a full molecular picture of the solvation structure and thermodynamics from the first principles, with proper account of chemical specificities of both solvent and biomolecules, such as hydrogen bonding, hydrophobic interactions, salt bridges, etc. We test the method on the binding of seven biotin analogues to avidin in aqueous solution and show it to work well in predicting the ligand-binding affinities. We have compared the results of 3D-RISM-KH with four different generalized Born and two Poisson-Boltzmann methods. They give absolute binding energies that differ by up to 208 kJ/mol and mean absolute deviations in the relative affinities of 10-43 kJ/mol.
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| 9. |
- Genheden, Samuel
(författare)
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Are homology models sufficiently good for free-energy simulations?
- 2012
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Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-960X .- 1549-9596. ; 52:11, s. 3013-3021
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Tidskriftsartikel (refereegranskat)abstract
- In this paper, I evaluate the usefulness of protein homology models in rigorous free-energy simulations to determine ligand affinities. Two templates were used to create models of the factor Xa protein and one template was used for dihydrofolate reductase from Plasmodium falciparum. Then, the relative free energies for several pairs of ligands were estimated using thermodynamic integration with the homology models as starting point of the simulation. These binding affinities were compared to affinities obtained when using published crystal structures as starting point of the simulations. Encouragingly, the differences between the affinities obtained when starting from either homology models or crystal structure were not statistical significant for a majority of the considered pairs of ligands. Differences between 1 and 2 kJ/mol were observed for the dihydrofolate reductase ligands and differences between 0 and 8 kJ/mol were observed for the factor Xa ligands. The largest difference for factor Xa was caused by an erroneous modeling of a loop region close to two of the ligands, and it was only observed when using one of the templates. Therefore, it is advisible to always use more than one template when creating homology models if they should be used in free-energy simulations.
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| 10. |
- Genheden, Samuel, et al.
(författare)
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Binding affinities by alchemical perturbation using QM/MM with a large QM system and polarizable MM model.
- 2015
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Ingår i: Journal of Computational Chemistry. - : Wiley. - 1096-987X .- 0192-8651. ; 36:28, s. 2114-2124
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Tidskriftsartikel (refereegranskat)abstract
- The most general way to improve the accuracy of binding-affinity calculations for protein-ligand systems is to use quantum-mechanical (QM) methods together with rigorous alchemical-perturbation (AP) methods. We explore this approach by calculating the relative binding free energy of two synthetic disaccharides binding to galectin-3 at a reasonably high QM level (dispersion-corrected density functional theory with a triple-zeta basis set) and with a sufficiently large QM system to include all short-range interactions with the ligand (744-748 atoms). The rest of the protein is treated as a collection of atomic multipoles (up to quadrupoles) and polarizabilities. Several methods for evaluating the binding free energy from the 3600 QM calculations are investigated in terms of stability and accuracy. In particular, methods using QM calculations only at the endpoints of the transformation are compared with the recently proposed non-Boltzmann Bennett acceptance ratio (NBB) method that uses QM calculations at several stages of the transformation. Unfortunately, none of the rigorous approaches give sufficient statistical precision. However, a novel approximate method, involving the direct use of QM energies in the Bennett acceptance ratio method, gives similar results as NBB but with better precision, ∼3 kJ/mol. The statistical error can be further reduced by performing a greater number of QM calculations. © 2015 Wiley Periodicals, Inc.
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