| 1. |
- Lind, Christoffer, et al.
(författare)
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Codon-reading specificities of mitochondrial release factors and translation termination at non-standard stop codons
- 2013
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- A key feature of mitochondrial translation is the reduced number of transfer RNAs and reassignment of codons. For human mitochondria, a major unresolved problem is how the set of stop codons are decoded by the release factors mtRF1a and mtRF1. Here we present three-dimensional structural models of human mtRF1a and mtRF1 based on their homology to bacterial RF1 in the codon recognition domain, and the strong conservation between mitochondrial and bacterial ribosomal RNA in the decoding region. Sequence changes in the less homologous mtRF1 appear to be correlated with specific features of the mitochondrial rRNA. Extensive computer simulations of the complexes with the ribosomal decoding site show that both mitochondrial factors have similar specificities and that neither reads the putative vertebrate stop codons AGA and AGG. Instead, we present a structural model for a mechanism by which the ICT1 protein causes termination by sensing the presence of these codons in the A-site of stalled ribosomes.
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| 2. |
- Mishra, Sushil Kumar, et al.
(författare)
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Computational prediction of monosaccharide binding free energies to lectins with linear interaction energy models
- 2012
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Ingår i: Journal of Computational Chemistry. - : Wiley. - 0192-8651 .- 1096-987X. ; 33:29, s. 2340-2350
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Tidskriftsartikel (refereegranskat)abstract
- The linear interaction energy (LIE) method to compute binding free energies is applied to lectin-monosaccharide complexes. Here, we calculate the binding free energies of monosaccharides to the Ralstonia solanacearum lectin (RSL) and the Pseudomonas aeruginosa lectin-II (PA-IIL). The standard LIE model performs very well for RSL, whereas the PA-IIL system, where ligand binding involves two calcium ions, presents a major challenge. To overcome this, we explore a new variant of the LIE model, where ligandmetal ion interactions are scaled separately. This model also predicts the saccharide binding preference of PA-IIL on mutation of the receptor, which may be useful for protein engineering of lectins.
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| 3. |
- Satpati, Priyadarshi, et al.
(författare)
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Structure-Based Energetics of mRNA Decoding on the Ribosome
- 2014
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 53:10, s. 1714-1722
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Tidskriftsartikel (refereegranskat)abstract
- The origin of high fidelity in bacterial protein synthesis on the ribosome remains a fundamental unsolved problem despite available three-dimensional structures of different stages of the translation process. However, these structures open up the possibility of directly computing the energetics of tRNA selection that is required for an authentic understanding of fidelity in decoding. Here, we report extensive computer simulations that allow us to quantitatively calculate tRNA discrimination and uncover the energetics underlying accuracy in code translation. We show that the tRNA-mRNA interaction energetics varies drastically along the path from initial selection to peptide bond formation. While the selection process is obviously controlled by kinetics, the underlying thermodynamics explains the origin of the high degree of accuracy. The existence of both low- and high-selectivity states provides an efficient mechanism for initial selection and proofreading that does not require codon-dependent long-range structural signaling within the ribosome. It is instead the distinctly unequal population of the high-selectivity states for cognate and noncognate substrates that is the key discriminatory factor. The simulations reveal the essential roles played both by the 30S subunit conformational switch and by the common tRNA modification at position 37 in amplifying the accuracy.
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| 4. |
- Sund, Johan, 1980-
(författare)
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From Structure to Function with Binding Free Energy Calculations for Codon Reading, Riboswitches and Lectins
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Molecular association is part of many important processes in living cells. Computational methods for calculating binding free energies allows for a quantitative examination of biomolecular structures and hypotheses drawn from biochemical experiments. Here, binding free energy calculations for tRNAs and release factors binding to mRNA codons on the ribosome, sugars binding to lectins and purine analogs binding to the purine riboswitch are presented.The relative affinities between cognate and non-cognate tRNAs for different states involved in codon reading on the ribosome were determined. The calculations show that tRNA discrimination varies between different conformations of the 30S subunit, where the existence of both low and high selectivity states provides an efficient common mechanism for initial selection and proofreading. The simulations reveal a desolvation mechanism for the 30S conformational switch with which the accuracy of peptide bond formation can be amplified.When an mRNA stop codon (UAA, UAG or UGA) is located in the ribosomal A-site release factors bind to the ribosome and the synthesized protein is released. RF1 is specific for UAA and UAG whereas RF2 is specific for UAA and UGA. The free energy calculations and an analysis of the performed simulations show the mechanisms for how RF1 and RF2 are able to read the stop codons with different specificities. Also mitochondrial release factors were investigated. Vertebrate mitochondria have four stop codons, UAA, UAG, AGA and AGG and two release factors mtRF1 and mtRF1a. The calculations show how the specificities of both mtRF1 and mtRF1a agree with RF1 and that none of them are likely to read the non-standard stop codons AGA and AGG.The linear interaction energy method has also been examined for the RSL and PA-IIL lectins and for the purine riboswitch. The standard parameterization of the method works well for RSL, but fails for PA-IIL and the purine riboswitch due to compositions of the active sites in these systems. The development of new parameterizations to overcome these problems leads to a better understanding of both the method and the binding mechanisms in these systems.
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| 5. |
- Sund, Johan, et al.
(författare)
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Principles of stop-codon reading on the ribosome
- 2010
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 465:7300, s. 947-U12
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Tidskriftsartikel (refereegranskat)abstract
- In termination of protein synthesis, the bacterial release factors RF1 and RF2 bind to the ribosome through specific recognition of messenger RNA stop codons and trigger hydrolysis of the bond between the nascent polypeptide and the transfer RNA at the peptidyl-tRNA site, thereby releasing the newly synthesized protein. The release factors are highly specific for a U in the first stop-codon position 1 and recognize different combinations of purines in the second and third positions, with RF1 reading UAA and UAG and RF2 reading UAA and UGA. With recently determined crystal structures of termination complexes(2-4), it has become possible to decipher the energetics of stop-codon reading by computational analysis and to clarify the origin of the high release-factor binding accuracy. Here we report molecular dynamics free-energy calculations on different cognate and non-cognate termination complexes. The simulations quantitatively explain the basic principles of decoding in all three codon positions and reveal the key elements responsible for specificity of the release factors. The overall reading mechanism involves hitherto unidentified interactions and recognition switches that cannot be described in terms of a tripeptide anticodon model. Further simulations of complexes with tRNA(Trp), the tRNA recognizing the triplet codon for Trp, explain the observation of a 'leaky' stop codon 5 and highlight the fundamentally different third position reading by RF2, which leads to a high stop-codon specificity with strong discrimination against the Trp codon. The simulations clearly illustrate the versatility of codon reading by protein, which goes far beyond tRNA mimicry.
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| 6. |
- Svebrant, Johan, et al.
(författare)
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Fire safety in military vehicles evaluation of ISO 3795
- 2013
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Ingår i: Fire and Materials 2013 - 13th International Conference and Exhibition, Conference Proceedings. ; , s. 551-562
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Konferensbidrag (refereegranskat)abstract
- It has been shown in previous studies that the current standard used to fire test interior materials in civilian and military vehicles, ISO 3795 (FMVSS 302), has led to a low level of fire safety of interior materials in civilian vehicles. The purpose of this study was to evaluate the fire safety of interior materials in military vehicles. The aim was to suggest a fire test method to be used on interior materials in military vehicles, which correlate better to full scale fires. The aim was also to perform fire tests of a number of interior materials, and evaluate these. Eleven different interior materials from two military vehicles were tested in the cone calorimeter at 25 and 50 kW/m 2. Time to ignition was found to be short and the heat release rate to be high. The materials were evaluated according to criteria used for trains, high speed crafts and submarines. Only one material passed, but only for use on trains and only according to the criterion for 25 kW/m2. All other materials failed. A comparison was also made with materials from cars and buses, which have been tested in previous studies. It was found that materials from military vehicles ignite just as fast, or faster, compared to the results from previous studies on car and bus interior for civil purposes. The rate of heat release rate was however lower. It was found that ISO 3795 is not a suitable method to test interior materials in military vehicles and its use has led to a low level of fire safety of interior materials in military vehicles. A different test method to be used for fire testing interior materials in military vehicles is suggested; ISO 5660 cone calorimeter.
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| 7. |
- Boukharta, Lars, 1979-
(författare)
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Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Accurate predictions of binding free energies from computer simulations are an invaluable resource for understanding biochemical processes and drug action. The primary aim of the work described in the thesis was to predict and understand ligand binding to several proteins of major pharmaceutical importance using computational methods.We report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 G-protein coupled receptor and a series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Site-directed mutagenesis, homology modelling and docking were further used to characterize agonist binding to the human neuropeptide Y2 receptor, which is important in feeding behavior and an obesity drug target. In a separate project, homology modelling was also used for rationalization of mutagenesis data for an integron integrase involved in antibiotic resistance.Blockade of the hERG potassium channel by various drug-like compounds, potentially causing serious cardiac side effects, is a major problem in drug development. We have used a homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations are in good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships with implications for design of new compounds. Docking, scoring, molecular dynamics, and the linear interaction energy method were also used to predict binding modes and affinities for a large set of inhibitors to HIV-1 reverse transcriptase. Good agreement with experiment was found and the work provides a validation of the methodology as a powerful tool in structure-based drug design. It is also easily scalable for higher throughput of compounds.
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| 8. |
- Boukharta, Lars, et al.
(författare)
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Computational prediction of alanine scanning and ligand binding energetics in G-protein coupled receptors
- 2014
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Ingår i: PloS Computational Biology. - : Public Library of Science (PLoS). - 1553-734X .- 1553-7358. ; 10:4, s. e1003585-
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Tidskriftsartikel (refereegranskat)abstract
- Site-directed mutagenesis combined with binding affinity measurements is widely used to probe the nature of ligand interactions with GPCRs. Such experiments, as well as structure-activity relationships for series of ligands, are usually interpreted with computationally derived models of ligand binding modes. However, systematic approaches for accurate calculations of the corresponding binding free energies are still lacking. Here, we report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 receptor and series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Author Summary G-protein coupled receptors constitute a family of drug targets of outstanding interest, with more than 30% of the marketed drugs targeting a GPCR. The combination of site-directed mutagenesis, biochemical experiments and computationally generated 3D structural models has traditionally been used to investigate these receptors. The increasing number of GPCR crystal structures now paves the way for detailed characterization of receptor-ligand interactions and energetics using advanced computer simulations. Here, we present an accurate computational scheme to predict and interpret the effects of alanine scanning experiments, based on molecular dynamics free energy simulations. We apply the technique to antagonist binding to the neuropeptide Y receptor Y1, the structure of which is still unknown. A structural model of a Y1-antagonist complex was derived and used as starting point for computational characterization of the effects on binding of alanine substitutions at thirteen different receptor positions. Further, we used the model and computational scheme to predict the binding of a series of seven antagonist analogs. The results are in excellent agreement with available experimental data and provide validation of both the methodology and structural models of the complexes.
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| 9. |
- Boukharta, Lars, et al.
(författare)
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Computer Simulations of Structure-Activity Relationships for hERG Channel Blockers
- 2011
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 50:27, s. 6146-6156
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Tidskriftsartikel (refereegranskat)abstract
- The hERG potassium channel is of major pharmaceutical importance, and its blockade by various compounds, potentially causing serious cardiac side effects, is a major problem in drug development. Despite the large amounts of existing biochemical data on blockade of hERG by drugs and druglike compounds, relatively little is known regarding the structural basis of binding of blockers to the channel. Here, we have used a recently developed homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations yield a remarkably good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships in terms of a number of key interactions. Two main interaction regions of the channel are thus identified with implications for further mutagenesis experiments and design of new compounds.
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| 10. |
- Diaz, Lucia, et al.
(författare)
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Computational Prediction of Structure-Activity Relationships for the Binding of Aminocyclitols to beta-Glucocerebrosidase
- 2011
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Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 51:3, s. 601-611
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Tidskriftsartikel (refereegranskat)abstract
- Glucocerebrosidase (GCase, acid beta-Glucosidase) hydrolyzes the sphingolipid glucosylceramide into glucose and ceramide. Mutations in this enzyme lead to a lipid metabolism disorder known as Gaucher disease. The design of competitive inhibitors of GCase is a promising field of research for the design of pharmacological chaperones as new therapeutic agents. Using a series of recently reported molecules with experimental binding affinities for GCase in the nanomolar to micromolar range, we here report an extensive theoretical analysis of their binding mode. On the basis of molecular docking, molecular dynamics, and binding free energy calculations using the linear interaction energy method (LIE), we provide details on the molecular interactions supporting ligand binding in the different families of compounds. The applicability of other computational approaches, such as the COMBINE methodology, is also investigated. The results show the robustness of the standard parametrization of the LIE method, which reproduces the experimental affinities with a mean unsigned error of 0.7 kcal/mol. Several structure activity relationships are established using the computational models here provided, including the identification of hot spot residues in the binding site. The models derived are envisaged as important tools in ligand-design programs for GCase inhibitors.
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