| 1. |
- Allnér, Olof
(författare)
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Biomolecular simulations, from RNA to protein : thermodynamic and dynamic aspects
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The process of transforming the information stored in the DNA of genes into functional RNA molecules and proteins via transcription and translation is the most fundamental process of all known life. Even though these processes involve large macromolecules and dynamics on long time scales they all ultimately rely on atomic level interactions between nucleic acids or amino acids. Only a few experimental techniques are available that can study the large systems involved in atomic detail. Computer simulations, modeling biological macromolecules, are therefore an important tool in investigating fundamental biological processes. In this thesis, Molecular Dynamics (MD) simulations have been used to study the translation of mRNA by tRNA and the function of the regulatory riboswitches. The thesis also covers the improvement of methodology by the development of a new representation of the important Mg2+ ions and an improvement of the understanding of the connection between MD and experimental NMR data. In Paper I, the effect of post transcriptional modifications of the tRNA anti codon on the decoding of mRNA in the ribosome is studied. All atom MD simulations have been performed of the ribosomal A site with and without modifications present, including extensive free energy calculations. The results show two mechanism by which the decoding is affected: The further reach provided by the modifications allows an alternative outer conformation to be formed for the non cognate base pairs, and the modifications results in increased “catalytic” contacts between tRNA, mRNA and the ribosome. In Paper II, the folding mechanism of the add A riboswitch is studied under different ionic conditions and with and without the ligand bound. In addition to standard simulations, we simulated the unfolding by umbrella sampling of distance between the L2 and L3 loops. In the results, no significant effect of Mg2+ or Na+ ion environments or ligand presence can be seen. But a consistent mechanism with the P3 stem being more flexible than P2 is observed. More data might however be needed to draw general conclusions. In Paper III, the parameters describe Mg2+ ions in MD simulations are improved by optimizing to kinetic data of the H2O exchange. Data from NMR relaxation experiments was used as optimization goal. The newly developed parameters do not only display better kinetic properties, but also better agreement with experimental structural data. In Paper IV, the dynamical data, obtained from NMR relaxation experiment of a protein is related to dynamics seen in an MD simulation. The analysis provides important information for the interpretation of experimental data and the development of simulation methods. The results show, among other things, that significant parts of the entropy are not seen by NMR due to a limited time window and inability to account for correlation of motions.
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| 2. |
- Allnér, Olof, et al.
(författare)
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Magnesium ion–water coordination and exchange in biomolecular simulations
- 2012
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Ingår i: Journal of Chemical Theory and Computation. - Stockholm : Karolinska Institutet, Dept of Biosciences and Nutrition. - 1549-9618 .- 1549-9626.
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Tidskriftsartikel (refereegranskat)abstract
- Magnesium ions have an important role in the structure and folding mechanism of ribonucleic systems. To properly simulate these biophysical processes, the applied molecular models should reproduce, among others, the kinetic properties of the ions in water solution. Here, we have studied the kinetics of the binding of magnesium ions with water molecules and nucleic acids systems using molecular dynamics simulation in detail. We have validated the parameters used in biomolecular force fields, such as AMBER and CHARMM, for Mg2+ ions, and also for the biological relevant ions, Na+, K+ and Ca2+ together with three different water models (TIP3P, SPC/E and TIP5P). The results show that Mg2+ ions have a slower exchange rate than Na+, K+ and Ca2+ in agreement with experimental trend, but the simulated value underestimates the experimentally observed Mg2+-water exchange rate with several orders of magnitudes, irrespective of force field and water model. A new set of parameters for Mg2+ was developed to reproduce the experimental kinetic data. This set also leads to better reproduction of structural data than existing models. We have applied the new parameters set to Mg2+ binding with a mono-phosphate model system and with the purine riboswitch, add A-riboswitch. In line with the Mg2+-water results, the newly developed parameters show a better description of the structure and kinetic of the Mg2+-phosphate binding than all other models. The characterization of the ion binding to the riboswitch system shows that the new parameter set does not affect the global structure of the ribonucleic acid system or the number of ions involved in direct or indirect binding. A slight decrease in the number of water-bridged contacts between A-riboswitch and Mg2+ ion is observed. The results support the ability of the newly developed parameters to improve the kinetic description of the Mg2+ and phosphate ions and their applicability in nucleic acid simulation.
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| 3. |
- Svedendahl, Maria, 1975-, et al.
(författare)
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Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory
- 2008
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 9:15, s. 2443-2451
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Tidskriftsartikel (refereegranskat)abstract
- Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of alpha,beta-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated that the reaction proceeds through a two step-mechanism with formation of an oxyanionic intermediate. The active-site residue His224 functions as a general acid-base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.
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