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The high intensity of new x-ray sources such as Free Electron Lasers (FEL) offers the possibility to do single-shot molecule diffraction experiments. Even for small molecules, the dynamics induced by the radiation damage in such experiments are not yet fully understood. In particular, double core-ionized molecules are expected to be created in considerable quantity.We have therefore studied the electronic and nuclear dynamics of water molecules in single and double core ionized states by means of electronic transition rates and ab initio molecular dynamics (MD) simulations. From MD trajectories Auger transition rates were computed based on continuum electronic wavefunctions obtained by explicit integration of the coupled radial Schrodinger equations. The calculated spectra for different molecular geometries were accumulated to account for the effects of nuclear dynamics during the core-hole lifetime.In contrast to the single core ionized water molecule, we found that dissociation dynamics of double core-ionized water have strong effect on the resulting electron emission spectra. In addition, we found that the single core hole lifetime is slightly smaller than the value obtained in earlier theoretical works. Finally, we predict that the lifetime of double core ionized states is significantly lower than half of the lifetime of a single core hole.

In our work the influence of chemical modificatons and ions on RNA structure and dynamics has been tested. The effect of nucleotide modifications on E. coli and yeast tRNA in solution has been examined with a molecular dynamics approach. Simulations show a decrease of helical content in RNA secondary structure due to those modifications. In another step magnesium ion binding effects on the same tRNA were looked at by performing simulations with and without ions bound to tRNA. Ions coordinating nucleotides in those simulations show them highly affecting local secondary structure motifs. Thus the simulations performed give new hints on the function of nulceotide modifications and ion binding to RNA.

Phytochromes are photosensor proteins in plants and bacteria. The biological response is mediated by structural changes that follow photon absorption in the protein complex. The initial step is the photoisomerization of the biliverdin chromophore. How this leads to large-scale structural changes of the whole complex is, however, poorly understood. In this work, we use molecular dynamics (MD) simulations to investigate the structural changes after isomerization. In particular, we perform MD simulations at constant pH, using a recently developed method, to explore the effect of chromophore isomerization on the protonation (pKa) of nearby residues. In addition, we use a hybrid quantum mechanics/molecular mechanics approach to investigate the effect of isomerization, protonation and protein conformational changes on the absorption spectrum of the protein, for which experimental data are available. Here, we will first describe the constant pH MD simulations, and then compare the calculated spectra to experiment, and discuss the implications of our results for the photo-switching mechanism.

Fatty acids are crucial biomolecules, important for lipid metabolism, signaling, models for protocell membranes, soaps, industrial applications, and drug delivery. Oleic acid has complex phase behavior with respect to the protonation state of the carboxylic head group, which depends on the pH of the solution. Oils form at low pHs, vesicles at intermediate pHs, and micelles at high pHs. We use constant pH molecular dynamics with the MARTINI coarse-grained model to investigate oleic acid aggregates at different pH conditions. We determine titration curves for the oleic acid monomers in different aggregates, and observe a shift in the microscopic pKa. In agreement with experimental results, the pKa of a monomer in bulk water is ca. 4 and shifts to ca. 5.5 in a small micelle and ca. 8-9 in a fatty acid bilayer. There is strong anti-cooperative protonation behavior between monomers within an aggregate. This work presents a proof of concept for using constant pH simulations with the MARTINI model, and provides a physiochemical basis for the phase behavior of fatty acids in different aggregate environments.

Regulation of (Bio)chemical reactions by mechanical force has been proposed to be fundamental to cellular functions[1]. Atomic force microscopy and molecular force probe experiments suggested an enhancement on the reactivity of thiol/disulfide exchange[2] and ring-opening of cyclobutene[3], respectively. Recently, we have performed hybrid quantum mechanical molecular mechanical simulations in combination with transition path sampling on the thiol/disulfide exchange. We could show that stretching a molecule can significantly shift the transition state, and also affects degrees of freedoms other than sole bond stretching [4].In order to understand into which degrees of freedom the force goes, we have developed a force distribution analysis method for ab initio simulations, a simple scheme to deduce pairwise forces from non-pairwise quantum mechanical descriptions, which is transferable to any other (bio)chemical molecule for which internal stresses are of interest. The application to the ring-opening of cyclobutene shows how mechanical stretching forces propagate into the bonds of the reactive system, leading to both compressive and tensile forces in the strained cyclobutene. The force distribution allows to directly relate internal forces in bonds to mechanochemical events of bond scission.[1] Bustamante C, Chemla Y, Forde N, Izhaky D. Annu. Rev. Biochem. (2004) 73: 705-748.[2] Wiita AP, Ainavarapu SR, Huang HH, Fernandez JM. Proc.Natl. Acad. Sci. U.S.A. (2006) 103: 7222-7227.[3] Yang QZ, Huang Z, Kucharski TJ, Khvostichenko D, Chen J, Boulatov R. Nat. Nano. (2009) 4: 302-306[4] Li W, Graeter F. J. Amer. Chem. Soc. (2010) 132: 16790-5.

Triosephosphate Isomerase (TIM) is a glycolytic enzyme catalyzing the interconversion of Dihydroxyacetone phosphate (DHAP) to Glyceraldehyde-3-phosphate (GAP). TIM is a non-allosteric dimeric enzyme with three distinct loops (loop-6, 7 & loop-8) enveloping the active site. Loop-6 acts as a lid on the active site and residues from loop-7 and loop-8 stabilize the substrate in the active site. Depending on the orientation of loop-6 and loop-7, TIM can be classified into various conformational states. These different conformations of TIM suggest that for each task i.e. substrate binding, catalysis and product release, the protein adopts to a specific conformation suited for that particular task. Various NMR, X-ray and MM experiments have studied the conformational flexibility of loop-6 in presence or absence of natural substrates, various inhibitors and have suggested that the conformational exchange rate of loop-6 is similar to the catalytic rate of the enzyme (104/s). These studies have provided glimpses of individual events of what essentially is a dynamic process.We study the sequence of events from the binding of the ligand to the release of the product by molecular dynamics simulations. Our simulations revealed that loop-6 opens and closes in both apo and holo enzymes at microsecond time scale and also that N-termini (168P-169V-170W) and C-termini (176K-177V-178A) hinges of the loop-6 move independently which was also reported by Berlow et.al [Biochemistry 46 2007 6001]. Loop-7 on the other hand samples the closed state only when the active site is occupied by the ligand or an inhibitor. It was also observed that the conformational preference of loop-6 and loop-7 is independent of the conformation of the other.

The pKa's of the ionizable amino acids are crucial for the function of many proteins as they are key factors that determine their electrostatic potential and its spatial distribution, often controlling and optimizing enzymatic catalysis. Further, during conformational motions pKa's and protonation states particularly of histidines may change. In established force field simulation, however, this effect is typically not included, and protonation states must therefore be either guessed or derived from experiment. There have been a number of approaches to include protonation effects within simulations, mainly based on continuum electrostatics or implicit solvent molecular dynamics [1--3].However, these methods lack the effect of the hydrogen bonding and the entropy contribution that comes from the solvent. Here we present the implementation and application of a dynamic protonation atomistic simulation method with explicit solvent, which also allows for explicit solvent constant pH MD simulations, previously developed also in our group [4]. This method is used here to calculate the pKa's of the ionzable groups in proteins. In order to validate it, we selected a number of prototypic proteins and calculated titration curves and pKa values from constant pH simulations at a range of different pH values.The results compare favorably with measured values, and explain atomistically the strong deviations of some of the calculated pKa values from the solution ones.1. Lee, M. S., Salsbury, F. R., Jr., and Brooks, C. L., III (2004), Proteins 56, 738–752.2. Khandogin J, Brooks C. L., 3rd., Biophys J. 2005 Jul;89(1):141-57. Epub 2005 Apr, 29.3. Mongan, J., Case, D. A., McCammon, J. A. J., Comput. Chem. 2004, 25,2038–20484. Donnini S, Tegeler F, Groenhof G, Grubmuller H., J. Chem Theory and Comp 7: 1962–1978 (2011).

The pH is an important parameter in macromolecular systems as it determines the protonation state of ionizable groups and consequently influences the structure, dynamics and function of molecules in solution. In most force field simulation protocols, however, the protonation state of a system (rather than its pH) is kept fix and cannot adapt to changes of the local environment. Here, we present a method to perform molecular dynamics simulations in explicit solvent at constant pH. During the simulation the protonation states of titratable groups are allowed to change dynamically, and the titration curves agree with experiment. Our method is based on the lambda-dynamics approach, in which the dynamics of the titration coordinate lambda is driven by generalized forces between the protonated and deprotonated states. Constant pH simulations can be achieved by accounting for the pH dependence of the hydration free energy. As a benchmark, titration curves of amino acid analogues and a di-peptide, as well as of turkey ovomucoid inhibitor protein were calculated.

pH is an important parameter in condensed phase systems as it determines the protonation state of ionizable groups and consequently influences the structure, dynamics and function of molecules in solution. In the past ten years, few approaches have been applied to model the pH of a solution in the framework of Molecular Dynamics (MD) and Monte Carlo (MC) simulation methods. These include stochastic and mean field approximation methods to model the (de)protonation events and methods based on the lambda-dynamics approach, where the dynamics of the titration coordinate lambda is driven by the free energy gradient between the protonated and deprotonated states. In particular, the latter approach was so far limited to implicit solvent. We present here a method for constant pH simulations in explicit solvent that is based on the lambda-dynamics approach. The method has been implemented in the MD package Gromacs. The titration curve of single amino acids and small peptides and the shift in the pKa of an active site glutamic acid in the enzyme triosephosphate isomerase were correctly predicted. This preliminary tests suggest that the approach can be applied to simulate (bio)molecules with multiple titrating sites at constant pH.