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In this work, the development of parameters for a series of imidazolium-based ionic liquids molecules, now included in the AMOEBA force field, is discussed. The quality of obtained parameters is tested in a variety of calculations to reproduce structural, thermodynamic, and transport properties. First, it is proposed a novel method to parameterize in a faster, and more efficient way parameters for the AMOEBA force field that can be applied to any imidazolim-based cation. Second, AMOEBA-IL polarizable force field is applied to study the N-tert-butyloxycarbonylation of aniline reaction mechanism in water/[EMIM][BF4] solvent via QM/MM approach and compared with the reaction carried out in gas-phase and implicit solvent media. Third, AMOEBA-IL force field is applied in alchemical calculations. Free energies of solvation for selected solutes solvated in [EMIm][OTf] are calculated via BAR method implemented in TINKER considering the effect of polarization as well as the methodology to perform the sampling of the alchemical process. Finally, QM/MM calculations using AMOEBA to get more insights into the catalytic reaction mechanism of horseradish peroxidase enzyme, particularly the structures involved in the transition from Cp I to Cp II.

Biochemical reactions make up most of the activity in a cell. There is inherent stochasticity in the kinetic behavior of biochemical reactions which in turn governs the fate of various cellular processes. In this work, the precision of a method for dimensionality reduction for stochastic modeling of biochemical reactions is evaluated. Further, a method of stochastic simulation of reaction kinetics is implemented in case of a specific biochemical network involved in maintenance of long-term potentiation (LTP), the basic substrate for learning and memory formation. The dimensionality reduction method diverges significantly from a full stochastic model in prediction the variance of the fluctuations. The application of the stochastic simulation method to LTP modeling was used to find qualitative dependence of stochastic fluctuations on reaction volume and model parameters. / text

Dehydroquinate synthase (DHQS) catalyses the five-step transformation of the seven carbon sugar 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) to the carbacycle dehydroquinate (DHQ). Multiple studies have described in detail the mechanism of most of the steps carried out by DHQS with the exception of the final cyclisation step. In this study, (3S)-3-fluoro-DAH7P and (3R)-3-fluoro-DAH7P (fluorinated analogues of DAH7P) were produced and assayed across three phylogenetically distinct sources of DHQS in order to determine the role of the enzyme during the cyclisation step of the reaction. Incubation of (3S)-3-fluoro-DAH7P with DHQS from Escherichia coli, Pyrococcus furiosus, and Kiwifruit resulted in the production of different ratios of (6S)-6-fluoro-DHQ and 1-epi-(6S)-6-fluoro-DHQ for each enzyme. In addition, enzyme catalysis showed a slowing of reaction rates when (3S)-3-fluoro-DAH7P was used, suggesting that the fluorine at C-3 is stabilising the enol pyranose. An increase in the stabilisation of the fluoro-enol pyranose would allow release of this substrate intermediate from the enzyme to compete with the on-going on-enzyme reaction. The differences in the ratio of products formed suggest that the cyclisation occurs in part on the enzyme and that the epimeric product arises only by an abortive reaction pathway where the (3S)-3-fluoro-enol pyranose is prematurely released and allowed to cyclise free in solution. Once in solution, the (3S)-3-fluoro-enol pyranose could undergo a conformational change in the ring leading to the formation of the epimeric product. Furthermore, it is suspected that the position of fluorine influences the likely transition-state in carbacycle formation leading to the production of the epimeric product. This research has illuminated the role of the enzyme in guiding the correct stereochemistry of the product and illustrates the important molecular interplay between the enzyme and substrate.

Abstract The myelin sheath is a crucial component of vertebrate nervous systems. Myelin is formed as the plasma membrane of a glial cell is wrapped around a neuronal axon. The presence of myelin enables the fast transmission of neuronal impulses, and degradation or dysfunction of myelin results in severe neurological symptoms. Molecular composition of myelin is unique, and many myelin proteins are not present elsewhere in the body. A myelin enzyme, 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), is found in specific regions within the myelin sheath and is one of the most abundant proteins in the brain. Substrates for CNPase catalytic activity are formed during brain damage. CNPase also interacts with the cytoskeleton and cell membranes, and it is thought to play a role during myelin formation. Mice that lack CNPase suffer from axonal degeneration and die early. The aim of this study was to characterise CNPase structure and function. To this end, a system was first developed to produce the protein for subsequent analyses. The aim was to characterise the catalytic mechanism of CNPase by determining its three-dimensional molecular structure at different stages of the catalytic reaction. The interactions between CNPase and other molecules related to its function would also be characterised. Finally, the structure of the full-length protein would be used to understand of the function of the uncharacterised N-terminal domain. Using X-ray crystallography, the structure of the CNPase catalytic domain was determined in the presence of substrate and product molecules. These data, complemented with analyses of mutationally inactivated enzyme variants, were used to examine the catalytic reaction at the molecular level. The catalytic domain structure was compared to homologous enzymes from diverse organisms. The interaction between CNPase and the calcium-sensing protein calmodulin was characterised. The solution structure of full-length CNPase was determined using small-angle X-ray scattering, and protein sequence databases were utilised to determine CNPase conservation during animal evolution. The results provide novel information on the catalytic activity and overall function of CNPase. Further studies will be necessary to determine its specific role, but it is increasingly clear that CNPase can perform multiple important tasks within the nervous system. / Tiivistelmä Myeliinituppi on tärkeä osa selkärankaisten hermostoa. Myeliiniä muodostuu, kun gliasolun solukalvo kiertyy hermosolun aksonin ympärille. Myeliini mahdollistaa hermoimpulssien nopean välityksen, ja sen tuhoutuminen ja vajaatoiminta aiheuttavat vakavia neurologisia oireita. Myeliinin molekyylikoostumus on ainutlaatuinen, ja monet myeliiniproteiineista eivät esiinny muualla elimistössä. Myeliinissä esiintyvää entsyymiä, 2′,3′-syklisten nukleotidien 3′-fosfodiesteraasia (CNPaasi), esiintyy runsaasti tietyillä myeliinialueilla, ja se on yksi aivojen runsaslukuisimmista proteiineista. Substraatteja CNPaasin katalyyttiselle aktiivisuudelle muodostuu aivovaurion aikana. CNPaasi on myös vuorovaikutuksessa solun tukirangan ja solukalvon kanssa, ja sen uskotaan vaikuttavan myeliinin muodostumiseen. Hiiret, joilta puuttuu CNPaasi, kärsivät aksonien rappeumista ja kuolevat ennenaikaisesti. Tämän tutkimuksen tavoite oli karakterisoida CNPaasin rakennetta ja toimintaa. Tätä tarkoitusta varten ensin kehitettiin menetelmä analysoitavan proteiinin tuottamiseksi. Tavoitteena oli karakterisoida CNPaasin katalyyttinen mekanismi määrittämällä sen kolmiulotteinen molekyylirakenne katalyysireaktion eri vaiheissa. Myös CNPaasin vuorovaikutuksia sen toimintaan liittyvien molekyylien kanssa tutkittiin. Lopuksi kokopitkän proteiinin rakenteen avulla selvitettiin karakterisoimattoman aminoterminaalisen alayksikön toimintaa. CNPaasin katalyyttisen alayksikön rakenne määritettiin käyttäen röntgenkristallografiaa substraatti- ja tuotemolekyylien läsnäollessa. Rakennetta, täydennettynä mutaatioilla inaktivoitujen entsyymimuunnosten analyysillä, käytettiin katalyyttisen reaktion molekyylitason karakterisointiin. Katalyyttisen alayksikön rakennetta verrattiin eri organismeissa esiintyviin homologisiin entsyymeihin. CNPaasin ja kalsiumia sitovan kalmoduliinin vuorovaikutusta karakterisoitiin. Kokopitkän CNPaasin liuosrakenne selvitettiin pienkulmaröntgensironnan avulla, ja CNPaasin sekvenssin säilymistä eläinten evoluution aikana tarkasteltiin proteiinisekvenssitietokantoja käyttämällä. Tulokset antavat uutta tietoa CNPaasin katalyyttisestä aktiivisuudesta ja tämän arvoituksellisen entsyymin toiminnasta. Jatkotutkimukset ovat tarpeen sen täsmällisen roolin selvittämiseksi, mutta on kasvavassa määrin selvää, että CNPaasi pystyy suorittamaan useita tärkeitä tehtäviä hermostossa.