Structure and Dynamics of Coil-like Molecules by Residual Dipolar Couplings

Nuclear magnetic resonance (NMR) spectroscopy provides us with many means to study biological macromolecules in solution. Proteins in particular are the most intriguing targets for NMR studies. Protein functions are usually ascribed to specific three-dimensional structures but more recently tails, long loops and non-structural polypeptides have also been shown to be biologically active. Examples include prions, -synuclein, amylin and the NEF HIV-protein. However, conformational preferences in coil-like molecules are difficult to study by traditional methods. Residual dipolar couplings (RDCs) have opened up new opportunities; however their analysis is not trivial. Here we show how to interpret RDCs from these weakly structured molecules. The most notable residual dipolar couplings arise from steric obstruction effects. In dilute liquid crystalline media as well as in anisotropic gels polypeptides encounter nematogens. The shape of a polypeptide conformation limits the encounter with the nematogen. The most elongated conformations may come closest whereas the most compact remain furthest away. As a result there is slightly more room in the solution for the extended than for the compact conformations. This conformation-dependent concentration effect leads to a bias in the measured data. The measured values are not arithmetic averages but essentially weighted averages over conformations. The overall effect can be calculated for random flight chains and simulated for more realistic molecular models. Earlier there was an implicit thought that weakly structured or non-structural molecules would not yield to any observable residual dipolar couplings. However, in the pioneering study by Shortle and Ackerman RDCs were clearly observed. We repeated the study for urea-denatured protein at high temperature and also observed indisputably RDCs. This was very convincing to us but we could not possibly accept the proposed reason for the non-zero RDCs, namely that there would be some residual structure left in the protein that to our understanding was fully denatured. We proceeded to gain understanding via simulations and elementary experiments. In measurements we used simple homopolymers with only two labelled residues and we simulated the data to learn more about the origin of RDCs. We realized that RDCs depend on the position of the residue as well as on the length of the polypeptide. Investigations resulted in a theoretical model for RDCs from coil-like molecules. Later we extended the studies by molecular dynamics. Somewhat surprisingly the effects are small for non-structured molecules whereas the bias may be large for a small compact protein. All in all the work gave clear and unambiguous results on how to interpret RDCs as structural and dynamic parameters of weakly structured proteins.

European aspen and hybrid aspen under changing environment : Leaf traits, growth and litter decomposition

Understanding the responses of species and ecosystems to human-induced global environmental change has become a high research priority. The main aim of this thesis was to investigate how certain environmental factors that relate to global change affect European aspen (Populus tremula), a keystone species in boreal forests, and hybrid aspen (P. tremula × P. tremuloides), cultivated in commercial plantations. The main points under consideration were the acclimatization potential of aspen through changes in leaf morphology, as well as effects on growth, leaf litter chemistry and decomposition. The thesis is based on two experiments, in which young aspen (< 1 year) were exposed either to an atmospheric pollutant [elevated ozone (O3)] or variable resource availability [water, nitrogen (N)]; and two field studies, in which mature trees (> 8 years) were growing in environments exposed to multiple environmental stress factors (roadside and urban environments). The field studies included litter decomposition experiments. The results show that young aspen, especially the native European aspen, was sensitive to O3 in terms of visible leaf injuries. Elevated O3 resulted in reduced biomass allocation to roots and accelerated leaf senescence, suggesting negative effects on growth in the long term. Water and N availability modified the frost hardening of young aspen: High N supply, especially when combined with drought, postponed the development of frost hardiness, which in turn may predispose trees to early autumn frosts. This effect was more pronounced in European aspen. The field studies showed that mature aspen acclimatized to roadside and urban environments by producing more xeromorphic leaves. Leaf morphology was also observed to vary in response to interannual climatic variation, which further indicates the ability of aspen for phenotypic plasticity. Intraspecific variation was found in several of the traits measured, although intraspecific differences in response to the abiotic factors examined were generally small throughout the studies. However, some differences between clones were found in sensitivity to O3 and the roadside environment. Aspen leaf litter decomposition was retarded in the roadside environment, but only initially. By contrast, decomposition was found to be faster in the urban than the rural environment throughout the study. The higher quality of urban litter (higher in N, lower in lignin and phenolics), as well as higher temperature, N deposition and humus pH at the urban site were factors likely to promote decay. The phenotypic plasticity combined with intraspecific variation found in the studies imply that aspen has potential for withstanding environmental changes, although some global change factors, such as rising O3 levels, may adversely affect its performance. The results also suggest that the multiple environmental changes taking place in urban areas which correspond closely with the main drivers of global change can modify ecosystem functioning by promoting litter decomposition, mediated partly by alterations in leaf litter quality.

Discovery of oxidative enzymes for food engineering : Tyrosinase and sulfhydryl oxidase

Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.

Computational Methods for Studies of Quantum Dots and Boson Condensates

This thesis presents ab initio studies of two kinds of physical systems, quantum dots and bosons, using two program packages of which the bosonic one has mainly been developed by the author. The implemented models, \emph{i.e.}, configuration interaction (CI) and coupled cluster (CC) take the correlated motion of the particles into account, and provide a hierarchy of computational schemes, on top of which the exact solution, within the limit of the single-particle basis set, is obtained. The theory underlying the models is presented in some detail, in order to provide insight into the approximations made and the circumstances under which they hold. Some of the computational methods are also highlighted. In the final sections the results are summarized. The CI and CC calculations on multiexciton complexes in self-assembled semiconductor quantum dots are presented and compared, along with radiative and non-radiative transition rates. Full CI calculations on quantum rings and double quantum rings are also presented. In the latter case, experimental and theoretical results from the literature are re-examined and an alternative explanation for the reported photoluminescence spectra is found. The boson program is first applied on a fictitious model system consisting of bosonic electrons in a central Coulomb field for which CI at the singles and doubles level is found to account for almost all of the correlation energy. Finally, the boson program is employed to study Bose-Einstein condensates confined in different anisotropic trap potentials. The effects of the anisotropy on the relative correlation energy is examined, as well as the effect of varying the interaction potential.}

Timotein (Phleum pratense ssp. pratense L.) alkuperän vaikutus kylmänkestävyyteen, vernalisaatiovaatimukseen sekä kasvuun ja kehitykseen

Nurmiheinien merkitys maailmanlaajuisesti on merkittävä, sillä noin 69 % maapallon peltopinta-alasta on pysyvää laidunmaata tai niittyä. Suomessa nurmien osuus on noin 29 %, ja tuotanto perustuu pääosin intensiiviseen säilörehuntuotantoon. Yleisin nurmiheinälaji Suomessa on timotei (Phleum pratense ssp. pratense L.). Timotei on talvenkestävä ja soveltuu siksi pohjoisiin kasvuoloihin. Timoteilajikkeita jalostettaessa pohjoista alkuperää olevia vanhempaislinjoja käytetään hyvän talvenkestävyyden varmistamiseksi, eteläisiä tavoiteltaessa nopeaa kasvurytmiä. Ilmaston muutoksen ennustetaan lisäävän erilaisia äärioloja kuten myrskyjä ja sateita. Vuorokauden keskilämpötila nousee ja kasvukausi pidentyy. Lisäksi talvet muuttuvat sateisemmiksi. Muutokset näkyvät erityisesti pohjoisissa kasvuympäristöissä. Tutkimuksessa haluttiinkin selvittää eri alkuperää edustavien timoteilajikkeiden ja linjojen kylmänkestävyyttä, kasvu-, ja kehitysnopeutta sekä vernalisaation vaikutusta. Lisäksi tutkittiin syysviljojen vernalisaatiovasteen mittaamiseen käytettyjen menetelmien soveltuvuutta nurmille. Tutkimukseen kuului kaksivuotinen peltokoe sekä kasvatuskaappikoe. Vernalisaatio nopeutti timotein kasvua ja kehitystä. Tutkimuksen perusteella eteläistä alkuperää olevilla lajikkeilla kasvu- ja kukintavalmius oli olemassa ilman vernalisaatiota. Pohjoisilla lajikkeilla oli suurempi vernalisaatiovaste ja niiden kukkiminen ja kasvu nopeutui vernalisaation myötä. Vernalisaatiolla oli vaikutusta myös kasvuston rakenteeseen. Generatiivisten versojen määrä lisääntyi vernalisaation myötä, kun taas vegetatiivisten versojen määrä väheni. Kylmänkestävyys oli tutkimuksen perusteella riippuvainen syksyn karaistumisjakson pituudesta sekä jakson lämpösummasta (FH-COLD). Korkea keskilämpötila ja lyhyt karaistumisjakso heikensivät kylmänkestävyyttä. Vastaavasti karaistumiskauden lämpötilan ollessa välillä 0 °C:ta ja + 5 °C:ta ja jakson pituuden kasvaessa kylmänkestävyys lisääntyi. Tutkimuksen perusteella vernalisaatiolla oli selvä vaikutus timotein kasvuun ja kehitykseen. Pohjoista alkuperää olevat timoteit reagoivat vernalisaatioon eteläisiä enemmän. Osa pohjoisista linjoista vaati vernalisaation generatiivisten versojen muodostumiseen. Syysviljojen vernalisaatiovasteen mittausmenetelmät soveltuvat osin myös puhtaiden timoteilajikkeiden vernalisaation seurantaan.