A presente tese trata da síntese e a análise conformacional de seis séries de compostos: 2-feniltioN;N-dietilacetamidas 4\'-substituídas (I); 2-fenilsulfinil-N,N-dietilacetamidas 4\'-substituídas (II); 2-fenilsulfonil-N,N-dietilacetamidas 4\'-substituídas (III); 2-feniltio-cicloexanonas 4\'-substituídas (IV); 2-fenilsulfonil-cicloexanonas 4\'-substituídas (V); 2-fenilsulfinil-cicloexanonas 4\'substituídas (IV). O estudo conformacional foi realizado por intermédio das espectroscopias no IV e RMN de hidrogênio, apoiados por cálculos teóricos. A análise de vCO no infravermelho para as séries I, II e III, de forma geral, se correlaciona bem com os cálculos em HF e B3LYP os quais demonstram a ocorrência de duas conformações estáveis para os compostos da série (I), tendo o confôrmero gauche mais estável em relação ao confôrmero cis. O efeito dos substituintes da série (I), observado experimentalmente, é mais concordante com os resultados calculados em B3LYP do que em HF. Com relação à série (II), há apenas uma conformação estável, ou seja, a cis, no gás e em solventes de diferentes polaridades. A série (III) apresentou a existência de três conformações estáveis, sendo duas conformações gauche de maior estabilidade que a terceira conformação cis (no gás e no solvente de baixa polaridade: CCl4). Nas séries das cicloexanonas (IV, V e VI) a análise de vCO no infravermelho e RMN de hidrogênio apresentou a conformação axial como preferencial em CCl4, sendo que esta preferência passou para equatorial em CH3CN (CD3CN). Os cálculos ab initio (HF) reproduziram o resultado experimental em CCl4, apenas para os derivados 2-fenilsulfonil-cicloexanonas 4\'-substituídas (V). Por outro lado, para estas cicloexanonas (IV - VI) os cálculos em B3LYP estão mais concordantes com os dados experimentais. O método NBO (Natural Bond Orbital) confirmou que as conformações gauche das séries (I, II e III) são estabilizadas por pelas interações: nN/π*C=O, πC=O/σ*C-S, σC-S/π*C=O, n(S)/π*C=O, π*C=O/σ*C-S, e as séries (IV, V e VI) pelas interações πC=O/σ*C-S, σC-S/π*C=O, n(S)/π*C=O, π*C=O/σ*C-S. Em contraste, estes resultados de NBO demonstraram também a importância das interações: nO(CO)/σC-S, nO(CO)/σ*S-O e nO(CO)/σ*C-H para a estabilização da conformação cis. Adicionalmente, a análise das cargas calculadas sugeriu a participação de interações Coulombicas na estabilização das conformações cis e gauche. Para todas as séries de compostos (I - VI), os cálculos incluindo o efeito do solvente (Onsager e PCM), para alguns compostos representativos, facilitaram a atribuição de cada conformação em solução. / This thesis reports the synthesis and conformational analysis of six sedes of compounds: N,N diethyl-2-[( 4-substituted)phenylthio] acetamides (I); N,N-diethyl-2-[(4-substituted)phenylsulfinyl] acetamides (II); N,N-diethyl-2-[(4-substituted)phenylsulfonyl] acetamides (III); [4 = OMe, Me, H, Cl, Br and N02]; 2-[(4-substituted)phenylthio] cyclohexanones (IV); 2-[(4-substituted)phenylsulfonyl] cyclohexanones (V) and 2-[(4-substituted)phenylsulfinyl] cyclohexanones (VI), [4 = OMe, H and N02]. The conformational study was performed through the IR and 1H NMR spectroscopies, supported by theoretical calculations. The vCO infrared analysis for the series I, II and III in general matches well with the HF and B3LYP calculations which showed the occurrence of two stable conformations for compounds of series (I), being the gauche the most stable and the cis the second one. As for the substituent effect of series (I), the agreement between the experimental results with B3LYP is better than with HF. calculations. Considering the serries (II), there is only one stable conformation, the cis one, in gas and in solvents of different polarity. The series (III) showed the existence of three stable conformations, being the two gauche the most stable and the cis the least stable (both in the gas and in the low polarity solvent, CCl4). In the cyclohexanones series (IV, V and VI) the vCO infrared and 1H NMR analyses showed a preference for the axial conformation, in CCl4, changing the preference to equatorial one in CH3CN (CD3CN). The ab initio (HF) calculations reproduced the experimental preference, in CCl4, for the 2-[(4-substituted)phenylsulfonyl] cyclohexanones only. On the other hand, for these cyclohexanones (IV-VI) the B3LYP calculations improved the agreement with the experimental data. The NBO (Natural Bond Orbital) method confirmed that the gauche conformations of the series (I, II and III) are stabilized by: nN/π*C=O, πC=O/&#963*C-S, σC-S/π*C=O, n(S)/π*C=O, π*C=O/σ*C-S, and for the series (IV, V and VI) by the interactions: πC=O/σ*C-S, σC-S/π*C=O, N(S)/π*C=O, π*C=O/σ*C-S. In contrast these NBO results also revealed the importance of the interactions nO(CO)/σ*C-S, nO(CO)/σ*S-O and nO(CO)/σ*C-H for the stabilization of the cis conformation. Additionally, the calculated charge analysis suggested the stabilization of both cis and gauche conformations due to Coulombic interactions. The inclusion of the solvent effect in the computations (Onsager and PCM methods) for some representative compounds of series (I-VI) facilitated the conformer attribution in solution.

<p>Protein conformational transitions are fundamental to the functions of many proteins, and computational methods are valuable for elucidating the transitions that are not readily accessible by experimental techniques. Here we developed accelerated sampling methods to calculate optimized all-atom protein conformational transition paths. Adaptively biased path optimization (ABPO) is a computational simulation method to optimize the conformational transition path between two states. We first examined the transition paths of three systems with relatively simple transitions. The ways to define reduced variables were explored and transition paths were built at convergence of the optimizations. We constructed the all-atom conformational transition path between the active and the inactive states of the Src kinase domain. The C helix rotation was identified as the main free energy barrier in the all‑atom system, and the intermediate conformations and key interactions along the transition path were analyzed. This is the first demonstration of the robustness of a computational method for calculating protein conformational transitions without restraints to a specified path. We also evaluated protein‑peptide interactions using both molecular dynamics simulations and peptide docking. Long unbiased simulations were used to evaluate Src‑SSP interactions and complex stability in both implicit and explicit solvent. Molecular docking was used to build possible protein‑peptide interaction models, using both Src regulatory domain SH2 and the kinase domain. Possible Src‑SSP complexes were built as the first Src‑substrate complex structure models.</p>

Conformations of phenyl and alkyl substituted phosphines were studied by using an empirical computer program, CAMSEQ. The computer program employs potential energy functions for calculating non-bonded interactions. The three potential energy functions employed are: steric interactions, represented by Lennard-Jones 6-12 potentials; electrostatic interactions, computed from a Coulomb's Law function; and torsional barriers, approximated by a two-term cosine function. P-C and C-C torsional functions were parameterized for a variety of phosphorus and carbon substituents from published experimental data. Phenyl and alkyl substituent conformations as affected by length, Position and number of alkyl chain substituents are described and related to effective sizes of the phosphines.Ball State UniversityMuncie, IN 47306

No és difícil trobar exemples que mostrin la inqüestionable importància de la estereoquímica en temes com la salut o l'economia: D'una banda, la quiralitat és tristament ben coneguda a causa del desastre de la Talidomida. Per altra banda, varem trobar recentment un altre exemple no menys important dins el camp de les conformacions de macromolècules: La Malaltia de Creutzfeld-Jacob. Per això, creiem que és rellevant examinar amb més detall aquells temes relacionats amb els estudis conformacionals.A la present Tesi Doctoral es proposa un procés en dues etapes per a estudiar espais conformacionals de macromolècules mitjançant Simulated Annealing (SA) i Dinàmica Molecular (DM). Ambdues metodologies són ben conegudes dins el camp de la Modelització Molecular; no obstant això, la principal contribució aportada per aquest treball és el desenvolupament d'eines metodològiques millorades -descriptors moleculars adequats, anàlisi de saturació de conformacions i grau de solapament de trajectòries- per mesurar quantitativament l'evolució i convergència dels càlculs SA i MD. / No es difícil encontrar ejemplos que muestren la incuestionable importacia de la estereoquímica en temas como la salud o la economía: Por una parte, la quiralidad es tristemente bien conocida debido al desastre de la Talidomida. Por otra parte, encontramos recientemente otro ejemplo no menos importante dentro del campo de las conformaciones de macromoléculas: La Enfermedad de Creutzfeld-Jacob. Por ello, creemos que es relevante examinar más detenidamente aquellos temas relacionados con los estudios conformacionales.En la presente Tesis Doctoral se propone un proceso en 2 etapas para estudiar espacios conformacionales de macromoléculas mediante Simulated Annealing (SA) y Dinámica Molecular (DM). Ambas metodologías son bien conocidas dentro del campo de la Modelización Molecular; sin embargo la principal contribución aportada por este trabajo es el desarrollo de herramientas metodológicas mejoradas -descriptores moleculares adecuados, análisis de saturación de conformaciones y grado de solapamiento de trayectorias- para medir cuantitativamente la evolución y convergencia de los cálculos SA y MD. / It is not difficult to find examples that show the unquestionable importance of stereochemistry in human life and economy: On the one hand, chirality is unfortunately the most well known one due to the Thalidomide Disaster. On the other hand, there is a no less important example in recent years in the field of molecular conformations: the Creutzfeldt-Jakob Disease. In this sense, we think that it is worth paying more attention to conformational studies due to their indisputable relevance.A 2-stage process for studying Conformational Spaces of large macromolecules involving Simulated Annealing (SA) Conformational Search followed by series of Molecular Dynamics (MD) calculations is proposed in this PhD Thesis. Both methodologies are well-known ones in the Molecular Modelling area of knowledge; nevertheless, the main contribution made by this research work is the development of enhanced methodological techniques -suitable molecular descriptors, saturation analysis and trajectory overlapping ratio- for monitoring quantitatively how SA and MD calculations evolve.

Better understanding of protein folding process can provide physical insights on the function of proteins and makes it possible to benefit from genetic information accumulated so far. Protein folding process normally takes place in less than seconds but even seconds are beyond reach of current computational power for simulations on a system of all-atom detail. Hence, to model and explore protein folding process it is crucial to construct a proper model that can adequately describe the physical process and mechanism for the relevant time scale. We discuss the reduced off-lattice model that can express <em>&alpha;</em>-helix and <em>&beta;</em>-hairpin conformations defined solely by a given sequence in order to investigate a protein folding mechanism of conformations such as a <em>&beta;</em>-hairpin and also to investigate conformational conversions in proteins. The first two chapters introduce and review essential concepts in protein folding modelling physical interaction in proteins, various simple models, and also review computational methods, in particular, the Metropolis Monte Carlo method, its dynamic interpretation and thermodynamic Monte Carlo algorithms. Chapter 3 describes the minimalist model that represents both <em>&alpha;</em>-helix and <em>&beta;</em>-sheet conformations using simple potentials. The native conformation can be specified by the sequence without particular conformational biases to a reference state. In Chapter 4, the model is used to investigate the folding mechanism of <em>&beta;</em>-hairpins exhaustively using the dynamic Monte Carlo and a thermodynamic Monte Carlo method an effcient combination of the multicanonical Monte Carlo and the weighted histogram analysis method. We show that the major folding pathways and folding rate depend on the location of a hydrophobic. The conformational conversions between <em>&alpha;</em>-helix and <em>&beta;</em>-sheet conformations are examined in Chapter 5 and 6. First, the conformational conversion due to mutation in a non-hydrophobic system and then the conformational conversion due to mutation with a hydrophobic pair at a different position at various temperatures are examined.

The structure, potential energy functions and vibrational spectra of several cyclic and bicyclic molecules have been investigated using several spectroscopic techniques and high-level ab initio and density functional theory (DFT) calculations. Laser induced fluorescence and Raman spectroscopies were used to study the conformation of 2- indanol in the electronic ground and excited states. These, along with detailed ab initio calculations, confirmed the existence of four different stable conformations with the one undergoing an intermolecular hydrogen bonding being the most stable. A theoretical two-dimensional surface in terms of the ring-puckering and the hydroxyl group internal rotation vibrations was constructed. This work was extended to obtain preliminary insights on the conformations and ring-puckering frequencies of 3-cyclopenten-1-ol using ab initio and DFT calculations. Infrared and Raman spectra were also utilized to study the structures and vibrational spectra of -crotonolactone and 2,3-cyclopentenopyridine (pyrindan). Ab initio results showed that -crotonolactone is rigidly planar in the electronic ground state and has a nearly harmonic ring-puckering potential function. The calculated vibrational levels were shown to be in very good agreement with the experimental ring-puckering frequency from vapor-phase Raman observations. The structures, vibrational spectra, and potential energy functions of several cyclic molecules were reinvestigated using high-level ab initio computations, and detailed vibrational analyses based on DFT-B3LYP calculated frequencies were also carried out. A number of new insights were presented by re-evaluating the available experimental data for several cyclopentenes, silacyclobutanes and silacyclopentenes. It was found that the vibrational spectra of some deuterated cyclopentenes possess extensive coupling between several ring modes and other low-frequency modes. Reassignments of these spectra have been proposed. Frequencies from DFT-B3LYP calculations showed very good agreement with the experimental values for silacyclobutane and its derivatives. The presence of silicon and halogen atoms did not affect the accuracy of the DFT calculations. In addition, the ring-puckering potential energy function for silacyclopent-2-ene was studied and alternative assignments of the far-infrared results were proposed. The new assignments are in good agreement with computational results. Silacyclopent-2-ene and its -1,1-d2 isotopomer were shown to be slightly puckered with barriers of less than 50 cm-1.

Tyrosine hydroxylase (TyrH) is the first and rate-limiting enzyme of catecholamine synthetic pathway, and its regulation is critical for controlling catecholamine synthesis. The well recognized regulatory mechanisms are inhibition by catecholamine binding and re-activation upon Ser40 phosphorylation. Catecholamines bind to TyrH tightly, while phosphorylation of TyrH at Ser40 decreases the binding affinity by several hundred-fold. Regulation of TyrH is accompanied by conformational changes of the protein. This study focuses on the identification of the conformational changes of TyrH upon dopamine binding and Ser40 phosphorylation, using hydrogen deuterium exchange mass spectrometry (HDMS) and fluorescence spectroscopy. HDMS identifies three peptides undergoing conformational changes upon dopamine binding, peptide 35-41, 42-71 and 295-299. Peptides 35-41 and 42-71 are on the regulatory domain, while peptide 295-299 is at the active site entrance. Upon dopamine binding, all three peptides are protected from exchange; phosphorylation of TyrH at Ser40 has opposite effects on the exchange kinetics of peptide 295-299, but peptides 35-41 and 42-71 could not be detected by MS after phosphorylation. This suggests that the structural effects of dopamine binding and Ser40 phosphorylation are opposite. The fluorescence spectroscopy of mutant enzymes containing a single tryptophan at position 14, 34 or 74 was performed before and after phosphorylation. F34W/F3W TyrH has a significant decrease in steady-state fluorescence anisotropy, an increase in the bimolecular quenching rate constant kq and dynamic anisotropy upon phosphorylation at Ser40, while F14W/F3W TyrH and F74W/F3W TyrH exhibit much smaller differences. This suggests that phosphorylation of TyrH at Ser40 increases the flexibility of the regulatory domain. The results are consistent with TyrH existing in two conformations, a closed conformation stabilized by dopamine in which the N-terminal regulator domain of TyrH covers the active site entrance and an open conformation stabilized by phosphorylation in which the regulatory domain has moved away from the active site entrance.

Conformational ensembles are models of proteins that capture variations in conformation that result from thermal fluctuations. Ensemble based models are important tools for studying Intrinsically Disordered Proteins (IDPs), which adopt a heterogeneous set of conformations in solution. In order to construct an ensemble that provides an accurate model for a protein, one must identify a set of conformations, and their relative stabilities, that agree with experimental data. Inferring the characteristics of an ensemble for an IDP is a problem plagued by degeneracy; that is, one can typically construct many different ensembles that agree with any given set of experimental measurements. In light of this problem, this thesis will introduce three tools for characterizing ensembles: (1) an algorithm for modeling ensembles that provides estimates for the uncertainty in the resulting model, (2) a fast algorithm for constructing ensembles for large or complex IDPs and (3) a measure of the degree of disorder in an ensemble. Our hypothesis is that a protein can be accurately modeled as an ensemble only when the degeneracy of the model is appropriately accounted for. We demonstrate these methods by constructing ensembles for K18 tau protein, \(\alpha\)-synuclein and amyloid beta - IDPs that are implicated in the pathogenesis of Alzheimer's and Parkinson's diseases.

Investigation and control of alkylsilane stationary phase structure in reversed phase liquid chromatography is presented. Raman spectroscopy is used to probe the alkyl chain conformational order and interchain coupling as a function of various chromatographic conditions. A new method is further developed to fabricate alkylsilane stationary phases with controlled surface coverage. The alkyl chain conformational order and interchain coupling of a series of high-density docosylsilane (C22) bonded stationary phases is shown as a function of temperature, surface coverage, polymerization method, common solvents and solutes. The conformational order of C22 stationary phases is compared to that of octadecylsilane (C18) stationary phases to understand the chain length effect on stationary phase structure. The conformational order information as indicated by Raman spectral order indicators for a C22 phase are correlated with the capacity factor and separation efficiency for each solute studied to gain insight into the retention mechanism. These studies help to understand the origin of stationary phase shape selectivity and the separation process in general. Based on these results, the molecular pictures at the stationary phase/solvent interface are proposed. The effect of pressurized solvent environments on two C18 phases is studied to obtain direct evidence for changes in stationary phase structure due to pressure. These changes are compared to effects of solvation relative to air in the same solvents. In addition, Raman spectral order indicators are identified for perdeuterated alkyl-containing system. This study provides a foundation for studying stationary phase structure in complex systems comprised of long alkyl-containing solutes.A further development of a new method is presented as well for synthesizing alkylsilane stationary phases with precisely controlled surface coverage by using a displaceable surface template monolayer of n-alcohol. A mechanism for this process is proposed based on the studies of n-alcohol concentration and chain length effect on the stationary phase surface coverage. The utility of these new stationary phases as chromatographic support is demonstrated. The shape selectivity for these new phases is comparable to or better than similar phases prepared by conventional methods.

The focus of this thesis is to examine the structural properties of polysaccharides produced by bacteria, as well as the dynamic and conformational behavior of a synthetically derived oligosaccharide. The primary structures of the O-polysaccharide repeating units of four different Escherichia coli (E. coli) strains, namely O175, O177, O103 and TD2158, as well as the first report of a capsular polysaccharide produced by lactic acid bacteria Leuconostoc mesenteroides ssp. cremoris PIA2 are reported in paper I–V. Structural analyses have been performed using a combination of nuclear magnetic resonance spectroscopy and chemical component analysis. The elucidated structures in paper I–III, as well as paper V, are composed of linear repeating units of varying composition and length. In paper IV, the structure of the O-polysaccharide repeating unit of E. coli TD2158 is determined to be a branched hexasaccharide structure with a heterogeneous substitution pattern, with either a β-GlcpNAc or β-Glcp residue branching to the backbone chain. Incubation with bacteriophage HK620 tailspike protein shows that the polysaccharide is selectively cleaved at the α-GlcpNAc-(1→2)-α-Rhap-linkage of the backbone chain, yielding a 9:1 ratio of β-GlcpNAc/β-Glcp containing hexasaccharides after digestion. In paper VI the conformational properties of a trisaccharide, which constitutes an internal epitope of the LeaLex hexasaccharide over-expressed on the surface of squamous lung cancer cells, have been analyzed using NMR spectroscopy and molecular dynamics simulations. The β-(1→3)-linkage of the trisaccharide was shown to be highly flexible. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Submitted. Paper 6: Submitted.</p>