The sensitivity of biological studies performed between 190 and 650 nm is greatly reduced due to the autofluorescence of biomolecules and impurities in this region. Therefore, the enhanced signal-to-noise ratios encountered at longer wavelengths makes biological analysis within the near infrared (NIR) region from 650 nm to 1100 nm far more advantageous. This dissertation describes the noncovalent binding interactions of near-infrared (NIR) carbocyanine dyes with human serum albumin (HSA) and poly-L-lysine (PLL) using UV-Vis/NIR absorption spectroscopy, emission spectroscopy, circular dichroism (CD), and fluorescence detected circular dichroism (FDCD). The optical spectroscopy methods used in this work are described in detail in Chapter 1. The various applications of NIR dyes in protein analysis are introduced in Chapter 2. In general, the sensitivity of cyanines to the polarity of their local environment makes them quite suitable for protein labeling schemes. In aqueous media, cyanines have a high propensity for self-association. Yet in the hydrophobic binding sites of globular proteins, these aggregates often dissipate. Absorption and emission spectroscopy can be utilized to observe the differential spectral properties of monomer, intra-molecular and intermolecular aggregates. In Chapter 3, the photophysical properties of bis(cyanine) NIR dyes containing di-, tri-, and tetraethylene glycol linkers were each examined in the presence of HSA are discussed. Variations in chain length as well as probe flexibility were demonstrated through distinct differences in absorption and emission spectra. The observed changes in the spectral properties of the NIR dyes in the presence and absence of HSA were correlated to the physical parameters of the probes' local environment (i.e., protein binding sites and self-association). All three bis-cyanines examined exhibited enhanced fluorescence in the presence of HSA. The bis-cyanine dye containing the tri(ethylene glycol) spacer allowed for a complete overlap of the benzene rings, to form π-π interactions which were observed as intra-molecular H-aggregate bands. The dye exhibited no fluorescence in buffer, owing to the H-aggregation observed in the absorption data. In the presence of HSA, the intra-molecular dimers were disrupted and fluorescence was then detected. The "cut-on" fluorescence displayed by the dye in the presence of HSA made it ideal for noncovalent labeling applications. The utility of several NIR dyes for use as secondary structural probes was investigated in Chapter 4. NIR dyes were screened thoroughly using UV-Vis/NIR absorption spectroscopy dyes with spectral properties which were sensitive to protein secondary structure models of such as PLL in basic solution. Two NIR dyes were found to be quite sensitive to the structural features of uncharged α- and β-PLL. The chiral discrimination of these probes for basic protein secondary structures was also evaluated through CD measurements within the NIR probes' absorption bands.

In quantum chemistry, scientists aim to solve the time-independent Schrödinger equation by employing a variety of approximation techniques whose accuracy are typically inversely proportional to their computational cost. This problem is amplified when it comes to chiral molecules, whose stereochemical assignments and associated chiroptical properties can be incredibly sensitive to small changes in their three-dimensional structure, requiring highly accurate theoretical methods. On the other hand, due to the polynomial scaling with system size, it is sometimes impractical to apply such methods to chemical compounds of broad scientific interest, especially when a multitude of low-energy conformations have to be accounted for as well. As a result, the assignment of absolute configurations to chiral compounds remains a tedious task. However, the characterization of these compounds is something that many different scientists are significantly invested in. The ultimate goal, then, is twofold: to gain useful insight by utilizing the electronic structure methods at your disposal while simultaneously developing new approximation techniques that can be used to push the boundaries on what is currently capable in computational chemistry. Therefore, we start by applying widely accepted density functional theory methods to predict optical rotations and electronic circular dichroism for naturally occurring antiplasmodial and anticancer drug candidates. We find that by comparing the computational results directly with those obtained through experimental measurement, we can provide reliable absolute config- uraitonal assignments to a variety of chiral compounds with numerous stereogenic centers. We also present the first ever prediction of vibrational circular dichroism with second-order Møller-Plesset perturbation theory. This extension opens the door to systematically improvable correlated wave function methods that can be employed when density functional theory fails or when higher accuracy results are required. / Doctor of Philosophy / Theoretical chemistry aims to draw a line from a molecule's three-dimensional structure to a set of physical observables, allowing for the efficient prediction of such properties. One family of chemical compounds for which this task becomes increasingly difficult is known as chiral molecules. A chiral compound is defined as one that has a non-superimposable mirror image. The concept of chirality is most tangibly seen with a pair of human hands, which demonstrate this same mirror-like behavior. In the same way that a person has left and right hands, a three-dimensonal handedness can be used to characterize many compounds that are essential to life including enzymes, sugars, and proteins. Although procedures have been developed to consistently isolate pure samples of such compounds, the correct identification of each hand poses a much larger task and costs the global pharmaceutical industry tens to hundreds of millions of dollars every year. As such, gaining insight about these incredibly valuable compounds and their associated properties is a never ending goal for many scientists. One such way to gain insight is through the direct comparison of experimental and calculated properties, namely chiroptical properties. These unique properties define how chiral compounds interact with light. While experimental scientists are limited in the degree to which they can probe a molecule's structure, theoretical chemists have the advantage of knowing the exact three-dimensional structure for the compound they are studying. On the other hand, theoretical chemists rely on comparison to experimental results to develop new methods or apply the available techniques to predict molecular properties. This work begins by attempting to match calculated properties to experimentally measured ones in order to confirm the detailed molecular structure of natural product drug candidates. Through multiple such computational studies, it is shown that the current methods are sometimes limited in the knowledge that they can provide. As a result, it is absolutely necessary to continue to improve on the existing methods. We go on to provide a first-of-its-kind implementation that allows for theoretical chemists to compare their results to experiment in a way that was not previously possible.

Title: Magnetic circular dichroism and aromatic compounds Author: Petr Štěpánek Department/Institute: Institute of Organic Chemistry and Biochemistry AS CR, v.v.i. Supervisor: prof. RNDr. Petr Bouř, DSc., Institute of Organic Chemistry and Biochemistry AS CR, v.v.i. Abstract: The thesis presents a series of studies concerning magnetic circular dichroism (MCD), a spectroscopic method, which experienced an intense theo- retical development in the recent years. New computational codes opened possi- bilities to calculate MCD spectra of larger and more varied molecules than was possible in the past. In the presented studies, we took the advantage of the new computational codes to broaden the possible span of applications of the MCD technique. As an example, we present MCD as a method useful for obtaining information about the structure of fullerenes. We also studied the influence of the molecular conformation and the explicit and implicit solvent models on the MCD spectra of aromatic amino acids using the newly implemented alterna- tive computational protocol based on sum-over-states calculations. We have also theoretically predicted spectra of the nuclear spin circular dichroism (NSCD), a potential new high-resolution spectroscopy. Keywords: magnetic circular dichroism, quantum-chemical calculations, density...

The enzyme inosine triphosphate pyrophosphatase (ITPase) is responsible for controlling the levels of the by-products guanosine monophosphate (GMP) and adenosine monophosphate (AMP) through their precursor inosine monophosphate (IMP). ). Human ITPase consists of a 194-amino acid homodimer which relies upon either an Mg2+ ion or a Mn2+ ion for catalytic activity, and orthologs of this protein have been found in many different organisms. The purpose of this project was to try out methods learned throughout the education and to use this knowledge to gather new data about the human protein inosine triphosphate pyrophosphatase (ITPase). The protein was expressed in BL21/DE3 cells from a pre-made vector. Experiments performed during this project include secondary- and tertiary stability measurements, tryptophan fluorescence spectra, binding curve and thermic stability to ITPase with ANS and methotrexate. The Tm-value of human ITPase was examined with Trp-Fluorescence, ANS-fluorescence and Near-UV and Far-UV circular dichroism (CD). The stability of ITPase monitored by Near-UV as well as Far-UV coincides, indicating that secondary- and tertiary-unfolding occur simultaneously without any intermediates. The results of Trp-fluorescence showed that the tryptophans were already exposed and thus it did not yield a reliable result. The binding properties of ANS and MTX to ITPase were also examined.

In the present work the vacuum polarization and the circular dichroism of hydrogen-like atoms are studied. We derive equations for the Fourier transforma- tion of the vacuum expectation value of the charge density. We use it to derive Uehling potential and calculate energy shifts caused by it. Then we discuss effects of vacuum polarization in higher orders of α. In second part we define circular dichroism and we express it in terms of reduced matrix elements. Then we derive the formula for parity vilating potential which is generated by weak interaction and together with other results we use it to find the expression for circular dichro- ism in terms of hydrogen radial functions. 1

by Clifford Sanders. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web. / Ethylene is the simplest alkene. The carbon-carbon double bond is ubiquitous in the field of chemistry. Ethylene serves as the basis for understanding these molecules. Thus, the assignment of the electronic transitions in ethylene is an important endeavor that many scientists have undertaken, but are yet to decipher theoretically or experimentally. Synchrotron Radiation in the vacuum ultraviolet region allows for magnetic circular dichroism (MCD) measurements of ethylene and other simple alkenes. Studies of ethylene and propylene revealed that the páap* (AgáaB1u ethylene notation) transition is not the lowest energy transition. The páa3s(R) (AgáaB3u ethylene notation) is the lowest energy transition. To further this investigation, MCD and absorption measurement were carried out on isobutene. The isobutene spectra clearly showed four electronic transitions in the 156 to 212 nm wavelength region. These four isobutene transitions have been assigned as páa3s, páap*, páa3p(Sv (Band páa3px proceeding from lower energy to higher energy. The present results support the assignments in ethylene and propylene.

Neste trabalho, apresentamos resultados da caracterização Magneto-Óptica de íons terras-raras (Nd3+ e Yb3+) em monocristais de niobato de lítio (LiNbO3). Medidas de Dicroísmo Circular Magnético (MCD) e de Emissão Circularmente Polarizada em Presença de Campo Magnético (MCPE) foram realizadas pela primeira vez nesses sistemas. Os resultados foram obtidos à temperatura de 2K, e em campos magnéticos de até 5 T. Através desses estudos, foi possível identificar os números quânticos cristalinos (&#956) dos subníveis Zeeman desses íons. A partir da dependência do sinal de MCD com a intensidade de campo magnético, determinou¬se o fator giromagnético efetivo g// do estado fundamental de cada íon, obtendo-se os valores: g//Nd = (1,4 &#177 0,1) e g//Yb = (4,7 &#177 0,1). Esses valores foram confirmados através de medidas de espectroscopia de Ressonância Paramagnética Eletrônica (EPR), realizadas a baixa temperatura (4-8 K), em banda X. Os espectros de EPR foram tomados em função da orientação relativa do campo magnético externo com o eixo c cristalino dos cristais, em 3 planos perpendiculares entre si. Os espectros de EPR mostram a existência de diferentes sítios ocupados pelos íons terras-raras. O sítio mais populado tem simetria axial, e para esse centro determinaram-se os fatores g efetivos g//Nd = (1,440 &#177 0,005) e g//Nd = (2,959 &#177 0,004), para o íon Nd3+, e g//Yb = (4,705 &#177 0,008) e g//Yb = (2,693 &#177 0,005) para o íon Yb3+. Espectros de MCD e MCPE obtidos para um cristal de rubi ilustram as convenções utilizadas e atestam que o sistema experimental funciona adequadamente. Os espectros obtidos nessa amostra também são originais, tendo sido resolvidas as transições permitidas com luz circularmente polarizada entre os subníveis Zeeman correspondentes aos níveis de energia 4A2 e &#8254E (2E) do íon Cr3+. / In this work we present Magneto-Optícal characterizations of rare-earth ions (Nd3+ e Yb3+) in lithium niobate (LiNbO3) single crystals. Magnetic Circular Dichroism (MCD) and Magnetic Circularly Polarized Emission (MCPE) measurements were performed for the first time on those systems. Spectra were obtained at 2K and at magnetic field strength up to 5T. From these studies, it was possible to assign the crystal quantum number (&#956) of the Zeeman sublevels of these ions, so that the sign and allowance of the electronic transitions could be predicted. From the dependence of suitable MCD spectral lines on the magnetic field strength, the effective parallel gyromagnetic factor (g//) of the ground state for each of the rare earth ions has been determined to be: g//Nd = (1,4 &#177 0,1) e g//Yb = (4,7 &#177 0,1). These values are in dose agreement to those obtained by means of Electron Paramagnetic Resonance (EPR) spectroscopy, at 4-8 K, and at X-band frequency. EPR spectra were recorded as a function of the external magnetic field orientation relative to the c crystalline axis in three mutual perpendicular planes. These spectra show evidence of multiple sites occupied by the rare-earth ions. For the most intense line seen in the spectra of each ion, it could be clearly assigned a site with axial symmetry, with effective g factors of g//Nd= (1,440 &#177 0,005) and g//Nd = (2,959 &#177 0,004), for the Nd3+3+ ion, and g//Yb = (4,705 &#177 0,008) and g//Yb= (2,693 &#177 0,005) for the Yb3+ ion. MCD and MCPE spectra recorded for a ruby crystal shows the experimental conventions used so far in this work, as well as assure that the experimental system works properly. These results are original ones, by means of which, the spectral transitions between the Zeeman sublevels of the 4A2 and &#8254E (2E) of the Cr3+ ions in ruby could be resolved.

Utilizando cristais de KCl:SH- dopados com centros F2+ na ausência de centros F e F2, permitiu-nos estudar o Dicroísmo Circular Magnético (DCM) em absorção das transições 1s ?g(493?m) e 1s ?g - 2py?w (509 ?m) como função do campo magnético de 0 < H < 48 KG e temperatura entre 1.5 < T < 77K. A transição 1s ?g, 2p? (1.4 ?m) em absorção não apresentou DCM dentro do limite de detecção de nosso equipamento (1.2 X 10-4); o mesmo aconteceu com a transição (2p?w- 1s ?g) em emissão (1.2 X 10-4). Irradiando com luz polarizada na banda ?, os centros F2+ se .reorienta ao longo da direção [110] em até 1.5 K, apresentando uma forte birrefringência. Medidas em absorção com centros F2+ alinhados em várias geometrias, permitiu estudar a contribuição ao DCM de cada orientação do defeito. Apresentamos um modelo teórico em bom acordo com os resultados experimentais. Utilizando uma técnica de Detecção óptica de EPR, determinamos o fator de Landé para o estado fundamental (g =1.965 ± 0.007) e o tempo de relaxação spin-rede do estado fundamental a H = 3.2 KG, que é típico do processo direto T1-1 = 4.3 X 10-2cotgh(g?H/2kT). / Using KCl:SH- doped with F2+ centers without F and F2, we studied the Magnetic Circular Dichroism (MCD), in absorption of yhe transition 1s ?g(493?m) and 1s ?g - 2py?w (509 ?m) as a function of the magnetic field 0 < H < 48 KG and temperature 1.5 < T < 77K. The transition 1s ?g, 2p? (1.4 ?m) does not present any MCD within the limit of detection of our equipament (1.2 X 10-4); No dichroism has been observed in emission in the 2p?w- 1s ?g transition (1.2 X 10-4). F2+ centers reorient along the [110] direction down to 1.5 K by polarized light excitation in the ? bands and present a strong birefringence. Measurements in absorption with aligned F2+ in various geometry allows as to determine the contribution of each orientation to the DCM. A theorical model is presented in good agrement with the experimental results. The optical detection of the EPR in X-band give the Landé factor af the graund state is g =1.965 ± 0.007) and the spin-lattice relaxation measured at H = 3.2 KG is typical of a direct process T1-1 = 4.3 X 10-2cotgh(g?H/2kT).

Os estudos sobre o mecanismo de enovelamento das proteínas é o resultado de um estudo intenso utilizando métodos bioquímicos, biofísicos e teóricos. \"In vitro\", o estado inicial deste estudo é a proteína desnaturada. Neste trabalho, temos estudado o reenovelamento, após desnaturação térmica, de uma glicoproteína denominada frutalina; da família das lectinas. A característica principal desta classe de proteínas é sua habilidade para interagir com carboidratos e, portanto, combinar-se com glicocomponentes da superfície da célula, induzindo suas propriedades biológicas. A frutalina é uma lectina tetramérica extraída das sementes de Artocarpus incisa. Ela é ligante de D-galactose e o espectro de CD (dicroísmo circular) de sua estrutura nativa foi identificado como sendo dominado por folhas ?. A desnaturação térmica e as etapas do reenovelamento foram monitoradas por espectroscopia de CD, fluorescência e também pela perda da atividade hemaglutinante. As condições de desnaturação utilizadas foram aquecimento à 60°C por 30 a 60 minutos, dependendo do tempo de estocagem (a -18°C) da proteína na forma nativa. Os resultados indicaram que o reenovelamento é promovido por um processo de congelamento na presença de PBS contendo 0,l M de D-galactose seguida por centrífugoconcentração em Centriprep 3. A hemaglutinação positiva ocorreu tanto para a fração nativa quanto para a fração reenovelada. O reenovelamento da frutalina desnaturada também ocorreu com PBS contendo 0,1 M de solução de D-glicose. Quando a forma desnaturada foi concentrada antes do congelamento em PBS sem D-galactose ou em PBS contendo xilose, o reenovelamento não ocorreu. Estes resultados mostraram que o reenovelamento da frutalina foi dependente da ligação com a D-galactose ou D-glicose, bem como a importância do congelamento para obter a forma biologicamente ativa. A análise da estrutura secundária utilizando o programa CCA forneceu um resultado importante: para a forma nativa da frutalina obtivemos 85% de folhas ? paralelas e antiparalelas, incluindo voltas ?, enquanto que para a forma reenovelada obtivemos 73%, mostrando que a estrutura reenovelada, a nível secundário, se aproximou satisfatoriamente da nativa, concordando com os resultados obtidos nos testes de hemaglutinação. / Our current understanding of the protein folding mechanism is the result of intense study employing biophysical, biochemical and theoretical methods. \"In vitro\", the initial state of the protein in this puzzle is its unfolded form. In the present work we have studied the refolding, after thermal denaturation, of the glycoprotein frutalin, a member of the lectin class. The main characteristic of these proteins is their ability to interact with carbohydrates and thus combine with glycocomponents of the cell surface, leading to their biological properties. Frutalin is a tetrameric lectin extracted from the seeds of Artocarpus incisa. It is D-galactose specific and its native CD spectrum was identified as being dominated by ? -sheet. The thermal unfolding and refolding steps were measured by CD and fluorescence spectroscopies together with the loss of hemagglutinating activity. The unfolding conditions used were 60°C for 30 to 60 minutes, depending on the protein storage time. The results indicate that refolding is promoted by the freezing process in the presence of 0,1 M D-galactose-PBS followed by three-fold concentration in a Centriprep 3. Positive hemagglutination occurred for both the native and refolded forms. Refolding of denatured frutalin also occurred with PBS containing 0,1 M D-glucose. When the unfolded form was concentrated before freezing in PBS without D-galactose or in PBS containing xylose, refolding did not occur. These results show that the refolding of frutalin is dependent on the binding of D-galactose or D-glucose, and demonstrate the importance of freezing in order to obtain the biologically activity form. An analysis of secondary structure using the CCA program showed an important result: the native form, presented 85% ? -sheet/ ?-turns, while in the refolded form, this content fell to 73%. These results show that the refolded form is very similar to the native protein, which is in agreement with the hemagglutination results.

O estudo de enovelamento de proteínas tem sido um problema de fundamental importância em biofísica e biologia molecular. Neste trabalho, estudamos os processos de desnaturação e renovelamento das lectinas jacalina e frutalina. Estas lectinas são tetraméricas, apresentam alta homologia estrutural, porém diferem em atividade biológica, sendo a frutalina mais potente. Apesar desta homologia, estas lectinas diferem também nos processos de desnaturação e renovelamento como função da temperatura e o comportamento frente ao desnaturante químico hidrocloreto de guanidina (GndHCl). Ambas proteídas foram desnaturadas pela ação de GndHCl e suas curvas de desnaturação medidas por espectroscopia de fluorescência e CD. As medidas de fluorescência da frutalina deram valores de estabilidade conformacional de 17,12 kJ/mol e 12,34 kJ/mol, na presença e na ausência de D-Galactose, enquanto a jacalina forneceu valores de 8,12 kJ/mol para a transição NI e 5,61 kJ/mol para a transição IU em PBS. Os valores na presença de açúcar foram similares. NOs estudos da frutalina foram separadas as formas nativa, desnaturada, renovelada e uma forma molecularmente distinta chamada mal-enovelada, por cromatografia de exclusão molecular. Estas formas foram analisadas por atividades hemaglutinante e espectroscopias de CD e fluorescência. Todos os resultados obtidos confirmaram a ocorrência do renovelamento de ambas lectinas e que os monômeros renovelados, depois de alcançarem sua estrutura tridimensional, se associam espontaneamente para a formação dos tetrâmeros. / Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of jacalin and frutalin. They are tetrameric lectins that present structural homology, buti jacalin shows a more marked biological activity than the latter. These proteins, despite their homology, have different unfolding/refolding behaviors as function of temperature and chemical agents. Both proteins were unfolded induced by guanidine hydrochlroide and their dnaturation curves mesuared by fluorescence emission and CD. Fluorescence measurments of frutalin gave values of conformational stability of 17.12 kJ/mol and 12.32 kJ/mol, in the presence and absence of D-Galactose, while jacaline gave values of 8.12 kJ/mol for NI transition and 5.61 kJ/mol for IU transition in PBS. In sugar presence the values are similar. In the frutalin studies were separeted the native, unfolded, refolded and a distinct molecular form denoted misfolded, by Size Exclusion Chromatography. These forms were analyzed for hemagglutination activity, CD and fluorescence spectroscopy. All the results obtained confirmed the successful refolding of the both lectins and the refolded monomers, after adopting their native three-dimensional structures, spontaneously assembled to form tetramers.