Structure-specific recognition of nucleic acids is a promising method to reduce the size of the recognition unit required to achieve the necessary selectivity and binding affinity for small molecules. It has been demonstrated recently that G-quadruplex DNA structures can be targeted by organic cations in a structure-specific manner. Structural targets of quadruplexes include the planar end surfaces of the G-tetrad stacked columns as well as four grooves. The significant structural differences between quadruplex DNA and duplex DNA make quadruplex DNA a very attractive target for highly selective, structure-specific drug design. We have used a variety of biophysical techniques including circular dichroism, surface plasmon resonance, thermal melting and absorbance spectroscopy to investigate small molecules that can selectively bind to the ends of human telomeric DNA as well as the ends of the G-quadruplex structure formed by the purine-rich promoter region of the c-MYC oncogene. We have also screened a library of heterocyclic diamidines, and identified one that binds selectively in the grooves of human telomeric quadruplex DNA. This compound is an excellent starting point for the design of new anti-cancer and anti-parasitic compounds with high affinity and selectivity for human telomeric DNA.

It has been shown that heterocyclic diamidines, a class of minor groove binders, are promising antimicrobial agents. These compounds bind none covalently to the minor groove of A/T rich regions of the kinetoplast DNA and kill the parasite. The mechanism of action of these compounds is not well understood, yet many hypotheses have been proposed. One of the methods that improve the specificity is cooperative binding. Since there are many binding sites available in k-DNA thus the cooperativity in adjacent binding sites is desirable. A library of compounds has been scanned and few of those compounds identified that are able to bind to two adjacent A/T binding sites separated by a single G. Many biophysical methods such as isothermal titration calorimetry, surface Plasmon resonance, circular dichroism and thermal melting have been used to explore the thermodynamic profiles and binding mode of these compounds. The pulsed field gradient NMR was used to investigate the structural changes to the DNA sequence upon binding of the minor groove binders and find a correlation between their biological difference and structural changes. The molecular dynamics was applied to look at the interaction of some of the heterocyclic diamidines to the DNA with more details and predict the unknown structures.

The rotavirus NSP4 protein is the first described viral enterotoxin. Abundant data from our laboratory reveals that NSP4 binds both the N- and C-termini of caveolin- 1 (aa2-31 and 161-178, respectively). Yeast two-hybrid and peptide binding analysis mapped the caveolin-1 binding site to three hydrophobic residues within the amphipathic a-helix, enterotoxic peptide domain (aa114-135). The research studies herein utilized peptides to investigate the interaction between NSP4 and caveolin-1. Peptides were synthesized corresponding to the amphipathic a-helix and caveolin-1 binding domain of NSP4 (aa112-140) and to the N- (aa2-20 and 19-40) and C- (161-178) termini of caveolin-1, and were utilized in structural and functional studies. Fluorescence binding assays revealed that NSP4 (aa112-140) binds to the N-terminus (aa19-40) of caveolin-1 with a stronger affinity than the C-terminus (aa161-178). In addition, this assay further delineated the NSP4 binding domain on caveolin-1 to aa19-40. Secondary structural changes following NSP4-caveolin-1 peptide-peptide interactions were investigated by circular dichroism analysis. Changes in a-helix formation were observed only upon interaction of the NSP4112-140 peptide with the C-terminal caveolin-1 peptide (C-Cav161- 178). The NSP4112-140 peptide contains a potential cholesterol recognition amino acid consensus (CRAC) sequence. Therefore this peptide was examined for cholesterol binding. Results of the binding assay revealed NSP4 binds cholesterol with a Kd of 7.67 +/- 1.49nM and this interaction occurs via aa112-140. Mutation of amino acid residues within the CRAC motif resulted in weaker binding affinities between each of the corresponding mutant peptides and cholesterol. NSP4 peptides containing mutations within the hydrophobic and charged faces of the amphipathic a-helix, enterotoxic peptide and caveolin-1 binding domain of NSP4 were examined for changes in secondary structure as well as diarrhea induction in mouse pups. Circular dichroism analysis revealed that mutation of hydrophobic residues resulted in a decrease in a-helix formation, whereas mutation of acidic and basic charged residues caused little to no change in a-helical content. When tested for diarrhea induction in mouse pups, the peptides containing mutations of either the hydrophobic or basic charged residues did not cause diarrhea. Taken together, the results of this research suggest a complex interplay between NSP4 secondary structure, caveolin-1 and cholesterol binding and diarrheagenic function.

In chapter 1 the principles behind circular dichroism spectroscopy and exciton coupled circular dichroism spectroscopy are outlined, and examples are cited that illustrate the utility of these methods in the determination of absolute configuration and ee of chiral amines. This provides background and context for this thesis, which mostly pertains to the sensing of chirality in amines. An exciton coupled circular dichroism method based on the induction of helical chirality in an organometallic host for sensing chiral amines is presented in chapter 2. The method can be used to determine absolute configuration by relating the sign of the first Cotton effect of the host-amine complex to the handedness of the amine. Analysis of the primary circular dichroism optical data is by principal component analysis allows for differentiation of the analytes based on their idendity and handedness. A novel circular dichroism method for detecting chiral amines is discussed in chapter 3. The method uses a highly efficient derivatization method to convert the primary amine into a bidentate imine. Three equivalents of the imine are then assembled together by coordination to Fe(II). The proximity and chiral orientation of the imines leads to exciton coupled circular dichroism, which is of utility in the determination of absolute configuration. Additionally, there is a metal-to-ligand charge transfer band in the visible region that can be used to develop calibration curves, which allow for the determination of the enantiomeric excess of unknown samples with an absolute error of ±5%. Chapter 4 details another imine based circular dichroism method for chiral amines. The method uses a commercially available aldehyde, Fe(II), and circular dichroism spectrometry to sense chirality in amines. It is shown that the circular dichroism signals in the ultraviolet spectrum vary predictably with the handedness of the chiral amine, which has potential applications in the determination of absolute configuration. By developing calibraton curves, signals in the visible spectrum can be used to determine enantiomeric excess with an absolute error of ±6%. Analyzing the primary circular dichroism optical data with linear discriminant analysis allows for differentiation between amines based on their identity and handedness. Finally, chapter 5 illustrates the potential of using the thermodynamic parameters of partitioning between water and octanol as a predictive tool for estimating the contributions of hydrophobicity to host-guest binding events. This is done by showing a relationship between the thermodynamics of partitioning and thermodynamics of hydrophobic binding events for a series of guests and cyclodextrin. A plot of the thermodynamic parameters of binding of a variety of guests to cyclodextrin as a function of the thermodynamic parameters of partitioning between water and octanol shows a linear relationship for a series of alcohols. / text

To understand protein stability and the mechanism of protein folding, it is essential that we gain a better understanding of the ensemble of conformations that make up the denatured state of a protein. The primary goal of the research described here was to see what we might learn about the denatured state using fluorescence. To this end, tryptophan was introduced at five sites in Ribonuclease Sa (RNase Sa): D1W, Y52W, Y55W, T76W, and Y81W. The fluorescent properties of the denatured states of these five proteins were studied and compared to the fluorescent properties of eight model compounds: N-acetyl-tryptophan-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and five pentapeptides based on the sequence around the original tryptophan substitutions in RNase Sa. Regardless of the denaturant, λmax for the proteins and model compounds differed very little, 349.3 ± 1.2 nm. However, significant differences were observed in the fluorescence intensity at λmax (IF), suggesting that IF is more sensitive to the immediate environment than λmax. The differences in IF are due in part to quenching by neighboring side chains. More importantly, IF was always significantly greater in the protein than in its corresponding pentapeptide, indicating that the protein exerts an effect on the tryptophan, which cannot be mimicked by the pentapeptide models. Acrylamide and iodide quenching experiments were also performed on the model compounds and proteins. Significant differences in the Stern-Volmer quenching constant (KSV) were also observed between the proteins and between the proteins and their corresponding pentapeptides. Importantly, the KSV for the protein was always less than in its corresponding pentapeptide. These data along with the IF data show that non-local structure in the unfolded state influences tryptophan fluorescence and accessibility. In summary, these and our other studies show that fluorescence can be used to gain a better understanding of the denatured states of proteins.

En el curso de esta Tesis Doctoral, se sintetizaron librerías de (1,2)-diaminas enantiopuras y 2,2’-bifenoles pro-quirales. La transferencia de quiralidad desde la diamina al bifenol, mediante puentes de hidrógeno o coordinación a un metal (ZnII, CuII), fue demostrada por dicroísmo circular (DC). El comportamiento en disolución de los complejos supramoleculares (usando puentes de hidrógeno), así como sus constantes de asociación, fue estudiado mediante valoraciones de RMN, UV-vis y ITC. La determinación de las configuraciones absolutas de los complejos de ZnII se consiguió mediante la resolución de las estructuras de rayos-X y los estudios de DC, tanto a un nivel teórico como experimental. Un nuevo ligando fosforado, potencialmente catalítico, fue preparado a partir de 2,2’-bifenol, mostrando el camino para el desarrollo de nuevos catalizadores supramoleculares. / During the course of the thesis libraries of chiral (1,2)-diamines and prochiral 2,2’-biphenol derivatives were synthesised. The transfer of chirality from the diamine to the biphenol moiety mediated either by hydrogen bonding or coordination to a metal centre (ZnII, CuII) was demonstrated by CD (circular dicroism). The behaviour in solution of the hydrogen bonded complexes was investigated by NMR spectroscopy, UV-vis and ITC titrations (determination of association constants. The determination of absolute configurations of the ZnII complexes was achieved by X-ray structure determination and CD analyses both on the experimental and theoretical level. Last but not least, a new potentially catalytic phosphane ligand was derived from a dynamically racemic 2,2’-biphenol derivative.

No description available.

Tat1-72, is an intrinsically disordered protein at pH 4.1 as previously indicated by NMR chemical shifts and coupling constants, and confirmed by 15N-relaxation parameters. The presence of SDS elicits a conformational change to α-helicity in Tat1-72. In the presence of the non-ionic DDM detergent and zinc, Tat was found to be soluble at pH 4 when bound to TAR RNA; TAR binding also elicits a conformational shift to α-helicity in Tat1-72. The β-sheet content of Tat1-72 is increased in the presence of NaCl. In similar conditions, Tat1-72 aggregates stained with Congo Red displayed a yellow-green birefringence and a red-shift in the Congo Red absorbance that is typical of β-amyloid fibril. The web-based algorithm “WALTZ” identifies the majority of the Tat1-72 hydrophobic core region as amyloidogenic. The helical propensity of Tat1-72 in TFE was determined by two-dimensional NMR spectroscopy.

No description available.

Tat1-72, is an intrinsically disordered protein at pH 4.1 as previously indicated by NMR chemical shifts and coupling constants, and confirmed by 15N-relaxation parameters. The presence of SDS elicits a conformational change to α-helicity in Tat1-72. In the presence of the non-ionic DDM detergent and zinc, Tat was found to be soluble at pH 4 when bound to TAR RNA; TAR binding also elicits a conformational shift to α-helicity in Tat1-72. The β-sheet content of Tat1-72 is increased in the presence of NaCl. In similar conditions, Tat1-72 aggregates stained with Congo Red displayed a yellow-green birefringence and a red-shift in the Congo Red absorbance that is typical of β-amyloid fibril. The web-based algorithm “WALTZ” identifies the majority of the Tat1-72 hydrophobic core region as amyloidogenic. The helical propensity of Tat1-72 in TFE was determined by two-dimensional NMR spectroscopy.