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Structure-Property Relationship of the Two-Photon Circular Dichroism of Compounds with Axial and Helical Chirality
Title
Structure-Property Relationship of the Two-Photon Circular Dichroism of Compounds with Axial and Helical Chirality.
Creator
Diaz, Carlos, Hernandez, Florencio, Uribe Romo, Fernando, Kuebler, Stephen, Masunov, Artem, Del Barco, Enrique, University of Central Florida
Abstract / Description
Back in 1894 Lord Kelvin coined the term (")chiral(") in order to refer to molecules whose mirror images were not superimposable with themselves. Over the years, research has demonstrated the important role that chiral molecules play in life, chemistry, and biology as well as their importance in the development of new drugs and technologies.The efforts to understand chiral systems have been mainly driven by spectroscopic methods that leverage on the opposite responses that enantiomers have to...
Show moreBack in 1894 Lord Kelvin coined the term (")chiral(") in order to refer to molecules whose mirror images were not superimposable with themselves. Over the years, research has demonstrated the important role that chiral molecules play in life, chemistry, and biology as well as their importance in the development of new drugs and technologies.The efforts to understand chiral systems have been mainly driven by spectroscopic methods that leverage on the opposite responses that enantiomers have to linear or circularly polarized light of both handedness. More specifically, Electronic Circular Dichroism (ECD) which measures the differences in linear absorption of left and right circularly polarized light has been the method par excellence for the spectroscopic characterization of chiral compounds. Unfortunately, the fact that ECD is based on linear absorption severely limits the use of this method in the near to far UV region. This is mainly due to the interferences generated by the strong linear absorption of common organic solvents and buffers in this portion of the light spectrum. Nevertheless, the fact remains that many chiral biomolecules of interest related to deceases like Alzheimer and Parkinson, exhibit most of their linear absorption in the near to far UV region where ECD cannot be employed for their study. Therefore, it has become an urgent necessity to develop spectroscopic methods to study chiral molecules that can circumvent the limitations of ECD at shorter wavelengths. In order to overcome the existent limitations in linear chiral spectroscopy, the nonlinear equivalent of ECD arises as a promising alternative, i.e. Two-Photon Circular Dichroism (TPCD). Although, this phenomenon was theoretically predicted in 1975, it was not until 2008, with the introduction of the double-L scan, that a reliable and versatile method for the measurement of TPCD was introduced. The high sensitivity of this method is based on the use of (")twin(") pulses that allow accounting for fluctuations in the excitation source that prevented the experimental realization of the measurement. The first measurement of a full TPCD spectrum was performed on BINOL enantiomers and the results were supported and discussed with the help of theoretical calculations. After that seminal work, we embarked in expanding the understanding of the structure-property relationship of TPCD by performing, systematically, a series of theoretical-experimental studies in chiral biaryl derivatives and compounds with helical chirality.In Chapter 2 we present the theoretical-experimental study of the effect of the ?-electron delocalization curvature on the TPCD of molecules with axial chirality. The targeted molecules for this part of our investigation were S-BINOL, S-VANOL, and S-VAPOL. Our findings revealed that an increase in the TPCD signal, within this series of compounds, was related to the curvature of the ?(-)electron delocalization. The contributions of the different transition moments to the two-photon rotatory strength support our outcomes. Then, in Chapter 3 we introduce the development of the Fragment-Recombination Approach (FRA) for the calculation of the TPCD spectra of large molecules. This simple but powerful method is based on the additivity of the TPCD signal, and is subject to a strict conditional fragmentation approach. FRA-TPCD is demonstrated, theoretically, in two hypothetical molecular systems from the biaryl derivatives family. Afterward, in Chapter 4 we show the first experimental demonstration of FRA-TPCD through the conformational analysis of an axially-chiral Salen ligand in solution (AXF-155). The FRA-TPCD spectra calculated for the different isomers of AXF-155 allowed narrowing the number of possible isomers of this complex molecule in THF solution to only two. This represents a significant improvement from previously reported results using ECD. Subsequently, in Chapter 5 we present the study of the effect of intramolecular charge transfer (ICT) in S-BINAP, an axially dissymmetric diphosphine ligand with strong ICT. The evaluation of the performance of two different exchange-correlation functional (XCF) confirmed that in order to properly predict the theoretical TPCD spectrum of a molecule exhibiting strong ICT, it is required to use an XCF such as CAM-B3LYP. In addition, our findings revealed the importance of considering an adequate number of excited states in order to be able to fully reproduce the experimental TPCD spectrum, thus avoiding wrong assignments of theoretical transitions to experimental spectral features. Finally, and expanding on our previous study, in Chapter 6 we investigated the effect of the nature of ICT on two hexahelicene derivatives. Our investigation demonstrated that the TPCD signal of chiral molecules with strong ICT does not only depend on the strength of this effect but on its nature, i.e. extension of the ?(-)electronic delocalization increasing beyond (EXO-ICT) or within (ENDO-ICT) the helicene core. In summary, with the results presented in this thesis we closed a first loop in the understanding of the structure-property relationship of TPCD. In the future, we expect to deepen in our knowledge of the structure-property relationship of this phenomenon by studying further helicene derivatives with donor-acceptor motif, and through the application of FRA-TPCD to the conformational analysis of amino acids in peptides. We foresee numerous applications of TPCD for the study of optically active molecules with implications in biology, medicine, and the drug and food industry, and applications in nanotechnology, asymmetric catalysis and photonics.
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Date Issued
2015
Identifier
CFE0005787, ucf:50067
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005787
BIOPHYSICAL CHARACTERIZATION OF THE MEMBRANE BINDING DOMAIN OF THE PRO-APOPTOTIC PROTEIN BAX
Title
BIOPHYSICAL CHARACTERIZATION OF THE MEMBRANE BINDING DOMAIN OF THE PRO-APOPTOTIC PROTEIN BAX.
Creator
Garg, Pranav, Tatulian, Suren, University of Central Florida
Abstract / Description
The BCL-2 family of proteins tightly regulates the delicate balance between life and death. The pore forming Bax is a pro-apoptotic member belonging to this protein family. At the onset of apoptosis, monomeric cytoplasmic Bax translocates to the outer mitochondrial membrane, forms oligomeric pores thereby letting mitochondrial cytochrome c enter the cytosol and initiate the apoptotic cascade. The C-terminal "helix 9" is thought to mediate the membrane binding of BAX. A 20-amino acid peptide...
Show moreThe BCL-2 family of proteins tightly regulates the delicate balance between life and death. The pore forming Bax is a pro-apoptotic member belonging to this protein family. At the onset of apoptosis, monomeric cytoplasmic Bax translocates to the outer mitochondrial membrane, forms oligomeric pores thereby letting mitochondrial cytochrome c enter the cytosol and initiate the apoptotic cascade. The C-terminal "helix 9" is thought to mediate the membrane binding of BAX. A 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with Glu (charge reversal mutant, EE) or Leu (charge neutralization mutant, LL) have been studied to elucidate the pore formation capabilities of Bax C-terminus and the underlying molecular mechanism. Interactions of the wild-type and the two mutant peptides with zwitterionic and anionic phospholipid membranes caused efficient membrane permeabilization, as documented by release of vesicle-entrapped fluorescent indicator calcein. Light scattering experiments showed that vesicles maintained their integrity upon peptide binding, indicating that the content leakage was due to pore formation and not vesicle degradation. Kinetics of calcein release at various peptide concentrations were used to determine the peptide-peptide association constants and the oligomeric state of the pore. The structure of membrane-bound peptides was analyzed by circular dichroism (CD) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. CD data indicated all three peptides reconstituted in lipid vesicles contained [alpha]-helical and [beta]-strand structures. ATR-FTIR experiments indicated that the minimally hydrated samples of peptides in stacked lipid bilayers (absence of bulk water) were mostly [alpha]-helical but adopted mostly [beta]-sheet conformation in the presence of excess water. Finally, the depth of membrane insertion of the peptides was analyzed using tryptophan fluorescence quenching by dibromo-phosphatidylcholines brominated at various positions of their acyl chains. In case of zwitterionc phospholipid membranes, the single Trp16 was located at ~9 Å from membrane center. In case of membranes containing 30% of an anionic phospholipid, the depth of membrane insertion of the EE mutant was not affected but the wild-type and the LL mutant peptides were embedded much deeper into the membrane, with Trp16 located at 3-4 Å from membrane center. These results will help achieve a better understanding of the molecular mechanism of membrane pore formation of Bax protein. In addition, they provide insight into the molecular details of membrane pore formation by peptides and could facilitate the design and production of cytotoxic peptides with improved capabilities to lyse cells such as bacteria or cancer cells.
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Date Issued
2011
Identifier
CFE0003983, ucf:48674
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003983
ROLE OF MEMBRANE LIPIDS IN MODULATING PROTEIN STRUCTURE & FUNCTION
Title
ROLE OF MEMBRANE LIPIDS IN MODULATING PROTEIN STRUCTURE & FUNCTION.
Creator
Ray, Supriyo, Tatulian, Suren, University of Central Florida
Abstract / Description
A-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated...
Show moreA-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated degradation (ERAD). Most ERAD substrates are purged out of the ER into the cytosol for proteasomal degradation. However, the low content of lysine amino acid residues allows the toxin to evade polyubiquitination and subsequent proteasomal degradation. The toxin A subunit refolds into an active conformation in the cytosol, setting off downstream toxic events. In the first part of my thesis, the hypothesis was tested that inhibiting the unfolding of the toxin A subunit inside the ER will prevent ERAD activation, toxin export to the cytosol and intoxication. The chemical chaperones glycerol and sodium 4-phenyl butyrate (PBA) were used to inhibit the toxin A chain unfolding. In vitro biophysical experiments indicated that both chemical chaperones indeed stabilize the cholera toxin A subunit and prevent cytotoxicity. In case of ricin, both chaperones stabilized the toxin A chain but only glycerol prevented cytotoxicity. Additional experiments showed that PBA-treated ricin A chain is destabilized when exposed to anionic lipid membranes mimicking the properties of the ER membrane. In contrast, anionic lipid did not prevent ricin A chain stabilization by glycerol. This explains why glycerol but not PBA blocked ricin intoxication, as only glycerol stabilizes ricin A chain in the presence of ER membranes. Cholera toxin in contrast, remained either unaffected or slightly stabilized in presence of anionic lipids both in presence and absence of PBA. This shows that destabilization by anionic lipids is a toxin-specific rather than a general effect. In the second part of my thesis, the effect of inner leaflet of plasma membrane on the structure of cholera toxin A chain (CTA1) was studied. Since CTA1 refolds into an active conformation in the cytosol in association with unidentified host factors, I hypothesized that inner leaflet of the plasma membrane might play a role to stabilization and/or refolding of CTA1. CTA1 was shown to be a membrane interacting protein, and membranes mimicking lipid rafts had a significant stabilizing effect on its structure. Lipid rafts helped in the regaining of the tertiary and secondary structure of CTA1, while non-raft lipids had a smaller stabilizing effect on CTA1 structure. In the next part of my thesis, I studied the effect of membrane binding on the structure and function of human pancreatic phospholipase A2 (PLA2). Lipid thermal phase transition was found to have a dramatic effect on PLA2 activity. It was also established that although membrane binding and insertion was essential for of PLA2 activity, lipid structural heterogeneity was more important than the depth of membrane insertion for enzyme activation. Most importantly, significant changes in PLA2 secondary and tertiary structures were identified that evidently contribute to the interfacial activation of PLA2. Overall, we conclude that the function of membrane binding enzymes can be significantly modulated via conformational changes induced by interactions with membranes. Thus, we have elucidated various roles of membrane lipids from unfolding and refolding to activation and modulation of membrane binding enzymes. Physical properties of lipids help in regulating various aspects of protein structure and function and their analysis helped us in appreciating the influence wielded by the membrane lipids in the enzyme's surrounding environment.
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Date Issued
2011
Identifier
CFE0004035, ucf:49184
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004035
Cavity-Coupled Plasmonic Systems for Enhanced Light-Matter Interactions
Title
Cavity-Coupled Plasmonic Systems for Enhanced Light-Matter Interactions.
Creator
Vazquez-Guardado, Abraham, Chanda, Debashis, Christodoulides, Demetrios, Abouraddy, Ayman, Moharam, Jim, Leuenberger, Michael, University of Central Florida
Abstract / Description
Light-matter interaction is a pivotal effect that involves the synergetic interplay of electromag- netic fields with fundamental particles. In this regard localized surface plasmons (LSP) arise from coherent interaction of the electromagnetic field with the collective oscillation of free electrons in confined sub-wavelength environments. Their most attractive properties are strong field en- hancements at the near field, highly inhomogeneous, peculiar temporal and spatial distributions and...
Show moreLight-matter interaction is a pivotal effect that involves the synergetic interplay of electromag- netic fields with fundamental particles. In this regard localized surface plasmons (LSP) arise from coherent interaction of the electromagnetic field with the collective oscillation of free electrons in confined sub-wavelength environments. Their most attractive properties are strong field en- hancements at the near field, highly inhomogeneous, peculiar temporal and spatial distributions and unique polarization properties. LSP systems also offer a unique playground for fundamental electromagnetic physics where micro-scale systemic properties can be studied in the macro-scale. These important properties and opportunities are brought up in this work where I study hybrid cavity-coupled plasmonic systems in which the weak plasmonic element is far-field coupled with the photonic cavity by properly tuning its phase. In this work I preset the fundamental understand- ing of such a complex systems from the multi-resonance interaction picture along experimental demonstration. Using this platform and its intricate near fields I further demonstrate a novel mech- anism to generate superchiral light: a field polarization property that adds a degree of freedom to light-matter interactions at the nanoscale exploited in advanced sensing applications and surface effect processes. Finally, the detection of non-chiral analytes, such as proteins, neurotransmit- ters or nanoparticles, and more complex chiral analytes, such as proteins and its conformation states, amino acids or chiral molecules at low concentrations is demonstrated in several biosensing applications. The accompanied experiential demonstrations were accomplished using the nanoim- printing technique, which places the cavity-coupled hybrid plasmonic system as a unique platform towards realistic applications not limited by expensive lithographic techniques.
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Date Issued
2018
Identifier
CFE0007418, ucf:52708
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007418