1,721,093 research outputs found
Optimization of a high-throughput fluorescent sensor for per- and polyfluoroalkyl substances (PFAS)
Get PDFPer- and polyfluoroalkyl substances (PFAS) are environmental contaminants that still lack accessible methods of detection. Due to their use in industrial production during the past couple decades, they have been released into waterways and continue to pose a threat to public health. As the current methods of PFAS sensing are time consuming, expensive, and low throughput, my project aims to address this discrepancy by optimizing a novel sensor for PFAS. Through template-directed synthesis that is used to self-assemble macrocyclic receptors, a high throughput sensing platform for PFAS was previously discovered. A series of monomer building block compounds with dithiol functional groups were synthesized and used to form a dynamic combinatorial library (DCL) combined with an environmentally sensitive dye. Through template-directed synthesis, the monomers self-assemble into receptors for the dye. When PFAS compounds are added, if they bind to the receptors the dye is displaced, and a change in fluorescence is observed via an indicator displacement assay. Principal component analysis (PCA) is then used to compare the various combinations of monomer DCLs for their ability to differentiate between PFAS samples. My research project focuses on further optimization of this novel PFAS sensing method by evaluating a series of additional building block combinations at new DCL concentrations to improve its sensitivity.Bachelor of Scienc
Investigating the Folding and Fibril-forming Behaviors of AAN- and NDI-Containing Foldamers
Get PDFAromatic foldamers containing 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) and 1,5-dialkoxynaphthalene (DAN) in alternation have been shown to assemble into supramolecular structures and, when heated, irreversibly aggregated fibrils. By replacing electron-rich DAN with similarly electron-rich aminoalkoxynaphthalene (AAN) in a similarly alternating peptide, we hypothesize that we can form similar stacked and fibril structures when the two monomers are connected by patterned charged and hydrophobic amino acids. Notably, AAN has a pKa of approximately 5, meaning pH can be used as a switch to alter the charge state of AAN, which we hypothesize can be used to alter the stacked or aggregated structure of the peptide as a whole. Presently, syntheses toward peptides with high fibril-forming propensities are being optimized, with the aim of studying their behaviors in response to pH modulation. In this work, we have also explored the π stacking and charge transfer behavior of an AAN- and NDI-containing peptide in both neutral and acidic environments.Bachelor of Scienc
Selective Classification of Citrullination and Acetylation on Histone Tails via Organic Receptor Molecules
Get PDFThe goal of this research is to determine whether an indicator displacement assay using organic small-molecule receptors and a fluorescent dye lucigenin (LCG) can correctly classify citrullination and acetylation in the context of histone tail peptides. Difference in fluorescent signal, dictated by the displacement of receptor-bound LCG, will be the benchmark by which the post-translational modifications (PTMs) of interest will be classified. Linear discrimination analysis (LDA) will provide the statistical means to visually separate and classify the fluorescence data for several species into respective categories. The proposed assay schema was shown to be effective in differentiating similarly citrullinated and acetylated peptides at low concentrations (15 μM) and was shown to be sensitive enough for 10 μM in citrullinated H3 tail peptides and 5 μM in acetylated H3 tail peptides.Bachelor of Art
Application of Dynamic Combinatorial Chemistry to Identify New Compounds that Bind G-Quadruplex DNA & Probing the Role of the Cation-π Interaction Between the HP1 Chromodomain and Methylated Lysine Using Unnatural Amino Acids
Get PDFThis dissertation discusses two different projects. The first project involves the
development of cyclic-peptide acridine conjugates in the effort to identify molecules that can
selectively bind to and stabilize G-quadruplex DNA. The second project seeks to probe the role
of the cation-π interaction in the binding of the HP1 chromodomain to trimethylated lysine 9 of
histone 3 (H3).
In recent years, interest in the G-quadruplex DNA structure has increased enormously
due to the unique physical properties of this secondary DNA structure as well as the presence of
guanine-rich sequences in biologically functional regions of many genomes. Given the
propensity of G-quadruplex structures to control many biological functions, it has become
desirable to identify small molecules that can bind to and stabilize G-quadruplexes. This work
aims to develop quadruplex ligands that exhibit selectively over not only duplex DNA but also
over various quadruplex sequences. It was believed that cyclic peptides could deliver selectivity
for the quadruplex structure over duplex DNA while also providing the added advantages of
mimicking native protein structure, displaying enhanced metabolic stability and possessing
structural preorganization. Using a strategy that has been developed in our lab, we propose
screening libraries of cyclic peptides generated in situ using thiol-thioester exchange for dynamic
combinatorial chemistry (DCC). These libraries can efficiently be screen against different
quadruplex sequences as well as duplex DNA in order to determine the selectivity of each
species.
In the second project, we sought to characterize the noncovalent interactions responsible
for the recognition of trimethylated lysine 9 of histone 3 by the aromatic pocket of the HP1
chromodomain. Lysine can exist in three distinct methylation states under the control of highly
specific methyl transferases or demethylases. These methylation states serve to turn on specific
protein-protein interactions with partners that specifically recognize the methylated side chain.
Recognition and affinity is mainly derived from cation-π interactions between the positively
charged cationic side chain and the electron rich π surfaces of nearby aromatic rings. This
interaction can be quantified by incorporation of fluorinated derivatives of the aromatic amino
acids responsible for the binding of H3K9Me3 to the HP1 chromodomain. The cation-π
interaction between the aromatic pocket of the HP1 chromodomain and H3K9Me3 can be
revealed by incorporation of a series of fluorinated amino acid analogues. A linear correlation
between binding affinity and the calculated magnitude of the cation-pi interaction of those
groups indicates a cation-π interaction. Because many reader proteins for methylated lysine have
an aromatic cage in their binding pockets, findings from the investigation of the HP1
chromodomain will provide broad insight into this class of proteins.Doctor of Philosoph
Evolving Chromodomain Reader Domains for Non-Natural Histone Mark Recognition
Get PDFThe development of new methods to bind and enrich histones that contain specific, non-natural histone PTMs has broad reaching implications to better understand many crucial biological processes, such as replication, transcription, and mitosis. To evolve a chromodomain reader protein, a Cbx1 gene III phage display strategy was developed and characterized in a phage ELISA system to replicate previously characterized binding data of a suite of Cbx1 binding probes ranging from Kd values of 320 µM to 130 nM. To accurately characterize these binding dynamics, it was necessary to incorporate at least 1/10th the amount of biotin to the amount of biotinylated peptide. Four parallel phage display selections were completed to attempt to evolve stronger binders to H3K9me3 and H3K9me3S10p from a naïve library of five amino acid randomized NNK codons in the EPEEN region rationally targeted by structural and computational information. The selection process showed greater than ten-fold enrichment for selections to the different histone tail peptides, and NGS data showed that the WT Cbx1 sequence was highly enriched in both libraries, and an additional RAWMI mutant was moderately enriched in the H3K9me3S10p selection and not as strongly selected against as other amino acids in the H3K9me3 selection. The Cbx1 RAWMI mutant protein has not shown any measurable binding via ITC to either of the H3 PTM peptides. This study sets the stage for future evolution of reader domains for improved function as a biological enrichment tool.Bachelor of Scienc
β-hairpin peptides and WW domains designed for selective recognition of oligonucleotides
Get PDFProtein-nucleic acid interactions are crucial in a variety of biological processes. Protein interactions with single-stranded DNA are particularly important in DNA replication, repair, and telomere regulation. The interactions involved in the binding of a designed β-hairpin dimer, (WKWK)2, to ssDNA and dsDNA were previously explored, and the peptide was found to bind ssDNA with a Kd of 3 μM via a combination of aromatic and electrostatic interactions, whereas binding to duplex DNA was driven primarily by electrostatic interactions. In this work, the effects of folding and chirality were studied to determine factors that contribute to affinity and selectivity for ssDNA versus dsDNA. Binding studies showed that (1) folding is crucial for binding to both ss- and dsDNA and (2) chirality affects binding for duplex DNA but not for ssDNA. Taken together, these studies reveal different modes of binding for ss- and duplex DNA, with different driving forces, but in each case peptide structure contributes significantly to binding.
In another study, a β-sheet peptide based on a WW domain sequence was redesigned for the molecular recognition of ssDNA. A previous report showed that (WKWK)2 binds ssDNA with low micromolar affinity but with little selectivity over dsDNA. This work extends those studies to a three-stranded β-sheet designed to mimic the OB-fold. The new peptide binds ssDNA with low micromolar affinity and shows enhanced selectivity for
ssDNA. The redesigned peptide no longer binds its native ligand, the polyproline helix. This indicates that the peptide has been redesigned for the function of binding ssDNA.
Structural studies indicate that this peptide consists of a structured β-hairpin made of Strands 2&3 with a less structured strand 1, which provides affinity for ssDNA but does not improve the stability of the full peptide. Both function and stability are gained by incorporating a novel binding pocket into the peptide, and the redesigned peptide successfully mimics the OB-fold domain. Further mutations were made to design a mutant with increased structure, affinity, and selectivity for ssDNA. Knowledge gained from these binding and structural studies may lead to better designs of β-sheet peptides designed to target nucleotides, damaged DNA, and ssDNA
Design of β-hairpins and β-sheets for Molecular Recognition
Get PDFProtein-nucleic acid interactions are essential in a multitude of biological processes. Protein interactions with single-stranded DNA are particularly important in DNA replication, repair, and telomere regulation. Previously, our laboratory had designed a β-hairpin dimer, (WKWK)2 which binds ssDNA with a Kd of 3 μM and dsDNA with a Kd of 5 μM. These results later led to the redesign of a β-sheet peptide from a native protein which displayed 10-fold selectivity for dsDNA but overall lower affinity for ssDNA at a Kd of 20 μM. In this work, with the insight gained from these studies, a third de novo β-sheet was designed, S123. This new system was found to bind ssDNA with a dissociation constant of 170 nM. Several derivatives were investigated to determine the origins of the marked improvement in binding affinity. It was found that high β-sheet structure was necessary to achieve the observed nanomolar affinity of S123 to ssDNA. In another study, the use of the copper(I)-assisted azide-alkyne cycloaddition as a method of β-hairpin stabilization was investigated at several different positions. It was determined that the CuAAC reaction was a suitable method for locking in β-hairpin structure in peptides possessing the type I' turn, VNGO and the type II' turn, VpGO. All cyclic variants exhibited improved thermal stability and resistance to proteolysis as compared to the non-cyclic peptides. Additionally, the function of the CuAAC cyclized peptides was not altered as exhibited by similar binding affinities for ATP as the WKWK peptide. These studies provided a comprehensive method for CuAAC cyclization of β-hairpin peptides, which could further be utilized in the inhibition of protein-protein interactions.Doctor of Philosoph
Interactions between chromodomains and trimethyllysine marks on histone H3 peptides
Get PDFRecent findings suggest that a variety of post-translational modifications (PTMs) found on N-terminal tails of histones are intricately involved in DNA packaging and directly control levels of gene expression. These modifications include methyllysine, methylarginine, phosposerine, phosphothreonine, and acetyllysine. Methyllysine marks on the N-terminal tail histone H3 are known to recruit effector proteins that can modify chromatin structure and regulate gene expression. Trimethyllysine 4 of histone H3 recruits CHD1 (chromo-ATPase/helicase-DNA binding domain 1), which is part of a chromatin-remodeling complex associated with active transcription. Additionally, trimethyllysine 9 of histone H3 recruits heterochromatin protein 1alpha(HP1alpha), which stabilizes heterochromatin, which is typically associated with repressed gene expression. I investigated the protein-protein interactions in each of these complexes to explore the driving force and the selectivity of recognition. The tandem chromodomain of CHD1 binds H3 K4Me3 with an aromatic cage consisting of two tryptophan residues (Trp64 and Trp67) forming cation-gamma interactions with the trimethyllysine. Arginine 2 of histone H3 is involved in an H-bond with the backbone of Gly66 of the tandem chromodomain and a cation-gamma interaction with Trp67. The effect of incorporating methylarginine and citrulline at position 2 in H3 K4Me3 on CHD1 binding affinity was explored. The results show that symmetric dimethylarginine and citrulline weakened binding affinity while asymmetric dimethylarginine enhanced binding affinity. This study demonstrates the significance of these three modifications and how they may play a role in regulating gene expression by affecting protein-protein interactions. The HP1alpha chromodomain binds to H3 K9Me2 with a KD of 20 muM H3 K9Me3 with a KD of 17 muM. The chromodomain contains a three-membered aromatic cage and a glutamate (Glu52) around di- and trimethyllysine. The aromatic residues are involved in a cation-gamma interaction with methyllysine and Glu52 forms a water-mediated H-bond to dimethyllysine. The histone tail is also inserted between two beta-strands of the chromodomain to form a 3-stranded beta-sheet. First, Glu52 was modified to enhance selectivity for H3 K9Me3 over H3 K9Me2. The E52Q mutant had a 2.5 fold weaker binding affinity to H3 K9Me2 (KD=52 muM) and maintained the same binding affinity to H3 K9Me3 (KD=15 muM) most likely because glutamine is a weak H-bond acceptor compared to glutamate. Second, beta-sheet interactions between HP1alpha chromodomain and H3 K9Me3 were investigated. Residue Thr6 of the histone tail forms cross-strand interactions with Ala25 and Asp62 of the chromodomain. Each of these three residues was systematically substituted for amino acids known to have high beta-sheet propensity and form favorable sidechain-sidechain interactions. These studies demonstrated the applicability of information gleaned from model systems and statistical studies to protein-protein recognition. Lastly, two PTM-recognition domains, derived from naturally occurring effector proteins were coupled together to create a coupled-receptor construct to visualize dual modifications on a single histone tail. Two HP1alpha chromodomains were coupled to determine if they were functional for detecting a synthetic peptide with two H3 K9Me2 sequences as proof that coupled receptors can have cooperative binding for a peptide with two dimethyllysine marks. Taken together, these studies provide a new mechanistic insight into the proteinprotein interactions between chromodomains and methyllysine marks on N-terminal histone H3 tails, which are important for sequence selectivity and binding affinity
Design of Well-Folded β-Hairpin Peptides for Molecular Recognition of RNA and Improved Resistance to Proteolysis
Get PDFRibonucleic acid (RNA) plays a vital role in many biological processes of the cell, which
makes it an attractive target for drug discovery. In this work, small β-hairpin peptides were
used to better understand RNA molecular recognition. Specifically, peptides were designed
with the capability to bind via aromatic, electrostatic, and hydrogen bonding interactions to
the single stranded RNA.
To investigate RNA recognition using a conjugated system of different binding motifs, a
β-hairpin peptide was connected to an RNA intercalator. It was found that the individual
parts did not bind well to RNA, but the conjugated system binds RNA with a dissociation
constant of 3.7 μM. RNase footprinting experiments showed that the intercalator threads the
stem region while the peptide interacts with an internal bulge region, and that the β-hairpin
structure of the peptide is important for binding.
Combinatorial chemistry and de novo peptide design were used to improve binding of
the BIV Tat (bTat) peptide for BIV TAR RNA (bTAR). A combinatorial library was
designed which incorporated natural and unnatural residues in the N-terminal region of bTat
with the purpose of inducing interactions with the bTAR hairpin loop region. Two peptides
were isolated from fluorescent library screens, one which had one less charge than bTat and
contained an unnatural homophenylalanine residue. It was determined by gel shift assays
that the two peptides bound equally as well to bTAR as the native sequence. In a separate
project, strong cation-π side chain interactions were used to promote β-hairpin structure of a
bTAR-binding peptide without the need for cyclization. The side-chain interactions were
enough to stabilize the hairpin structure; however some RNA binding affinity was lost.
To successfully develop peptide drugs that bind RNA, the receptors need to be stable to
proteolytic degradation. For this purpose, it was investigated whether stable β-hairpin
peptides have increased resistance to proteolysis due to their structure. A series of peptides
with ranging amount of thermodynamic stability were designed and digested using a variety
of specific and non-specific proteases. It was determined that increased thermodynamic
stability of the β-hairpin peptides does correlate to an increase in proteolytic stability
Non-covalent interactions in β-hairpin peptides and small molecule model systems
Get PDFPost-translational modifications of proteins are a key component of cell signaling. In particular, post-translational modifications of histone tails are known to modulate transcription of DNA. These modifications function via a number of different noncovalent interactions with both DNA and various nuclear proteins. Some relevant modifications include methylation or acylation of lysine and the methylation of arginine and are thought to trigger binding with aromatic rings (Trp, DNA bases) through cation-pi and amide-pi interactions. In order to characterize these biologically relevant interactions, they have been studied within the context of beta-hairpin peptide model systems, which enable the description and quantification of specific sidechain-sidechain interactions. In our system, the interaction between Trp and trimethylated Lys is shown to be worth 1.0 kcal/mol, a stabilization of about 0.7 kcal/mol over the nonmethylated Lys-Trp interaction. The methylated interaction occurs with an enhanced entropic driving force over the unmethylated interaction. Methylation of Arg is also shown to enhance its interaction with Trp, by about 0.5 kcal/mol. Additionally, the acyl Lys-Trp interaction is found to be equivalent in magnitude to the nonmethylated Lys-Trp interaction (0.3 kcal/mol). Acylation of lysine in our model system is shown to induce a switch from a cation-pi to an amide-pi interaction. Investigations into neutral analogues of trimethylated Lys reveals the critical nature of the cation-pi interaction to the interaction of histone tails with chromodomain proteins. This is confirmed both in our beta-hairpin model system and with binding studies with the HP1 chromodomain. Additional investigations in this thesis include mutational studies of a -hairpin receptor for ATP and the study of a cation-pi interaction within a small molecule model system. Through experimental and computational studies, the -hairpin receptor for ATP is shown to form a well-defined binding pocket with a number of important electrostatic contacts. The cation-pi interaction in the small molecule model system is found to prevail in both aqueous and organic solvent, despite the possible presence of competing noncovalent interactions. X-ray and computational evidence suggests the possible presence of an oxy-arene interaction in organic solvent, but the interpretation of conformational differences from NMR data is ambiguous
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