Repurposing triphenylmethane dyes to bind to trimers derived from Aß

Accepted author manuscriptSoluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimer’s disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ17–36. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N,N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe19, Phe20, and Ile31 were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe20 side chains and is clasped by the Ile31 side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.Ye

A tandem enzymatic sp2-C-methylation process : coupling in situ S-adenosyl-L-methionine formation with methyl transfer

A one-pot, two-step biocatalytic platform for the regiospecfic C-methylation and C-ethylation of aromatic substrates is described. The tandem process utilizes SalL (Salinospora tropica) for in situ synthesis of S-adenosyl-L-methionine (SAM), followed by alkylation of aromatic substrates using the C-methyltransferase NovO (Streptomyces spheroides). Application of this methodology is demonstrated by the regiospecific labelling of aromatic substrates via the transfer of methyl, ethyl and isotopically-labelled 13CH3, 13CD3 and CD3 groups from their corresponding SAM analogues formed in situ

Structural and functional study of Adenosine deaminase acting on RNA

Adenosine deaminases acting on RNA (ADAR) are a family of enzymes responsible for the conversion of adenosine to inosine in double stranded RNA. Inosine selectively base pairs with cytidine and is recognized like guanosine by cellular machinery. A-to-I editing has a myriad of downstream effects and dysregulated editing by ADARs is observed in multiple neurological and oncogenic diseases. In humans there are three ADAR enzymes, named ADAR1, ADAR2 and ADAR3, each with their own unique polypeptide sequences and RNA substrate preferences. There is great interest in uncovering the structural basis of RNA editing by ADAR with the potential to accelerate the development of targeted therapeutics to restore homeostatic levels of RNA editing in diseased patients. This thesis describes the effort to understand the structural basis behind ADAR and RNA interactions. Chapter 1 will provide an overview of what is known about RNA editing and describe the ways in which it is implicated in human disease. Chapter 2 describes the structural elucidation of an asymmetric protein-protein dimer of ADAR and how its formation aids in forming an RNA-bound complex. The biophysical characteristics of this protein-protein dimer will also be discussed, in addition to the identification of key conserved amino acid residues that are important for the formation of the protein-protein dimer. Chapter 3 describes the work towards the structural elucidation of the deaminase domain of ADAR1 bound to double stranded RNA. Current strategies to solve the crystal structure of ADAR1 bound to RNA will be discussed. A low resolution data set was collected. Information gained from the low-resolution model, how it was generated and strategies to optimize crystallization and diffraction to obtain a high-quality diffraction dataset will also be discussed. Chapter 4 will describe structural studies of ADAR2’s deaminase domain and an RNA duplex with a non-ideal nearest neighbor. Additionally, this chapter will discuss collaborative efforts towards the study of full-length ADAR2 bound to a double stranded RNA will be discussed

Structural and functional study of Adenosine deaminase acting on RNA

Adenosine deaminases acting on RNA (ADAR) are a family of enzymes responsible for the conversion of adenosine to inosine in double stranded RNA. Inosine selectively base pairs with cytidine and is recognized like guanosine by cellular machinery. A-to-I editing has a myriad of downstream effects and dysregulated editing by ADARs is observed in multiple neurological and oncogenic diseases. In humans there are three ADAR enzymes, named ADAR1, ADAR2 and ADAR3, each with their own unique polypeptide sequences and RNA substrate preferences. There is great interest in uncovering the structural basis of RNA editing by ADAR with the potential to accelerate the development of targeted therapeutics to restore homeostatic levels of RNA editing in diseased patients. This thesis describes the effort to understand the structural basis behind ADAR and RNA interactions. Chapter 1 will provide an overview of what is known about RNA editing and describe the ways in which it is implicated in human disease. Chapter 2 describes the structural elucidation of an asymmetric protein-protein dimer of ADAR and how its formation aids in forming an RNA-bound complex. The biophysical characteristics of this protein-protein dimer will also be discussed, in addition to the identification of key conserved amino acid residues that are important for the formation of the protein-protein dimer. Chapter 3 describes the work towards the structural elucidation of the deaminase domain of ADAR1 bound to double stranded RNA. Current strategies to solve the crystal structure of ADAR1 bound to RNA will be discussed. A low resolution data set was collected. Information gained from the low-resolution model, how it was generated and strategies to optimize crystallization and diffraction to obtain a high-quality diffraction dataset will also be discussed. Chapter 4 will describe structural studies of ADAR2’s deaminase domain and an RNA duplex with a non-ideal nearest neighbor. Additionally, this chapter will discuss collaborative efforts towards the study of full-length ADAR2 bound to a double stranded RNA will be discussed

A Glycal-Based Photoaffinity Probe That Enriches Sialic Acid Binding Proteins

To identify sialic acid binding proteins from complex proteomes, three photocrosslinking affinity-based probes were constructed using Neu5Ac (5 and 6) and Neu5Ac2en (7) scaffolds. Kinetic inhibition assays and Western blotting revealed the Neu5Ac2en-based 7 to be an effective probe for the labeling of a purified gut microbial sialidase (BDI_2946) and a purified human sialic acid binding protein (hCD33). Additionally, LC-MS/MS affinity-based protein profiling verified the ability of 7to enrich a low-abundance sialic acid binding protein (complement factor H) from human serum thus validating the utility of this probe in a complex context.</p

Ligand-Accelerated Cross-Coupling of C(sp²)–H Bonds with Arylboron Reagents

A ligand-accelerated Pd(II)-catalyzed C(sp²)–H/arylboron cross-coupling reaction of phenylacetic acid substrates is reported. Using Ac-Ile-OH as the ligand and Ag₂CO₃ as the oxidant, a fast, high-yielding, operationally simple, and functional group-tolerant protocol has been developed for the cross-coupling of phenylacetic acid substrates with aryltrifluoroborates. This ligand scaffold has also been shown to improve catalysis using 1 atm O₂ as the sole reoxidant, which sheds light on the path forward in developing optimized ligands for aerobic C–H/arylboron cross-coupling