Biochemistry Commons^™

Visible-Light Photooxidation In Water By 1o2-Generating Supramolecular Hydrogels, Sankarsan Biswas, Mohit Kumar, Andrew Levine, Ian Jimenez, Rein V. Ulijn, Adam Braunschweig

Advanced Science Research Center

We report supramolecular photocatalytic hydrogels, produced by the enzymatically driven self-assembly of low molecular weight gelators (LMWGs). These LMWG precursors are composed of the organic chromophore diketopyrrolopyrrole (DPP), which is bi-functionalized with a series of amino acid (Phe, Tyr, Leu) methyl esters. In situ enzymatic hydrolysis of these photoactive precursors results in supramolecular hydrogels that provide a high density of photocatalytic sites. Under visible light irradiation these hydrophobic fibers recruit the reaction substrates and also produce ¹O₂, which is used here for the photooxidation of thioanisole (aromatic substrate) and cyclohexyl methyl sulfide (aliphatic substrate), with yields as …

Parp1-Targeted Radiotherapies, Stephen Jannetti

Dissertations, Theses, and Capstone Projects

Poly-ADP-ribosylation reactions were first reported by Chambon in 1963 as enzymatic activity that increases incorporation of ATP in the presence of nicotinamide mononucleotide. In the decades since that publication, Poly(ADP-ribose)polymerase 1 (PARP1) and the PARP family enzymes have been widely studied. PARP enzymes are currently known to play various roles in mammals, including anti-aging processes, interactions with Breast Cancer Suppressor Protein-1 (BRCA1), and DNA damage repair. A significant focus of PARP1 research has been elucidating its role in DNA damage repair. PARP1 is recruited to repair single strand DNA (ssDNA) breaks, which can become double stranded DNA (dsDNA) breaks if …

Leveraging Antibodies For Positron Emission Tomography And Near-Infrared Fluorescence Imaging Of Cancer, Kimberly C. Fung

Dissertations, Theses, and Capstone Projects

The high specificity and affinity of antibodies make them attractive for developing drugs to diagnose and treat cancer. The overarching goal of this work is to explore the synthesis and use of antibody-based imaging agents in preclinical models of cancer. This work can be described as two-fold. In the first part, we investigated how the use of a glycans-specific bioconjugation strategy affects Fc gamma RI binding and why it results in improved in vivo performance of immunoconjugates. To do so, we used the clinically relevant positron emission tomography (PET) imaging agent, ⁸⁹Zr-DFO-pertuzumab, in mouse models of human breast cancer. …

Advanced Computational Methodologies To Study Binding Free Energies Of Biomolecular Complexes, Rajat Kumar Pal

Dissertations, Theses, and Capstone Projects

Molecular recognition is the basis of biological mechanisms and is a key element to consider while formulating effective and safe drugs. Pharmaceutical drugs are designed so as to bind a target protein even at very low concentrations to alter the diseased conditions without interfering with normal biological processes. In a rational drug design process, this is achieved by acquiring information about the chemical structure and the physical and chemical properties of the target protein receptor to gain insights on how changing the chemical composition of the substrate drug could affect the protein-drug interaction and binding affinities. Computational models are used …

Hur Ubiquitination By Brca1/Bard1 And Its Role In Controlling Gene Expression During Dna Damage Response, Gamage M. Aruggoda

Dissertations, Theses, and Capstone Projects

Despite our understanding of the biochemistry of the BRCA1/BARD1 E3 ligase, we know very little of the mechanism by which its cellular substrates, how they are chosen, how its E3 Ub ligase activity is regulated, which are the cofactors involved in this regulation, and how the substrates are modified during DDR. In this dissertation, I further evaluate BRCA1/BARD1-mediated E3 ligase activity and how CstF-50 and p97 regulate this reaction working as cofactors. My data show that BRCA1/BARD1 is an E3 ligase that ubiquitinates the RNA binding protein Human antigen R(HuR).

HuR is abundant in the nucleus and is transported to …

Mutational Analysis And Domain Characterization Of The Apolipoprotein L-1 Ion Channel, Charles M. Schaub

Dissertations, Theses, and Capstone Projects

The human innate immunity factor Apolipoprotein L-1 (APOL1) protects against Trypanosoma brucei brucei infection. Recent studies have shown recombinant APOL1 (rAPOL1) inserts into planar lipid bilayers at an acidic pH 5.6 and forms a cation-selective channel, which opens upon subsequent neutralization, pH 7.2. This corresponds with the pH changes APOL1 would encounter during endosome recycling, suggesting that APOL1 forms a pH-gated ion channel in the plasma membrane of the parasite, leading to uncontrolled ion flux and osmotic imbalance. However, structural and domain characteristics of the APOL1 channel are poorly understood, despite potential similarities to diphtheria and colicin toxins. Utilizing E. …

Through The Back Door: Proteins Escape Cells Without Conventional Permission, Michael J. Cohen

Dissertations, Theses, and Capstone Projects

Proteins secreted to the extracellular environment play a fundamental role as signals, in metabolism, and a variety of other processes. The process of secretion through the endoplasmic reticulum and Golgi to the plasma membrane is well documented, and all cargo in this pathway contains a signal peptide. However, a variety of proteins secreted from eukaryotes lack a signal peptide and are called unconventionally secreted proteins. Here we discuss known mechanisms of unconventional protein secretion, as well as model proteins which follow characterized pathways. Additionally, we summarize the roles various unconventionally secreted proteins play outside of cells and suggest criteria for …

A Review Of Small Molecule Inhibitors And Functional Probes Of Human Cathepsin L, Dibyendu Dana, Sanjai Kumar

Publications and Research

Human cathepsin L belongs to the cathepsin family of proteolytic enzymes with primarily an endopeptidase activity. Although its primary functions were originally thought to be only of a housekeeping enzyme that degraded intracellular and endocytosed proteins in lysosome, numerous recent studies suggest that it plays many critical and specific roles in diverse cellular settings. Not surprisingly, the dysregulated function of cathepsin L has manifested itself in several human diseases, making it an attractive target for drug development. Unfortunately, several redundant and isoform-specific functions have recently emerged, adding complexities to the drug discovery process. To address this, a series of chemical …

Hijacking The Fusion Complex Of Human Parainfluenza Virus As An Antiviral Strategy, T. C. Marcink, E. Yariv, K. Rybkina, V. Más, F. T. Bovier, A. Des Georges, A. L. Greninger, C. A. Alabi, M. Porotto, N. Ben-Tal, A. Moscona

Advanced Science Research Center

The receptor binding protein of parainfluenza virus, hemagglutinin- neuraminidase (HN), is responsible for actively triggering the viral fusion protein (F) to undergo a conformational change leading to insertion into the target cell and fusion of the virus with the target cell membrane. For proper viral entry to occur, this process must occur when HN is engaged with host cell receptors at the cell surface. It is possible to interfere with this process through premature activation of the F protein, distant from the target cell receptor. Conformational changes in the F protein and adoption of the postfusion form of the protein …

Correction: Visible-Light Photooxidation In Water By 1o2-Generating Supramolecular Hydrogels, Sankarsan Biswas, Mohit Kumar, Andrew Levine, Ian Jimenez, Rein V. Ulijn, Adam Braunschweig

Advanced Science Research Center

Correction for ‘Visible-light photooxidation in water by ¹O₂-generating supramolecular hydrogels’ by Sankarsan Biswas et al., Chem. Sci., 2020, 11, 4239–4245, DOI: 10.1039/C9SC06481H.

Polymer Brush Hypersurface Photolithography, Carlos Carbonell, Daniel J. Valles, Alexa M. Wong, Andrea S. Carlini, Mollie A. Touve, Joanna Korpanty, Nathan C. Gianneschi, Adam B. Braunschweig

Advanced Science Research Center

Polymer brush patterns have a central role in established and emerging research disciplines, from microarrays and smart surfaces to tissue engineering. The properties of these patterned surfaces are dependent on monomer composition, polymer height, and brush distribution across the surface. No current lithographic method, however, is capable of adjusting each of these variables independently and with micrometer-scale resolution. Here we report a technique termed Polymer Brush Hypersurface Photolithography, which produces polymeric pixels by combining a digital micromirror device (DMD), an air-free reaction chamber, and microfluidics to independently control monomer composition and polymer height of each pixel. The printer capabilities are …

A Kinesin Adapter Directly Mediates Dendritic Mrna Localization During Neural Development In Mice, Hao Wu, Jing Zhou, Tianhui Zhu, Ivan Cohen, Jason Dictenberg

Publications and Research

Motor protein-based active transport is essential for mRNA localization and local translation in animal cells, yet how mRNA granules interact with motor proteins remains poorly understood. Using an unbiased yeast two–hybrid screen for interactions between murine RNA-binding proteins (RBPs) and motor proteins, here we identified protein interaction with APP tail-1 (PAT1) as a potential direct adapter between zipcode-binding protein 1 (ZBP1, a β-actin RBP) and the kinesin-I motor complex. The amino acid sequence of mouse PAT1 is similar to that of the kinesin light chain (KLC), and we found that PAT1 binds to KLC directly. Studying PAT1 in mouse …

Inclusion Of Enclosed Hydration Effects In The Binding Free Energy Estimation Of Dopamine D3 Receptor Complexes, Rajat Kumar Pal, Satishkumar Gadhiya, Steven Ramsey, Pierpaolo Cordone, Lauren Wickstrom, Wayne W. Harding, Tom Kurtzman, Emilio Gallicchio

Publications and Research

Confined hydration and conformational flexibility are some of the challenges encountered for the rational design of selective antagonists of G-protein coupled receptors.We present a set of C3-substituted (-) -stepholidine derivatives as potent binders of the dopamine D3 receptor.The compounds are characterized biochemically, as well as by computer modeling using a novel molecular dynamics-based alchemical binding free energy approach which incorporates the effect of the displacement of enclosed water molecules from the binding site.The free energy of displacement of specific hydration sites is obtained using the Hydration Site Analysis method with explicit solvation. This work underscores the critical role of confined …

Bright Green Biofluorescence In Sharks Derives From Bromo-Kynurenine Metabolism, Hyun Bong Park, Yick Chong Lam, Jean P. Gaffney, James C. Weaver, Sara Rose Krivoshik, Randy Hamchand, Vincent Pieribone, David F. Gruber, Jason M. Crawford

Publications and Research

Although in recent years there has been an increased awareness of the widespread nature of biofluorescence in the marine environment, the diversity of the molecules responsible for this luminescent phenotype has been mostly limited to green fluorescent proteins (GFPs), GFP-like proteins, and fluorescent fatty acid-binding proteins (FABPs). In the present study, we describe a previously undescribed group of brominated tryptophan-kynurenine small molecule metabolites responsible for the green biofluorescence in two species of sharks and provide their structural, antimicrobial, and spectral characterization. Multi-scale fluorescence microscopy studies guided the discovery of metabolites that were differentially produced in fluorescent versus non-fluorescent skin, as …

Minimalistic Peptide-Based Supramolecular Systems Relevant To The Chemical Origin Of Life, Daniela Kroiss

Dissertations, Theses, and Capstone Projects

All forms of life are based on biopolymers, which are made up of a selection of simple building blocks, such as amino acids, nucleotides, fatty acids and sugars. Their individual properties govern their interactions, giving rise to complex supramolecular structures with highly specialized functionality, including ligand recognition, catalysis and compartmentalization. In this thesis, we aim to answer the question whether short peptides could have acted as precursors of modern proteins during prebiotic evolution. Using a combination of experimental and computational techniques, we screened a large molecular search space for peptide sequences that are capable of forming supramolecular complexes with adenosine …

Exploration Of The Inhibitory Properties Of The Nucleoside Antibiotic Salicyl-Ams And Analogues Targeting Siderophore Biosynthesis In Mycobacterium Tuberculosis, Glennon Bythrow

Dissertations, Theses, and Capstone Projects

Mycobacterium tuberculosis (Mtb) is a resilient, obligate bacterial pathogen responsible for pulmonary tuberculosis disease (TB), that has upheld a significant impact on global public health throughout history. The World Health Organization (WHO) approximates nearly 10 million new TB cases arose in 2017 alone, accounting for 1.6 million deaths. There has been a notable rise in TB cases produced by multidrug‑resistant (MDR) and extensively drug-resistant (XDR) strains of Mtb. This, along with the intrinsic resistance of Mtb to many standard drugs and poor patient compliance, is deeply impacting global control of TB. Among the several strategies currently in …

Developing A Dissociative Nanocontainer For Peptide Drug Delivery, Michael Patrick Kelly

Dissertations, Theses, and Capstone Projects

The potency and specificity of bioactive peptides have propelled these agents to the forefront of pharmacological research. However, delivery of peptides to their molecular target in cells is a major obstacle to their widespread application. A Trojan Horse strategy of packaging a bioactive peptide within a modified protein cage to protect it during transport, and releasing it at the target site, is a promising delivery method. Recent work has demonstrated that the viral capsid of the P22 bacteriophage can be loaded with an arbitrary, genetically-encoded peptide, and externally decorated with a cell-penetrating peptide, such as HIV-Tat, to translocate across in …

Innate Antibodies, Murine Models, And Evolution: A Study Of Trypanosome Lytic Factor Functions And Their Translational Applications, Joseph P. Verdi

Dissertations, Theses, and Capstone Projects

Trypanosome lytic factors (TLFs) are primate-specific antimicrobial protein complexes that lyse African trypanosome parasites by delivering the channel-forming toxin APOL1 to the invading microorganisms. Human serum contains two TLFs that are delivered to the parasite by separate mechanisms, only one of which has been characterized. TLF1 is endocytosed by a receptor that is typically blocked by other serum factors in vivo, suggesting that TLF2 is the more relevant lytic factor in the context of trypanosome immunity. TLF2 is non-covalently associated with polyclonal immunoglobulin M (IgM) antibodies, which we report here to be involved in the uptake mechanism. The TLF2-IgMs …

An Exploration Of Protein And Dna Components In Fingerprint Residue, Ashley Borrego

Student Theses

The main focus of this project was to investigate the protein and DNA components in both sebaceous and eccrine fingerprints. This study investigated the relative content of DNA and proteins in eccrine fingerprints to sebaceous fingerprints. All volunteers were instructed to wash and dry their hands prior to depositing parallel thumbprints. Twenty volunteers were instructed to touch their face to produce sebaceous prints, and 5 volunteers were instructed to wear gloves over a heat source to produce sweaty or eccrine prints. Microscopy was used to score the cellular debris of the right fingerprint on a scale of 1-4 based on …

Synthesis And Evaluations Of “1,4-Triazolyl Combretacoumarins” And Desmethoxy Analogs, Tashrique A. Khandaker, Jessica D. Hess, Renato Aguilera, Graciela Andrei, Robert Snoeck, Dominique Schols, Padmanava Pradhan, Mahesh K. Lakshman

Publications and Research

1,4-Triazolyl combretacoumarins have been prepared by linking the trimethoxyarene unit of combretastatin A4 with coumarins, via a 1,2,3-triazole. For this, 4-azidocoumarins were accessed by a sequential two-step, one-pot reaction of 4-hydroxycoumarins with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), followed by reaction with NaN₃. In the reaction with BOP, a coumarin-derived phosphonium ion intermediate seems to form, leading to an O⁴-(benzotriazolyl)coumarin derivative. For the CuAAC reaction of azidocoumarins with 5-ethynyl-1,2,3-trimethoxybenzene, catalytic [(MeCN)₄Cu]PF₆ in CH₂Cl₂/MeOH with 2,6-lutidine, at 50 ^oC, was suitable. The 4-azidocoumarins were less reactive as compared to PhN₃ and …

Reduced Alphabet Of Prebiotic Amino Acids Optimally Encodes The Conformational Space Of Diverse Extant Protein Folds, Armando D. Solis

Publications and Research

Background

There is wide agreement that only a subset of the twenty standard amino acids existed prebiotically in sufficient concentrations to form functional polypeptides. We ask how this subset, postulated as {A,D,E,G,I,L,P,S,T,V}, could have formed structures stable enough to found metabolic pathways. Inspired by alphabet reduction experiments, we undertook a computational analysis to measure the structural coding behavior of sequences simplified by reduced alphabets. We sought to discern characteristics of the prebiotic set that would endow it with unique properties relevant to structure, stability, and folding.

Results

Drawing on a large dataset of single-domain proteins, we employed an information-theoretic measure …

Global Biochemical And Structural Analysis Of The Type Iv Pilus From The Gram-Positive Bacterium Streptococcus Sanguinis, Jamie-Lee Berry, Ishwori Gurung, Jan Haug Anonsen, Ingrid Spielman, Elliot Harper, Alexander M. J. Hall, Vivianne J. Goosens, Claire Raynaud, Michael Koomey, Nicolas Biais, Steve Matthews, Vladimir Pelicic

Publications and Research

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis. In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis. We found that the two major pilins, PilE1 and PilE2, (i) follow …

Solvation Thermodynamic Mapping In Computer Aided Drug Design, Steven Ramsey

Dissertations, Theses, and Capstone Projects

The displacement of water from surfaces upon biomolecular recognition and association makes a significant contribution to the free energy changes of these processes. We therefore posit that accurate characterization of local structural and thermodynamic molecular water properties can improve computational model accuracy and predictivity of recognition and association processes. In this thesis, we discuss Solvation Thermodynamic Mapping (STM) methods that we have developed using inhomogeneous fluid solvation theory (IST) to better characterize active site water structural and thermodynamic properties on protein surfaces and the open source tools that we have developed, GIST-CPPTRAJ and SSTMap, which implement these methods which we …

The Determining Factors Of The Self-Assembly Of Collagen Mimetic Peptide, Fangfang Chen

Dissertations, Theses, and Capstone Projects

We recently reported that a designed triple helix Col108 self-associates to form collagen-like mini-fibrils having a d-periodicity of 35 nm. Recombinant protein Col108 consists of 378-residue triple helix domain organized into three repeating sequence units, and a C-terminal foldon domain. The 35 nm d-period of Col108 mini-fibrils is consistent with the one unit staggered arrangement of the associating helices. To further investigate if the sequence periodicity is crucial for collagen mimetic peptide fibril formation, we studied the self-assembly of four other designed triple helical peptides. Peptide 2U108 has two repeating sequence units; peptide Col877 consists of three repeating …

Pretargeted Radioimmunotherapy Utilizing The Inverse Electron Demand Diels-Alder Reaction, Rosemery Membreno

Dissertations, Theses, and Capstone Projects

Radioimmunotherapy capitalizes on the specificity and affinity of antibodies for their antigens to localize radioactivity at the tumor site. One limitation in using antibodies is their long circulation time, which can take days to weeks to fully clear from blood circulation. When a radioisotope is directly conjugated to this immunoglobulin vector, the red marrow and other healthy organs are receiving a constant radiation dose while it is in blood circulation. This not only harms healthy organs, but limits the dose of radioactivity that can be administered thus prolonging treatment. A promising alternative to limit the radiation dose to non-tumor tissue …

Bisthioether Stapled Peptides Targeting Polycomb Repressive Complex 2 Gene Repression, Gan Zhang

Dissertations, Theses, and Capstone Projects

Interactions between proteins play a key role in nearly all cellular process, and therefore, disruption of such interactions may lead to many different types of cellular dysfunctions. Hence, pathologic protein-protein interactions (PPIs) constitute highly attractive drug targets and hold great potential for developing novel therapeutic agents for the treatment of incurable human diseases. Unfortunately, the identification of PPI inhibitors is an extremely challenging task, since traditionally used small molecule ligands are mostly unable to cover and anchor on the extensive flat surfaces that define those binary protein complexes. In contrast, large biomolecules such as proteins or peptides are ideal fits …

Ck2 Negatively Regulates 5-Ht4 Receptor Signaling In The Prefrontal Cortex And Mediates Depression-Like Behaviors, Julia Castello Saval

Dissertations, Theses, and Capstone Projects

The serotonergic system has been the major candidate in the pathophysiology of mood related disorders such as anxiety and major depressive disorder (MDD). Unfortunately, current antidepressant drugs are ineffective in 50% of the population and require chronic administration for a period of 3-6 weeks before the onset of therapeutic response. 5-HT4 receptor (5-HT₄R) agonists have emerged as potential candidates for fast antidepressant action, since an antidepressant response can be achieved after 3 days of pharmacological administration in rodents.

This dissertation aims to investigate the role of casein kinase 2 (CK2) as a regulator of 5-HT₄R expression …

Characterization Of Immunomodulatory Microbial Factors In Medicinal Plants, Kriti Kalpana

Dissertations, Theses, and Capstone Projects

Medicinal plants are one of the biggest sources of natural products with therapeutic importance. There are currently over 28,000 plants with putative medicinal values. Plant-derived compounds have been explored extensively for various biological activities ranging from anti-cancer, immune-boosting to anti-inflammatory and anti-oxidant. Some of the most important therapeutic agents are of plant-origin, such as paclitaxel from Pacific yew (Taxus brevifolia) and artemisinin from qinghao su (a Chinese medicinal herb; a.k.a. Artemisia annua) to name a few.

The study presented in this thesis started out as classical pharmacognosy research, which focused on the identification of immunostimulatory factors in …

Structural Studies On Calcium/Calmodulin-Dependent Activation Of Eukaryotic Elongation Factor 2 Kinase, Kwangwoon Lee

Dissertations, Theses, and Capstone Projects

Eukaryotic elongation factor 2 kinase (eEF-2K) is a key modulator of the rate of protein synthesis. Activated by calcium-loaded calmodulin (Ca²⁺-CaM), eEF-2K phosphorylates its only known physiological substrate, eEF-2, on a specific threonine residue (Thr-56). Phosphorylated eEF-2 has reduced affinity for the ribosome, and results in a significant decrease in the rate of translation elongation. Modulation of the rate of translation elongation plays a crucial role in proteostasis – adequate regulation of protein synthesis, protein folding, and protein degradation that greatly influences cellular growth and survival. Binding of Ca²⁺-CaM triggers activation of eEF-2K and remains intact …

Deletion Of Mgr2p Affects The Gating Behavior Of The Tim23 Complex, Oygul Mirzalieva, Shinhye Jeon, Kevin Damri, Ruth Hartke, Layla Drwesh, Keren Demishtein-Zohary, Abdussalam Azem, Cory D. Dunn, Pablo M. Peixoto

Publications and Research

The TIM23 complex is a hub for translocation of preproteins into or across the mitochondrial inner membrane. This dual sorting mechanism is currently being investigated, and in yeast appears to be regulated by a recently discovered subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This result and other findings suggested that Mgr2p controls the lateral release of inner membrane proteins harboring a stop-transfer signal that follows an N-terminal amino acid signal. However, the mechanism of lateral release is unknown. Here, we used patch clamp electrophysiology …