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Natural products, such as fatty acids, polyketides, and non-ribosomal peptides, exhibit diverse biological functions toward human health and are constructed via many different pathways; however, these pathways often share the same synthetic logic. Central to these pathways is a carrier protein (CP). The role of a CP is to transfer elongating intermediates between catalytic domains and is mediated by protein-protein interactions between the CP and its partner enzymes. These interactions are transient, making it difficult to understand how they communicate with each other in recognizing a cargo or how the CP meets the right partner enzymes. The Burkart laboratory has been developing fluorescent and mechanistic probes to understand these CP-partner protein interactions. The CP requires post-translational modification to become an active form through the action of 4'-phosphopantetheinyl transferase (PPTase) loading 4'phosphopantetheine prosthetic (PPant) arm at the serine residue. The first part of this dissertation focuses on utilizing the PPTase function to search for (1) the minimum peptide substrate required for CP recognition via machine learning and (2) the biosynthetic pathway from unculturable microorganisms. Using the ability of some PPTases to recognize and transfer an unnatural PPant arm to a CP, we identified orthogonal peptide substrates that can be labeled by two different classes of PPTases. These peptide substrates can be appended to a protein and used as a peptide tag. Furthermore, we employed a PPTase to fluorescently label CPs in previously uncultured microorganisms. This enabled us to sequence single cells for the identification of a biosynthetic cluster with active PPTase-CP pairs. In the second part of this dissertation, we developed two mechanistic probes to study protein-protein interactions between epimerization (E) domain and peptidyl carrier protein in non-ribosomal peptide synthetases. D-amino acids are incorporated into non-ribosomal peptides, which contribute to their unique conformation and bioactivity. The E domains convert L- to D-amino acids by deprotonating/reprotonating C[alpha]-H. Despite the past research on the E domain, the mechanistic details remain unclear. Herein, with the help of molecular dynamics simulations and mutational studies, our research reveals more detailed evidence on which catalytic residues work as an acid/base in this mechanism.
Within the last decade, efforts have revealed remarkable detail in the machines that regulate primary metabolism with salient examples including the ribosome, protein folding and most recently the spliceosome. Our laboratory has taken interest in the macromolecular modular machines that construct fatty acids, polyketides and non-ribosomal peptides. The machines that prepare these classes of secondary metabolites share a common choreographed method. Small molecules such as acetyl-coenzyme A (CoA) and malonyl-CoA are assembled sequentially to form complex natural products, which have uses ranging from commodity chemicals to therapeutics. Although the starter units of these synthases are free floating molecules, their intermediates are tethered to acyl carrier protein (ACP). The ACP carries this cargo along the assemblyline of modifying enzymes (i.e., ketoreductase (KR), dehydratase (DH), enoyl-acyl carrier protein reductase (ER)) until it is release by a thioesterase (TE) domain. While this modular machinery appears ideal for metabolic engineering, many of the leading efforts, such as domain swapping, have been met with limited success. This arises, in part, from our lack in understanding the protein-protein interactions that guide the processivity between the CP and its associated partner domains (KS, KR, ER, DH and TE). Unfortunately, structural studies on these systems continue to pose challenges due to the transient nature of these interactions. The first couple chapters describes how new small molecule inhibitors were discovered to inhibit the ER in the fatty acid biosynthesis pathway in Plasmodium falciparum, the causitive agent responsible for malaria. These chapters describe in detail the collaborative work between in silico and in vitro methods to discover novel small molecules that have been from repositioned and commercial small libraries. The next chapter discuses an extension of the our laboratory's work dedicated to developing a suite of tools to study the interactivity between CP and associated partner domains. We first sought to leverage our previous work with covalent mechanism-based inactivators with the KS, TE and DH domains, and apply this to FabI. Because the reaction catalyzed by FabI is cofactor dependent and does not involve any active site residues in a covalent manner, designing a mechanistic based inactivator that covalently crosslinks AcpP and FabI poses a challenge. We hypothesized that if we attached a tight non-covalent FabI binder to AcpP via our chemoenzymatic methods, the binding affinity may be high enough to stabilize the complex for structural studies. The study extends our collection of chemoenzymatic AcpP tools with the first "inhibitor-based non-covalent probe" inspired by a well-characterized FabI tight-binding small molecule inhibitor. We synthesized a pantetheinamide derivative of TCL, appended it to AcpP and used this to study FabI-AcpP interactions. This approach shows that both protein-protein interactions and protein-substrate interactions are important for productive catalysis. We envision the use of this novel probe for structural characterization of the AcpP-FabI interaction, which has yet to be resolved in greater detail. The last chapter describes an expansion and discovery of new substrates for the the promiscuous 4'-phosphopantetheinyl transferase (PPTase) and acyl carrier protein hydrolase (AcpH) by artificial intelligence. With the development of conjugated-protein therapeutics over the last two decades, the need for robust protein labeling methods has intensified. Unlike conventional large fusion tag proteins, which can interfere with protein activity, a peptide tag only adds 8-20 amino acids to either terminus of a protein. Previous studies demonstrate that the post-translational enzyme, phosphopantetheinyl transferases (PPTase), can label YbbR, a short 11 amino acid sequence. More recently, our lab expanded on this system by demonstrating that the acyl carrier protein hydrolase (AcpH) can unlabel short peptide substrates. Our research highlights the discovery of peptide sequences that can be labeled selectively by either Sfp (PPTase 1) or AcpS (PPTase 2). and unlabeled by AcpH to obtain an orthogonal and reversible labeling system. Additionally, we have developed a statistical method, called Peptide Optimization with Optimal Learning (POOL), for efficiently discovering minimal peptide substrates from our high-throughput peptide screens. By leveraging post-translational modifying enzymes, our project will allow for site-selective functionalization.
Biology for AP® courses covers the scope and sequence requirements of a typical two-semester Advanced Placement® biology course. The text provides comprehensive coverage of foundational research and core biology concepts through an evolutionary lens. Biology for AP® Courses was designed to meet and exceed the requirements of the College Board’s AP® Biology framework while allowing significant flexibility for instructors. Each section of the book includes an introduction based on the AP® curriculum and includes rich features that engage students in scientific practice and AP® test preparation; it also highlights careers and research opportunities in biological sciences.
Chemistry and chemical engineering have changed significantly in the last decade. They have broadened their scopeâ€"into biology, nanotechnology, materials science, computation, and advanced methods of process systems engineering and controlâ€"so much that the programs in most chemistry and chemical engineering departments now barely resemble the classical notion of chemistry. Beyond the Molecular Frontier brings together research, discovery, and invention across the entire spectrum of the chemical sciencesâ€"from fundamental, molecular-level chemistry to large-scale chemical processing technology. This reflects the way the field has evolved, the synergy at universities between research and education in chemistry and chemical engineering, and the way chemists and chemical engineers work together in industry. The astonishing developments in science and engineering during the 20th century have made it possible to dream of new goals that might previously have been considered unthinkable. This book identifies the key opportunities and challenges for the chemical sciences, from basic research to societal needs and from terrorism defense to environmental protection, and it looks at the ways in which chemists and chemical engineers can work together to contribute to an improved future.
Proteins are indispensable players in virtually all biological events. The functions of proteins are coordinated through intricate regulatory networks of transient protein-protein interactions (PPIs). To predict and/or study PPIs, a wide variety of techniques have been developed over the last several decades. Many in vitro and in vivo assays have been implemented to explore the mechanism of these ubiquitous interactions. However, despite significant advances in these experimental approaches, many limitations exist such as false-positives/false-negatives, difficulty in obtaining crystal structures of proteins, challenges in the detection of transient PPI, among others. To overcome these limitations, many computational approaches have been developed which are becoming increasingly widely used to facilitate the investigation of PPIs. This book has gathered an ensemble of experts in the field, in 22 chapters, which have been broadly categorized into Computational Approaches, Experimental Approaches, and Others.
Introduces readers to the chemical biology of plant biostimulants This book brings together different aspects of biostimulants, providing an overview of the variety of materials exploited as biostimulants, their biological activity, and agricultural applications. As different groups of biostimulants display different bioactivity and specificity, advances in biostimulant research is illustrated by different examples of biostimulants, such as humic substance, seaweed extracts, and substances with hormone-like activities. The book also reports on methods used to screen for new biostimulant compounds by exploring natural sources. Combining the expertise of internationally-renowned scientists and entrepreneurs in the area of biostimulants and biofertilisers, The Chemical Biology of Plant Biostimulants offers in-depth chapters that look at: agricultural functions and action mechanisms of plant biostimulants (PBs); plant biostimulants from seaweed; seaweed carbohydrates; and the possible role for electron shuttling capacity in elicitation of PB activity of humic substances on plant growth enhancement. The subject of auxins is covered next, followed closely by a chapter on plant biostimulants in vermicomposts. Other topics include: exploring natural resources for biostimulants; the impact of biostimulants on whole plant and cellular levels; the impact of PBs on molecular level; and the use of use of plant metabolites to mitigate stress effects in crops. Provides an insightful introduction to the subject of biostimulants Discusses biostimulant modes of actions Covers microbial biostimulatory activities and biostimulant application strategies Offers unique and varied perspectives on the subject by a team of international contributors Features summaries of publications on biostimulants and biostimulant activity The Chemical Biology of Plant Biostimulants will appeal to a wide range of readers, including scientists and agricultural practitioners looking for more knowledge about the development and application of biostimulants.
Concepts of Biology is designed for the introductory biology course for nonmajors taught at most two- and four-year colleges. The scope, sequence, and level of the program are designed to match typical course syllabi in the market. Concepts of Biology includes interesting applications, features a rich art program, and conveys the major themes of biology. The images in this textbook are grayscale.
A new focus on glycoscience, a field that explores the structures and functions of sugars, promises great advances in areas as diverse as medicine, energy generation, and materials science, this report finds. Glycans-also known as carbohydrates, saccharides, or simply as sugars-play central roles in many biological processes and have properties useful in an array of applications. However, glycans have received little attention from the research community due to a lack of tools to probe their often complex structures and properties. Transforming Glycoscience: A Roadmap for the Future presents a roadmap for transforming glycoscience from a field dominated by specialists to a widely studied and integrated discipline, which could lead to a more complete understanding of glycans and help solve key challenges in diverse fields.
This volume focuses on applications of split inteins, and the progress that has been made in the past 5 years on discovery and engineering of fast and more efficient split inteins. The first few chapters in Split Inteins: Methods and Protocols explore new techniques on how to use split inteins for affinity purification of overproduced proteins, and split-intein based technologies to prepare cyclic peptides and proteins. The next few chapters discuss semisynthetic protein trans-splicing using one synthetic intein piece, synthetic intein-extein pieces used to deliver other cargos for chemical modification both of purified proteins and of proteins in living cells, as well as isotopic labeling of proteins for NMR studies, and a discussion on how protein block copolymers can be generated by protein trans-splicing to form protein hydrogels. The last few chapters deal with intein applications in transgenic plants and conditional inteins that can be regulated in artificial ways by small molecules or light, a cassette-based approach to quickly test many intein insertion positions, and a computational approach to predict new intein split sites (the approach also works for other proteins). Written in the highly successful Methods in Molecular Biology series format, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Split Inteins: Methods and Protocols is a valuable resource that will provide guidance toward possibilities of split intein applications, explore proven and detailed protocols adaptable to various research projects, and inspire new method developments.