UNDERSTANDING CARBOHYDRATE RECOGNITION MECHANISMS IN NON-CATALYTIC PROTEINS THROUGH MOLECULAR SIMULATIONS

Non-catalytic protein-carbohydrate interactions are an essential element of various biological events. This dissertation presents the work on understanding carbohydrate recognition mechanisms and their physical significance in two groups of non-catalytic proteins, also called lectins, which play key roles in major applications such as cellulosic biofuel production and drug delivery pathways. A computational approach using molecular modeling, molecular dynamic simulations and free energy calculations was used to study molecular-level protein-carbohydrate and protein-protein interactions. Various microorganisms like bacteria and fungi secret multi-modular enzymes to deconstruct cellulosic biomass into fermentable sugars. The carbohydrate binding modules (CBM) are non-catalytic domains of such enzymes that assist the catalytic domains to recognize the target substrate and keep it in proximity. Understanding the protein-carbohydrate recognition mechanisms by which CBMs selectively bind substrate is critical to development of enhanced biomass conversion technology. We focus on CBMs that target both oligomeric and non-crystalline cellulose while exhibiting various similarities and differences in binding specificity and structural properties; such CBMs are classified as Type B CBMs. We show that all six cellulose-specific Type B CBMs studied in this dissertation can recognize the cello-oligomeric ligands in bi-directional fashion, meaning there was no preference towards reducing or non-reducing end of ligand for the cleft/groove like binding sites. Out of the two sandwich and twisted forms of binding site architectures, twisted platform turned out to facilitate tighter binding also exhibiting longer binding sites. The exterior loops of such binding sites were specifically identified by modeling the CBMs with non-crystalline cellulose showing that high- and low-affinity binding site may arise based on orientation of CBM while interacting with non-crystalline substrate. These findings provide various insights that can be used for further understanding of tandem CBMs and for various CBM based biotechnological applications. The later part of this dissertation reports the identification of a physiological ligand for a mammalian glycoprotein YKL-40 that has been only known as a biomarker in various inflammatory diseases and cancers. It has been shown to bind to oligomers of chitin, but there is no known function of YKL-40, as chitin production in the human body has never been reported. Possible alternative ligands include proteoglycans, polysaccharides, and fibers such as collagen, all of which make up the mesh comprising the extracellular matrix. It is likely that YKL-40 is interacting with these alternative polysaccharides or proteins within the body, extending its function to cell biological roles such as mediating cellular receptors and cell adhesion and migration. We considered the feasibility of polysaccharides, including cello-oligosaccharides, hyaluronan, heparan sulfate, heparin, and chondroitin sulfate, and collagen-like peptides as physiological ligands for YKL-40. Our simulation results suggest that chitohexaose and hyaluronan preferentially bind to YKL-40 over collagen, and hyaluronan is likely the preferred physiological ligand, as the negatively charged hyaluronan shows enhanced affinity for YKL-40 over neutral chitohexaose. Collagen binds in two locations at the YKL-40 surface, potentially related to a role in fibrillar formation. Finally, heparin non- specifically binds at the YKL-40 surface, as predicted from structural studies. Overall, YKL-40 likely binds many natural ligands in vivo, but its concurrence with physical maladies may be related to the associated increases in hyaluronan

Inhibition of Mammalian Glycoprotein YKL-40 \u3cem\u3eIDENTIFICATION OF THE PHYSIOLOGICAL LIGAND\u3c/em\u3e

YKL-40 is a mammalian glycoprotein associated with progression, severity, and prognosis of chronic inflammatory diseases and a multitude of cancers. Despite this well documented association, identification of the lectin′s physiological ligand and, accordingly, biological function has proven experimentally difficult. YKL-40 has been shown to bind chito-oligosaccharides; however, the production of chitin by the human body has not yet been documented. Possible alternative ligands include proteoglycans, polysaccharides, and fibers like collagen, all of which makeup the extracellular matrix. It is likely that YKL-40 is interacting with these alternative polysaccharides or proteins within the body, extending its function to cell biological roles such as mediating cellular receptors and cell adhesion and migration. Here, we consider the feasibility of polysaccharides, including cello-oligosaccharides, hyaluronan, heparan sulfate, heparin, and chondroitin sulfate, and collagen-like peptides as physiological ligands for YKL-40. We use molecular dynamics simulations to resolve the molecular level recognition mechanisms and calculate the free energy of binding the hypothesized ligands to YKL-40, addressing thermodynamic preference relative to chito-oligosaccharides. Our results suggest that chitohexaose and hyaluronan preferentially bind to YKL-40 over collagen, and hyaluronan is likely the preferred physiological ligand, because the negatively charged hyaluronan shows enhanced affinity for YKL-40 over neutral chitohexaose. Collagen binds in two locations at the YKL-40 surface, potentially related to a role in fibrillar formation. Finally, heparin non-specifically binds at the YKL-40 surface, as predicted from structural studies. Overall, YKL-40 likely binds many natural ligands in vivo, but its concurrence with physical maladies may be related to associated increases in hyaluronan

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation

John Tavener's Choral Anthems, 1985--1990: Analysis of Style, Form and Performance Practice

124 p.Thesis (A.Mus.D.)--University of Illinois at Urbana-Champaign, 2003.This thesis explores the compositional characteristics of choral anthems composed by John Tavener between 1985 and 1990. Twenty-two anthems are examined to identify any common compositional characteristics both in melodic construction and large scale form. Particular attention is given to Tavener's affinity for Byzantine traditions and their influence on his compositional choices. Performance practice issues related to Tavener's works are explored through interviews with Sir John Tavener, Sir Martin Neary, and Patricia Rozario.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD