Tools for decoding the structure-function relationships of biopolymers in nanotechnology and glycobiology

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 232-252).In this thesis, new tools have been developed for decoding structure-function relationships governing complex biopolymers that have emerged as key players in biology, biotechnology, and medicine. Specifically: (1.) The first part of this thesis addresses the structure-function relationship of synthetic biodegradable plastics that are at the forefront of nanotechnology for spatiotemporally-regulated targeting and sustained release of drugs to treat Cancer and other chronic diseases. A Voxel-based 3-D platform for accurately simulating all phases of polymeric nanoparticle erosion and drug release is introduced. Using the developed Voxel platform, the release of anti-inflammatory and anti-cancer drugs such as doxorubicin and dexamethasone from poly lactic-co-glycolic acid (PLGA) nanoparticles is precisely predicted. The Voxel platform emerges as a powerful and versatile tool for deducing the dynamics in interplay of polymer, drug, and water molecules, thus permitting the rational design of optimal nanotechnology treatments for cancer. (2.) The second part of this thesis is focused on development of tools to elucidate structure-function relationships of complex polysaccharides (glycans) that specifically interact with proteins to modulate a host of biological processes including growth, development, angiogenesis, cancer, anticoagulation, microbial pathogenesis, and viral infections. First, towards the fine structure determination of complex linear glycans (glycosaminoglycans or GAGs), enzymatic tools are developed for both depolymerizing GAGs at specific linkages and for effectively modulating their functional groups. Specifically, the development and integrated biochemical-structural characterization of the Chondroitinase ABC-II enzyme that depolymerizes dermatan sulfate and chondroitin sulfate GAGs (CSGAGs), and the 6-0- Sulfatase and N-Sulfamidase enzymes that de-sulfate functional groups on heparin and heparan sulfate GAGs (HSGAGs) are described. Second, the interaction of branched glycans with proteins is analyzed using the interplay of Influenza virus surface proteins (mainly Hemagglutinin and Neuraminidase) with host cell surface sialylated glycan receptors as a model system. For this purpose, the novel triple reassortant "Swine Flu" pandemic virus (or 2009 HINI virus) is studied. Finally, in order to overcome the challenges facing protein structure prediction in the "Twilight Zone" of low homology that is rampant in glycan-binding protein (lectin) families, a new tool is introduced for modeling the 3-D structure of proteins directly from sequence. Specifically, it is identified that protein core atomic interaction networks (PCAINs) are evolutionarily non-tinkered and fold-conserved, and this finding is utilized towards assignment of folds, structures, and potential glycan substrates to lectin sequences. It is further demonstrated that the developed tool is effective universally; thus emerging as a promising platform for generic protein sequence-to-structure and function mapping in a broad spectrum of biological applications.by Venkataramanan Soundararajan.Ph.D

A Voxel-Based Monte Carlo Model of Drug Release from Bulk Eroding Nanoparticles

The use of polymeric nanoparticles as drug delivery devices is becoming increasingly prevalent in a variety of therapeutic applications. Despite their widespread clinical use, the factors influencing the release profiles of nanoparticle-encapsulated drugs are still not quantitatively understood. We present here a new, semi-empirical model of drug release from polymeric nanoparticles using a formulation of dexamethasone encapsulated within poly(lactic-co-glycolic acid) to set model parameters. We introduce a three-dimensional voxel-based framework for Monte Carlo simulations that enables direct investigation of the entire spherical nanoparticle during particle degradation and drug release. Due to implementation of this model at the nanoscale, we utilize assumptions that simplify the model while still allowing multi-phase drug release to be simulated with good correlation to experimental results. In the future, emerging mechanistic understandings of nanoparticle drug release may be integrated into this simulation framework to increase predictive power.National Institutes of Health (U.S.) (GM57073

Atomic Interaction Networks in the Core of Protein Domains and Their Native Folds

Vastly divergent sequences populate a majority of protein folds. In the quest to identify features that are conserved within protein domains belonging to the same fold, we set out to examine the entire protein universe on a fold-by-fold basis. We report that the atomic interaction network in the solvent-unexposed core of protein domains are fold-conserved, extraordinary sequence divergence notwithstanding. Further, we find that this feature, termed protein core atomic interaction network (or PCAIN) is significantly distinguishable across different folds, thus appearing to be “signature” of a domain's native fold. As part of this study, we computed the PCAINs for 8698 representative protein domains from families across the 1018 known protein folds to construct our seed database and an automated framework was developed for PCAIN-based characterization of the protein fold universe. A test set of randomly selected domains that are not in the seed database was classified with over 97% accuracy, independent of sequence divergence. As an application of this novel fold signature, a PCAIN-based scoring scheme was developed for comparative (homology-based) structure prediction, with 1–2 angstroms (mean 1.61A) Cα RMSD generally observed between computed structures and reference crystal structures. Our results are consistent across the full spectrum of test domains including those from recent CASP experiments and most notably in the ‘twilight’ and ‘midnight’ zones wherein <30% and <10% target-template sequence identity prevails (mean twilight RMSD of 1.69A). We further demonstrate the utility of the PCAIN protocol to derive biological insight into protein structure-function relationships, by modeling the structure of the YopM effector novel E3 ligase (NEL) domain from plague-causative bacterium Yersinia Pestis and discussing its implications for host adaptive and innate immune modulation by the pathogen. Considering the several high-throughput, sequence-identity-independent applications demonstrated in this work, we suggest that the PCAIN is a fundamental fold feature that could be a valuable addition to the arsenal of protein modeling and analysis tools

Engineering and optimising deaminase fusions for genome editing

Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications

Heparin/Heparan Sulfate N-Sulfamidase from Flavobacterium heparinum: STRUCTURAL AND BIOCHEMICAL INVESTIGATION OF CATALYTIC NITROGEN-SULFUR BOND CLEAVAGE*

Sulfated polysaccharides such as heparin and heparan sulfate glycosaminoglycans (HSGAGs) are chemically and structurally heterogeneous biopolymers that that function as key regulators of numerous biological functions. The elucidation of HSGAG fine structure is fundamental to understanding their functional diversity, and this is facilitated by the use of select degrading enzymes of defined substrate specificity. Our previous studies have reported the cloning, characterization, recombinant expression, and structure-function analysis in Escherichia coli of the Flavobacterium heparinum 2-O-sulfatase and 6-O-sulfatase enzymes that cleave O-sulfate groups from specific locations of the HSGAG polymer. Building on these preceding studies, we report here the molecular cloning and recombinant expression in Escherichia coli of an N-sulfamidase, specific for HSGAGs. In addition, we examine the basic enzymology of this enzyme through molecular modeling studies and structure-function analysis of substrate specificity and basic biochemistry. We use the results from these studies to propose a novel mechanism for nitrogen-sulfur bond cleavage by the N-sulfamidase. Taken together, our structural and biochemical studies indicate that N-sulfamidase is a predominantly exolytic enzyme that specifically acts on N-sulfated and 6-O-desulfated glucosamines present as monosaccharides or at the nonreducing end of odd-numbered oligosaccharide substrates. In conjunction with the previously reported specificities for the F. heparinum 2-O-sulfatase, 6-O-sulfatase, and unsaturated glucuronyl hydrolase, we are able to now reconstruct in vitro the defined exolytic sequence for the heparin and heparan sulfate degradation pathway of F. heparinum and apply these enzymes in tandem toward the exo-sequencing of heparin-derived oligosaccharides

Heparin/Heparan Sulfate 6-O-Sulfatase from Flavobacterium heparinum: INTEGRATED STRUCTURAL AND BIOCHEMICAL INVESTIGATION OF ENZYME ACTIVE SITE AND SUBSTRATE SPECIFICITY*

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes