ABSTRACT OF THE DISSERTATION

Proton NMR Studies of the Interaction of Heparin-Derived Oligosaccharide with Biological Molecules and the Cis/Trans Isomerization of Amide Bonds in Peptides and Peptide/Peptoid Hybrids

By

Khanh Trong Nguyen

Doctor of Philosophy, Graduate Program in Chemistry

University of California, Riverside, March 2009

Professor Dallas L. Rabenstein, Chairperson

Two diverse topics in biological chemistry are the subject of this dissertation. In part I, the research focuses on heparin and the interaction of histidine-containing peptides with heparin. Heparin is a linear polysaccharide, which is formed through the linkage of variously sulfated uronic acid-(14)-D-glucosamine repeating disaccharide subunits by glycosidic bonds. The structure of heparin is not yet completely understood due to its extreme complexity. Consequently, details of the binding of peptides and proteins by heparin is not well understood. One approach is to use well-defined heparin-derived oligosaccharides with different structures as models to probe the question of specificity of binding.

Three heparin-derived tetrasaccharides, one hexasaccharide and one octasaccharide were isolated, purified and characterized. A structurally defined tetrasaccharide was then used as a model to study heparin binding by histidine-containing peptides and related molecules. The imidazolium group of all the molecules studied was found to bind site specifically to a binding pocket formed by an iduronic acid-glucosamine-iduronic acid trisaccharide sequence. Binding constants were determined for the complexes by NMR. Binding constants and relative binding affinities were also determined for the binding of the same molecules by intact heparin by isothermal titration calorimetry and heparin affinity chromatography, respectively.

In part II, the kinetics and thermodynamics of cis/trans isomerization of Xaa-sarcosine tertiary amide bonds in peptide/peptoid hybrids were studied. Xaa represents an amino acid and sarcosine is N-methyl glycine. Specifically, the affect of amino acid sequence on the kinetics and equilibria of cis/trans isomerization across Xaa-sarcosine peptide bonds in three series of peptide/peptoid hybrids having the sequences Ac-Cys-Sar-His-Xaa-(Ala)3-Cys-NH2, where Xaa is His, Gly, Lys, Phe, Asp and Glu, Ac-Cys-Sar-(Ala)x-His-(Ala)y-Cys-NH2, x = 0-4 and y = 4-0, and Ac-Cys-Sar-His-(Ala)3-Cys-NH2, Ac-Cys-His-Sar-(Ala)3-Cys-NH2, etc., were studied. The populations of the cis and trans isomers and the kinetics of cis/trans interconversion were found to depend on the amino acid preceding sarcosine.

The kinetics and equilibria of cis/trans isomerization across the Xaa-Xaa secondary amide bonds in a series of peptides of the sequence Ac-Cys-Xaa-Xaa-Cys-His-NH2, where Xaa is Ala, Tyr and Phe, were also studied. These peptides were studied as model peptides for oxido-reductase enzymes with the Cys-Xaa-Xaa-Cys motif. The effects of the type and position of the central Xaa residues on the kinetics and equilibria of cis/trans isomerization were examined. Rate and equilibrium constants for cis/trans isomerization together with thermodynamic parameters were determined for all the peptides and peptide/peptoid hybrids studied in both their dithiol and disulfide forms. Rate constants for interconversion between the cis and trans configurational isomers were determined by inversion-magnetization transfer NMR methods and equilibrium constants for the cis/trans isomerization were determined from resonance intensities.

Proteins play major roles and participate in multiple pathways in the cell. Thus, cells have to rely on proper folding and localization of the proteome to avoid dysfunction. One of the key steps in maturation for secretory proteins is translocation into the endoplasmic reticulum (ER) rather than mistargeting to and accumulating in the cytosol. However, under ER stress, ER translocation becomes less efficient leading to higher accumulation of proteins in the cytosol. This potentially poses a threat to the cytosol. In our study, we identified an ER chaperone HSPA13 as a regulatory factor of protein translocation into the ER. Overexpression of HSPA13 in HEK293T cells impairs ER and cytosolic proteostasis by inhibiting ER translocation of substrates, disrupting their secretory pathway and promoting their intracellular aggregation. Additionally, we found that mistargeted protein in the cytosol is triaged on the basis of its stability. Highly destabilized mistargeted proteins are rapidly processed and degraded by the proteasome, while more stable proteins are degraded less efficiently. Furthermore, in the case of transthyretin, the processing is mediated by selective N-terminal proteolysis. Thus, we have found that mistargeted ER-directed proteins are subject to quality control in the cytosol.

Influenza infection requires viral escape from early endosomes into the cytosol, which is enabled by an acid-induced irreversible conformational transformation in the viral protein hemagglutinin. Despite the direct relationship between this conformational change and infectivity, label-free methods for characterizing this and other protein conformational changes in biological mixtures are limited. While the chemical reactivity of the protein backbone and side-chain residues is a proxy for protein conformation, coupling this reactivity to quantitative mass spectrometry is a challenge in complex environments. Herein, we developed electrophilic amidination coupled with reaction monitoring to detect the fusion-associated conformational transformation in recombinant hemagglutinin (rHA). We identified rHA peptides that are differentially amidinated between the pre- and post-fusion states, and validated that this difference relies upon the fusion-associated conformational switch. We further demonstrate that we can distinguish the fusion profile in a matrix of digested cellular lysate.

Large-scale data analytical applications such as social network analysis and web analysis have revolutionized modern computing. The processing demand posed by an unprecedented amount of data challenges both industrial practitioners and academia researchers to design and implement highly efficient and scalable system infrastructures. However, Big Data processing is fundamentally limited by inefficiencies inherent with the underlying programming languages.

While offering several invaluable benefits, a managed runtime comes with time and space overheads. In large-scale systems, the runtime system cost can be easily magnified and become the critical performance bottleneck. Our experience with dozens of real-world systems reveals the root cause is the mismatch between the fundamental assumptions based on which the current runtime is designed and the characteristics of modern data-intensive workloads.

This dissertation consists of a series of techniques, spanning programming model, compiler, and runtime system, that can efficiently mitigate the mismatches in real-world systems, and hence, significantly improve the efficiency of various aspects of Big Data processing. Specifically, this dissertation makes the following three contributions. The first contribution is the development of a framework named Facade aiming to reduce the cost of object-based representation without an intrusive modification of a JVM. Facade uses a compiler to generate highly efficient data manipulation code by automatically transforming classes in such a way that objects are created only as proxies. Facade advocates for the separation of data storage and data manipulation. The execution model enforces a statically-bounded total number of data objects in an application regardless of how much data it processes. The second contribution is the design and implementation of Yak, the first hybrid garbage collector tailored for Big Data systems. Yak provides high throughput and low latency for all JVM-based languages by adapting its algorithms to two vastly different types of object lifetime behaviors in Big Data applications. Finally, the third contribution is a JVM-based alternative to enable instantaneous data transfer across nodes in clusters called Skyway. Skyway optimizes away inefficiencies of relying on reflection – a heavy runtime operation, and handcrafted procedures in converting data format by transferring objects as-is, without changing much of their existing format.

We have extensively evaluated those compiler and runtime techniques in several real-world, widely-deployed systems. The results show significant improvement of the system over the baseline: faster execution, reduced memory management costs, and improved scalability. The techniques are also highly practical and easy to integrate without much user efforts, making the adoption in real setting possible. The work has inspired a line of several follow-up work from academia. Moreover, the Yak system has been adopted by a telecommunication company.

Oligomerization of G protein-coupled receptors (GPCRs) is a widespread phenomenon whose discovery generates a plethora of alternative targets for new therapeutic approaches towards human diseases. Nevertheless, challenges still exist in the characterization of these complexes, especially in terms of driving factors of formation, interfaces, and functional consequences. Despite their significance, structural and functional studies of GPCR oligomers have been hindered by their dynamic nature and their generally low suitability for biophysical techniques.

Among these receptors, the human adenosine A2A receptor (A2AR) serves as an excellent target to conduct modeling studies into oligomerization of GPCRs, as there is solid evidence that this receptor forms homo- and hetero-oligomers both in vitro and in vivo. Its intrinsically disordered C-terminus is removed in all structural studies of A2AR for stability and homogeneity purposes, but a C-terminal mutation has been shown to prevent A2AR oligomer formation. We first aimed to understand the role of the C-terminus in driving the oligomerization of A2AR. Size-exclusion chromatography (SEC) was applied as the primary method to quantify the oligomer levels of multiple variants of A2AR with strategic mutations and truncations on the C-terminus. We discovered that the C-terminus of A2AR drives receptor homo-oligomerization via multiple types of covalent and non-covalent interactions. Computational analysis revealed that A2AR dimers are formed via multiple interfaces, all involving the C-terminus. Variation of ionic strength of the buffer indicated depletion interactions via the C-terminus to be the main driving force of A2AR oligomerization. Experiments on the C-terminus sans the transmembrane (TM) helices demonstrated that A2AR C-terminus in and of itself can form insoluble aggregate at high salt concentrations.

The inclusion of the C-terminus enables the production and isolation of A2AR oligomers, yet also further complicates biophysical and structural studies of this receptor. Electron paramagnetic resonance (EPR) spectroscopy offers unique capability of probing how the dynamic C-terminus is involved at the multiple interfaces of A2AR oligomers. Much effort had been made using cell sorting in engineering properly folded A2AR variants void of free cysteines to facilitate biophysical characterization by EPR, but the structure and function of these variants needed to be thoroughly investigated. We discovered that these A2AR mutants, selected with an agonist-based assay, showed reduced binding activity to antagonist. Further characterization with EPR power saturation experiments demonstrated that various extracellular disulfide bonds were disrupted in these variants, suggesting that the removed transmembrane cysteines may serve a role in maintaining the proper structure and function of the receptor.

Moving forward, we next sought to visualize the oligomeric interfaces of A2AR and the structural role of the intrinsically disordered C-terminus by combining continuous-wave (CW) EPR with cryogenic electron microscopy (cryo-EM). CW-EPR revealed that a C-terminal residue was immobilized as A2AR formed oligomers, suggesting that the C-terminus is directly involved at the oligomeric interface of the receptor. The related technique double electron electron resonance (DEER) revealed a large intermolecular distance between two C-terminal cysteines, suggesting that A2AR oligomers are not stabilized by direct disulfide bonds between the C-termini. Early cryo-EM data collection yielded a low-resolution 3D structure of A2AR dimers that showed the involvement of the TM regions at the interfaces. Therefore, it appears that both the C-terminus and the TM helices of A2AR contribute to forming the oligomeric interfaces of the receptor.

Finally, in search of a membrane mimetic platform that can retain the native structure and function of A2AR oligomers, styrene maleic acid (SMA) lipid polymers were employed as a promising detergent-free method to isolate transmembrane proteins. We sought to assess the functional impact of extracting directly from the native host environment A2AR and proteorhodopsin (PR), a model bacterial transmembrane proton pump. We discovered that SMA-solubilized A2AR exhibited reduced binding activity to antagonist, likely due to the lack of functional cholesterol. For PR, SMA could only capture the monomeric form of the receptor and could not solubilize the functionally important hexameric form. Further analyses demonstrated that solubilizing PR with SMA severely reduced its active population and disrupt its photocycle properties. Taken together, despite retention of the native host membranes, SMA appeared to have negative impacts on the functional properties of both A2AR and PR.