Nuclear Magnetic Resonance (NMR) is a powerful spectroscopic technique to study the structure, molecular interactions, and dynamics of proteins. Modern NMR instrumentation, advancements in experimental techniques and revolutionary developments in recombinant DNA technology have made NMR a versatile and very convenient tool for biomolecule characterization.

My Dissertation work consists of several projects and focuses on the use of high resolution NMR spectroscopic techniques and molecular biology methods, to understand structural interactions between Spartin and Ubiquitin, and to study the role of Spartin as an adaptor molecule, elucidate the effectiness of palmitic acid as a therapeutic fusion inhibitor against HIV infection, and to study the backbone dynamics of cyclotide, MCoTI-I.

Hereditary spastic paraplegias (HSP) are a group of neurological disorders characterized by lower extremity spastic weakness. The HSP known as Troyer syndrome is caused by a mutation in the spartin gene (SPG20). Spartin harbors a novel Ubiqutin Binding Motif (UBM) that binds Ubiquitin. Since both mono-ubiquitination and binding to Ubiquitin are known to affect the function and/or localization of endocytic adaptor proteins, there is a need to determine the molecular components that are necessary for Spartin’s interaction with Ubiquitin. Here we characterized the structural interactions between Spartin and Ubiquitin by using NMR spectroscopy. We identified the region of Spartin which harbors two independent UBM within region 155-367.

Spartin is a multifunctional protein that associates with many cellular organelles, including atrophin-1-interacting protein 4 (AIP4). Spartin acts as an adaptor protein by recruiting AIP4 to lipid droplets and promotes ubiquitination of the lipid droplet associated protein, adipophilin which regulates turnover of lipid droplets. A deficiency of Spartin apparently causes lipid droplets to accumulate. We have show that Spartin has six times greater affinity for the WW region of AIP4 than for the WW region to the catalytic homologues of the E6-associated protein C-terminus (HECT) domain, as measured by enzyme linked immunosorbent assay (ELISAs).

The high rate of mutation in HIV-1 mutation highlights the need for novel therapeutic agents with broad activity against both CXCR4 and CCR5-tropic viruses. Dr. Cancki's group has identified Palmitic Acid (PA) as a natural, small, bioactive molecule with activity against HIV-1 infection. We performed captured ELISA assays and showed that PA inhibits CD4-gp120 complex formation in vitro. We used fluorescence spectroscopy to determine that PA binds directly to the CD4 receptor and we used one dimensional Saturational Transfer Difference NMR (STD-NMR) to determine that the PA binding epitope for CD4. These findings demonstrated a novel class of antiviral compound that binds directly to the CD4 receptor, blocking HIV-1 entry and infection. Based on an in silico model, these results support our STD and ELISA results and provides a framework for PA as a model scaffold for molecular modification to improve binding affinity and potency.

MCoTI-I cyclotide is a fascinating minprotein (34 amino acids) that is a powerful trypsin inhibitor (K_i ≈ 20-30 pM). It was recently isolated and characterized from the dormant seeds of Momordica cochichinesis, a plant member of the cucurbitaceae (melon) family. It has a head-to-tail, circular, cystine-knotted topology composed of three disulphide bridges, making it exceptionally resistant to thermal, chemical and enzymatic degradation. By using high resolution NMR, we reported for the first time the unusual internal dynamics of free biosynthesized cyclotide MCoTI-I and complexed with trypsin. The internal dynamics of cyclotide MCoTI-I was obtained from ¹⁵N spin-lattice and spin-spin relaxation times and {¹H}-¹⁵N heteronuclear Overhauser effect (NOE) enhancement measurements. The backbone dynamics of free cyclotide MCoTI-I confirm that MCoTI-I adopts a well-folded and highly compact structure with an average order parameter, < S² > = 0.83 ± 0.03. Surprisingly, the backbone flexibility of cyclotide MCoTI-I increases significantly upon binding to trypsin, < S² > =0.65 ± 0.07.