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Exploration of this hypothesis will further clarify the relevant functional form of the Grb7 protein. We report our progress towards establishing the binding strength of the Grb7-RA dimerization interaction and exploring the residue contact requirements at the domain interface.
CRYSTAL STRUCTURE OF THE G-PROTEIN RAC1 IN COMPLEX WITH THE REGULATORY SUBUNIT (RIIΒ) OF
CAMP-DEPENDENT PROTEIN KINASE ALuis Moreno Jr., Ping Zhang, Susan Taylor.
University of California San Diego, La Jolla, CA.
Rac1 is a small signaling G protein that belongs to the Ras superfamily of Rho proteins and acts as a molecular switch to control cytoskeletal rearrangements and cell growth via activation of various protein kinases. In recent studies, Rac1 was found to contain A-kinase anchoring protein properties (AKAP), which have the common function of binding to the regulatory subunit of cAMP-dependent protein kinase A (PKA) and restricting the holoenzyme to discrete locations within the cell. Additionally, Rac1 has been shown to bind to the regulatory subunit (RIIβ) of PKA, forming an inactive stable complex. The scope of this project is to express and purify Rac1 and PKA (RIIβ) and identity its interactions, and obtain a crystal structure of Rac1:PKA(RIIβ) complex.
IDENTIFYING INHIBITORS OF ZIPA THROUGH COMPUTATIONAL AND BIOCHEMICAL SCREENING
University of Kansas, Lawrence, KS.
Many formerly treatable bacterial infections are becoming harder to cure because widespread overuse of antibiotics has led to the development of antibiotic resistant strains. The aim of my project is to find a new antibiotic that will disable bacteria by a different mechanism than current antibiotics. A new possible pathway to inhibit bacterial growth is by interrupting the interaction of two proteins involved in cell division, ZipA and FtsZ. The interaction between ZipA and FtsZ is found in the vast majority of bacteria, so interfering with it with a small molecule should be effective for many different bacterial strains. The mechanism starts with multiple FtsZ proteins creating a loop of incomplete cytoskeletal filaments which are called the Z-ring. The Z-ring spans the equator of the cell just underneath the cytoplasmic membrane. During cell division, ZipA binds to FtsZ, which causes the filaments to connect and a solid loop to form across the middle of the bacterial cell. This loop then shrinks at the end of cell division, causing the telekinesis of the cell into two distinct cells, thus producing more bacteria. Without ZipA, the fragments do not connect or condense to divide the bacteria. An effective antibiotic would inhibit the interaction in between ZipA and FtsZ. A small molecule would be put in the binding site on FtsZ, hindering the ability of the ZipA to bind. The small molecule would outcompete the ZipA and effectively stop cell division in the bacteria.
BIOPHYSICAL CHARACTERIZATION OF MOUSE POLYAMINE OXIDASEDiana Zamora, Jose Tormos, Ahmad Galaleldeen.
St.Mary’s University, San Antonio, TX.
Mouse polyamine oxidase (mPAO) is a mammalian flavoprotein that is necessary for the catabolism of polyamines.
The mPAO oxidizes the endo carbon-nitrogen bonds of N1-acetylspermine and N1-acetylspermidine when oxidized to spermine and spermidine respectively. It is known that normal levels of polyamines are important for cell growth, yet their actual function is not well understood. The structure of mammalian PAO has yet to be determined; however, structures for maize and yeast PAO (Fms1) exist. Although the identity between mPAO, Fms1, and maize PAO is only 20%, it has been shown, through sequence comparison and mutational analysis, that a conserved histidine residue at position 67 of Fms1 and 64 of mPAO helps to properly position the amine substrate for oxidation. Determining
SYNTHESIS OF AGOUTI SIGNALLING PROTEIN FOR IMPROVING THE TREATMENT OF MELANOMARodrigo Andrade1 Jillian Miller2.
Hartnell College, Salinas, CA, 2University of California, Santa Cruz, Santa Cruz, CA.
1 Melanoma causes a large majority of skin cancer deaths. According to the American Cancer Society, approximately 10,000 people are expected to die of melanoma this year in the United States. Melanoma is difficult to treat because melanosomes, organelles containing the pigment eumelanin, prevent chemotherapy from working. Agouti signaling protein (ASIP) binds to melanocortin receptor 1 and suppresses the production of eumelanin; thus, fewer melanosomes are formed and their ability to absorb and inactivate chemotherapeutics is minimized. When cells are treated with ASIP, chemotherapy has been shown to be three times more successful in treating melanoma. In order to continue using ASIP to treat melanoma, ASIP is synthesized using solid phase peptide synthesis, oxidative folding, and purification using HPLC. It will then be further tested for its effectiveness in melanoma treatment in model organisms.
ANTIBACTERIAL PROPERTIES OF MICROCRYSTALLINE DIAMOND FILMS WITH SILVER NANOPARTICLES
INCORPORATEDZuania Cordero Badillo1, Javier Avalos2, Gerardo Morell2.
University of Puerto Rico, Rio Piedras, San Juan, PR, 2Universidad de Puerto Rico, Bayamón, Bayamón, PR.
1 Nosocomial infections are expensive and responsible for millions of deaths per year. To decrease this problem, innovative microcrystalline diamond films with silver nanoparticles incorporated (MCD-Ag) were successfully elaborated, characterized chemically and physically, and tested for antibacterial capacity. Recent studies demonstrated that pure silver films are more effective antibacterial agents compared to microcrystalline diamond films.
The incorporation of silver nanoparticles in the microcrystalline diamond films yielded a significant improvement in its antibacterial properties. In order to perform the bacterial characterization of these MCD-Ag films, a rigorous protocol for bacterial culture was executed and the development of the bacterial populations was assessed through growth curves and absorbance measurements with an ultraviolet-visible spectrophotometer. Furthermore, the technique of bacterial transfer was used to conduct a temporal quantitative analysis of the MCD-Ag bacterial inhibition properties resulting in zero bacterial growth within 24 hours. Additionally, scanning electron microscope (SEM) spectroscopy allowed us to obtain imaging of the colonial behavior of the P. Aeruginosa on the surfaces of the MCD-Ag films. The elaboration of the ground-breaking MCD-Ag films was achieved via the technique of hot filament chemical vapor deposition. The chemical and physical characteristics of the MCD-Ag films were assayed through transmission electron microscopy (TEM) and Raman spectroscopy.
CHARACTERIZING OLIGOSACCHARIDE STRUCTURE AFTER DIGESTION OF HEPARIN USING
HEPARITINASESScott Willie1, Caitlin Mencio2, Kuberan Balagurunathan2.
San Juan College, Farmington, NM, 2University of Utah, Salt Lake City, UT.
1 Heparin (HP) and heparan sulfate (HS) are glycosaminoglycan chains that have uronic acids and glucosamine disaccharides that can be sulfated in up to 4 positions. These chains bind to other proteins for vitally important cellular events including growth control, signal transduction, cell adhesion, hemostasis, and lipid metabolism. Medically, HP is used as an anticoagulant to prevent blood clots. In 2008, there were reports of heparin-related deaths in the US due to raw stock contaminated with chondronitin sulfate from an overseas factory. Our lab’s goal is to understand the interaction of HP with proteins for therapeutic purposes. We are assembling a library of oligosaccharides with diverse lengths and sulfation patterns. Previous studies have shown that heparitinase III digests sulfated chains to create
25 UNDERGRADUATE POSTER ABSTRACTS
specific oligosaccharides, and we want to understand whether different isoforms will create different oligosaccharides.
With this initial study, we will be using high-pressure liquid chromatography (HPLC) to separate the oligosaccharide chains and analyze sulfation and size using mass spectrometry. Our expected results for the experiments are for the digestion of HP by different heparitinases to produce oligosaccharides with different characters of structure and length.
STRUCTURE-FUNCTION STUDIES OF THE DIVERSE ADPGLUCOSE PYROPHOSPHORYLASE FROM
THERMODESULFOVIBRIO YELLOWSTONIIEric Yik1, Micheal Susoeff2, Christopher Meyer2, Andrew Orry3.
University of California, Fullerton, Fullerton, CA, 2California State University, Fullerton, Fullerton, CA, 3Molsoft LLC, La 1 Jolla, CA.
Currently, fossil fuel is the leading source of energy and is rapidly being depleted. A renewable supply of alternative fuel could be made from bacteria and from the conversion of starch to bioethanol. ADPGlucose pyrophosphorylase (ADPG PPase) glgC gene product catalyzes the rate-limiting step of glucan biosynthesis in plants and bacteria. Engineering of this enzyme family will allow for the increased production of renewable carbon. The Thermodesulfovibrio yellostonii (Td.y) glgC gene has been successfully cloned and the recombinant enzyme purified.
This enzyme displays only about 30% identity to other characterized ADPG PPases and harbors unusual sequences in regions involved in regulation. Molecular modeling studies revealed that the Td.y enzyme was most similar to a plant enzyme, in accord with 3-phosphoglycerate (3-PGA) activation. Heat stability up to 75 oC required the presence of ATP. Initial kinetic studies were performed at 37 oC (pH 7.5) and revealed S0.5 values for ATP and Mg of 8.0 mM and
13.9 mM, respectively, and a Vmax value of 5.33 units/mg. The metabolites PEP (2 mM), glucose-6-phosphate (2 mM), and 3-PGA (2 mM) were found to increase the apparent affinity for ATP by 4.2, 10.3, and 3.8-fold, respectively. PEP also increased the Vmax by 2.0 fold. Based on alignment studies and molecular modeling, the following site-directed mutants have been generated to probe regulatory properties: E15S, F18K, F23R, S25A, and S28A. Complete characterization of the native and altered proteins is underway.
UNDERSTANDING HOW THE DISTAL POCKET ENVIRONMENT AFFECTS THE LIGAND BINDING AFFINITY OF
NITRITE TO HEME PROTEINSAdriana Garcia, Raymond Esquerra.
San Francisco State University, San Francisco, CA.
Cardiovascular diseases are the leading causes of death worldwide. Nitric oxide plays a fundamental role in cardiovascular health, and disruptions in normal nitric oxide physiology are associated with the progression of cardiovascular diseases. Recently, it was shown that heme proteins can support vasodilatation during hypoxia by converting nitrite (NO2-) to nitric oxide (NO), and that this nitrite reductase activity of heme proteins plays an essential role in a variety of physiological processes. There is a large range in nitrite affinity and nitrite reductase activity in heme proteins with the same active site. This research seeks to understand more clearly how the protein environment controls the binding chemistry of nitrite to the heme active site. Our goal is to determine how the distal pocket environment affects the binding affinity of nitrite to metmyoglobin mutants. Our hypothesis is that the distal pocket environment can increase the binding affinity of nitrite by influencing the binding mode (o-nitrito vs. n-nitro) by way of electrostatic and steric interactions. We compare the binding affinity of a series of distal pocket mutants that affect hydrogen bonding, polarity, and the size of the distal pocket. We will correlate coordination chemistry and binding affinity in order to establish a clear picture of how the protein environment controls nitrite-binding affinity in heme proteins. Understanding how the protein environment influences nitrite binding in heme proteins helps to understand how these proteins generate NO physiologically and in designing therapeutics based on the nitrite reductase activity of heme proteins.
Circadian rhythms (CRs) evolved to align energy use with energy availability. The central clock in the brain is set by light while peripheral clocks, found in all major organs, can be independently set by environmental cues other than light. Feeding, for example, resets the liver clock. Dyssynchrony between the central and peripheral clocks leads to aberrant CRs that are associated with diabetes. We tested the hypothesis that creating dyssynchrony between the brain and liver clocks by restricted feeding (RF) will lead to impaired glucose use, a hallmark of diabetes. We restricted the feeding of wild-type C57BL/6J mice to the daytime, when mice normally sleep, and determined glucose tolerance and insulin sensitivity. Control mice that normally eat most of their food at night were fed ad libitum.
Following a glucose challenge, we observed a significant elevation in fasted blood glucose (p 0.05), increased insulin resistance (HOMA-IR, p = 0.02) and decreased beta-cell function (HOMA-B, p = 0.03) in the RF mice compared to controls. We investigated the molecular mechanisms of nutrient resetting of the liver clock in a HepG2 cell culture model and observed increased circadian expression of gluconeogenic enzymes PEPCK and G6Pase as well as modulation of circadian transcripts Rev-Erbα and Clock. These results will allow us to move toward examining the role of specific nutrients and micronutrients (iron) in feeding and circadian dyssynchrony. Our findings will also lead to a better understanding of how metabolic parameters affect the increased risk of diabetes seen in night-shift workers.
ASSEMBLY OF AMYLOID NANOSTRUCTURES FROM HYBRID PEPTIDE-POLYMER MOLECULESJose Dominguez IV, Sarah Warren, Tania Betancourt.
Texas State University-San Marcos, San Marcos, TX.