Biology and Biochemistry 2014-2015 Research Projects

1. Biology and Biochemistry2014-2015 Research Projects The Bio Group L to R: Rehill, Smith, Morse, Sweet, Schlessman, Isaac(CDR Kennedy not shown)

2. Biology and Biochemistry2014-2015 Research Projects • Assoc. Prof. Danny Morse biological roles and regulation of RNA editing, design of RNA aptamers to serve as sensitive and specific molecular probes • Asst. Prof. Charlie Sweet structural characterization of psychrophilic endotoxin, isolation and study of new arctic psychrophiles, bioprospecting for algal biofuel feedstocks • Asst. Prof Dan Isaac study of periplasmic proteins involved in the bacterial stress response • CDR Larry Kennedy -- identification of inhibitors of enzymes implicated in breast cancer • Assoc. Prof. Brian Rehill– insect - plant interactions, natural product isolation • Assoc. Prof. Jamie Schlessmanprotein structure-function studies using crystallography • Assoc. Prof. Virginia Smith –nanoparticle-membrane interactions, spectroscopic characterization of proteins, biochemistry of gingko leaves

3. Baby Steps in the March against Breast Cancer • Breast cancer is second only to skin cancer as the most common cancer in American women (Approx. 1 in 8 will develop breast cancer in their lifetime.) • Almost 40,000 women died in the US in 2012 from breast cancer. • Estrogen and its metabolites have been strongly linked to the etiology of both breast and uterine cancer.

4. An ounce of prevention is worth a pound of cure. X CYP1B1 • CYP1B1 and 4 hydroxyestradiol are more abundant in tumors of the uterus and breast tissue than normal healthy tissue. • Inhibitors of the CYP1B1 enzyme may eventually be used to prevent cancer in these tissues. • Preventing cancer from ever getting established in the body prevents the harmful side effects of treating cancer (chemotherapy, radiation, etc.)

5. What makes a good inhibitor? • Use spectrometry to measure the activity of the CYP1B1 enzyme • Looking for lowest effective concentration • Looking for high selectivity for the CYP1B1 enzyme • Further studies will pursue: • Docking sites for inhibitor on the enzyme • Effects of site directed mutagenesis Current research students: John Powers and Meghan Connor.

6. Gypsy moths, their virus, and red oak leaves Research in the Rehill Lab AY 2015

7. Gypsy Moth-Red Oak Projects • Isolation and Identification of Red Oak Tannins (in conjunction w/ Dr. Dillner) (liquid chromatography, MALDI-TOF and NMR spectroscopy) Currently: Awad Mohamed Previously: Jacob Cole (Research Award Winner 2012), Josh Kotler, Ian Eisenhauer 2. Feeding Studies: How do gypsy moth caterpillars respond to various tannins in their diets? (raising and handling caterpillars, statistical analysis of growth and development, dissection of caterpillars) Currently: Vacant Previously: Amanda Lau, Andrew Almonte

8. Also… • Feeding Studies: How do leaf defenses vary within the canopy of forest trees as well as among trees at different stages of development (seedling, sapling, canopy size tree)? (raising and handling caterpillars, diet preparation, statistical analysis of growth and development, tree planting???) Currently: Jenn Underhill Previously: Harold Hickey (Research Award Winner 2013)

9. Assoc. Prof. Jamie Schlessman Research Focus: Protein structure-function studies, primarily using x-ray crystallography. . Continuing project: Structural studies of Staphylococcal nuclease variants We determine protein crystal structures using x-ray diffraction methods in order to identify molecular determinants of protein electrostatics behavior. Midsh Midshipmen grow protein crystals, collect x-ray diffraction data, and use computer software to determine and analyze protein structures. This project is collaborative with The Johns Hopkins University. crystal crystallographic model diffraction image electron density map (Harms, Schlessman, Sue, and García-Moreno, PNAS(USA), 2011.) Recent students:with Asst. Prof. Isaac: Andrew Marthy ’12, Colin Kelly ‘12, Joe Gehrz,’11, Joe Georgeson ’10; with Assoc. Prof. Shirley Lin: Pat Wiedorn ’11

10. Schlessman lab research projects Research Focus: Protein structure-function studies, primarily using x-ray crystallography . Primary project: Structural studies of Staphylococcal nuclease (SNase) variants Determine protein X-ray crystal structures to identify molecular determinants of protein electrostatics behavior Techniques: grow protein crystals, collect X-ray diffraction data, & use computer software to determine & analyze protein structures Collaborative project with the Johns Hopkins University crystal crystallographic model diffraction image electron density map Harms, Schlessman, Sue, and García-Moreno, PNAS(USA), 2011.

11. Why should we care about protein electrostatics? • Classical view of proteins: • Nonpolar inside, polar/ionizable groups outside • Fundamental biochemical processes require electro- • static charge formation, movement or removal:catalysis, • H+ transport, e- transfer, ligand binding, & more • Current algorithms fail to reproduce pKa values for • internal ionizable groups based on atomic coordinates • SNase structural and spectroscopic studies have • identified numerous response modes to internal • ionizable groups (K, E, D, R, H): • local unfolding • water penetration • internal ion-pair formation • domain-swapping • Long-term goal: Better understanding of protein electrostatics may yield a more accurate dielectric constant for protein interiors & more robust algorithms for proteins electrostatics simulations T62R V66R Schlessman et al., in preparation

12. Applications of Protein Electrostatics Studies • Protein engineering studies include: • short- & intermediate-range interactions between internal & surface groups • design & creation of internal ion-pairs and water-binding sites to distinguish between high-strength hydrogen bonds & Coulomb interactions in the protein interior & as model to build an enzyme active site • creation & removal protein cavities to probe the effects on protein stability • "switches" that trigger protein unfolding (useful in drug delivery) or dimerization (potential model for neurodegenerative disease initiation) • What could you engineer? K36 E23 E23 / K36 Robinson et al., submitted CAVEAT: Is SNase unique? Other projects: 1. Collaboration with Prof. Isaac (Spy structure/function studies) 2. Development and structural studies of novel protein crystallization agents with Prof. Lin Recent students:with Asst. Prof. Isaac: Andrew Marthy ’12, Colin Kelly ‘12, Joe Gehrz,’11, Joe Georgeson ’10; with Assoc. Prof. Shirley Lin: Pat Wiedorn ’11

13. Associate Professor Daniel Morse Biological roles and regulation of A to I RNA editing Adenosine Deaminases that Act on RNA (ADARs) catalyze hydrolytic deamination of adenosines within double-stranded RNA A to I conversion changes the sequence and the structure of RNA. The biological consequences depend on the type of RNA and where editing occurs within the RNA. Figure shows possible consequences of mRNA editing.

14. complex structure In vitro selection of structure-switching RNA aptamers that can be used as very sensitive and specific molecular probes fluorophore fluorophore No ligand, fluorescence is quenched Fluorophore moves away from quencher, fluorescence increases The figure shows how a structure-switching aptamer works. Current efforts are focused on selecting aptamers that can detect inosine. This will provide a tool for studying ADAR function and regulation.

15. Smith Lab Research Projects How do small molecules and nanoparticles interact with biological membranes? We use a range of methods to answer this question, including fluorescence spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, microbiological growth studies, mutagenesis assays, and differential scanning calorimetry. Josh Sohn making liposomes. Liposomes derived from specific tissues or prepared from purified lipids are used to model lipid bilayers of cells. Students currently working on this : Josh Reyes, Ryan Gall

16. high How does the structure of a bifunctional metalloprotein change as switches between roles? Iron-responsive element binding protein-1 Low intracellular [Fe2+ ] Cytoplasmic aconitase High intracellular [Fe2+ ] We use spectroscopic and biochemical techniques to analyze the structural changes that occur as the protein transitions between its two functional states. Andrew Blank is currently working on this project.

17. There are changes to : • anti-oxidant levels • pigments, including chlorophyll, carotenoids • metal ions (Mg2+, Fe2+ Mo2+ , Cu2+ ) • total nitrogen • presence of photosynthetic proteins • total RNA (inc. mRNA) levels • and more What are the biochemical changes that occur in a deciduous leaf over the course of a season? Abbie Sigman (‘12) developed an anti-oxidant assay based on the oscillating reaction from Integrated Lab 3. Gingko biloba – a “living fossil” We use a variety of spectroscopic, chemical, and biochemical techniques to analyze the leaf samples. There are no students currently working on this project.

18. Sweet Lab: Structural characterization of psychrophilic endotoxin 1611.21 penta-acyl MALDI-TOF MS 1793.45 hexa-acyl 14 14 12 14 14 14 1976.01 hepta-acyl 1384.78 tetra-acyl FAME GC-MS Sweet, et al., submitted to BBA MCB Lipids

19. Sweet Lab: Isolation and study of new arctic psychrophiles image credit: Charles Sweet http://usnapsp.blogspot.com/2012/04/bromex-2012-wrap-up.html Nghiem, S.V., et al., Proc. Bionature 2013

20. Sweet Lab: Bioprospecting for algal biofuel feedstocks Estuarine algae isolated from the Severn in winter (cold-tolerant algae may have beneficial fuel oils) Umbach, B. E. Trident Scholar Project 2014 Image credit: Charles Sweet Next-gen processing and distillation (Bio-SPK) of drop-in biodistillate for boats and jets Image credit: Solix Biosystems 101022-N-9928E-917 NORFOLK (Oct. 22, 2010) Director of the Chief of Naval Operations Energy and Environmental Readiness Division, Rear Adm. Phillip Cullum displays an algae based fuel as part of an alternative fuels demonstration at Naval Station Norfolk. The Riverine Command Boat (Experimental) is powered by an alternative fuel blend of 50 percent algae-based and 50 percent NATO F-76 fuels to support the secretary of the Navy's efforts to reduce total energy consumption on naval ships. (U.S. Navy photo by Mass Communication Specialist 2nd Class Josue L. Escobosa/Released).

21. The Biochemistry Concentration • Requirements: • Complete all requirements for the chemistry major • Take SC336 • Take two semesters of biology (SB251 and higher) • And perform either • One year of biochemically-related independent research (SC495/496) • OR • One semester of research or Capstone and one biochemically-themed elective

22. What will qualify as biochemically-related research? • Uses biochemical methods or materials (e.g. materials science studies of silk, cellulose) • Has application to a biological or biochemical problem (e.g. environmental chemistry, medicinal chemistry, natural products chemistry) • Uses chemical or computational methods to study a biomolecule (e.g. X-ray crystallography, computational studies of fluorinated peptides, biofuels) • Others? – come talk to us!