Biology and Biochemistry2014-2015 Research Projects

L to R: Rehill, Smith, Morse, Sweet, Schlessman, Isaac (CDR Kennedy not shown)

The Bio Group

Biology and Biochemistry2014-2015 Research Projects

• 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 Schlessman protein structure-function studies using crystallography

• Assoc. Prof. Virginia Smith –nanoparticle-membrane interactions, spectroscopic characterization of proteins, biochemistry of gingko leaves

• 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 Isaacstudy of periplasmic proteins involved in the bacterial stress response

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.

An ounce of prevention is worth a pound of cure.

CYP1B1

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• 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.)

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.

Gypsy moths, their virus, and red oak leaves

Research in the Rehill Lab AY 2015

Gypsy Moth-Red Oak Projects 1. 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

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)

Assoc. Prof. Jamie Schlessman.

Continuing project: Structural studies of Staphylococcal nuclease variantsWe 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.

Research Focus: Protein structure-function studies, primarily using x-ray crystallography.

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

crystal

diffraction image crystallographic model

electron density map(Harms, Schlessman, Sue, and García-Moreno, PNAS(USA), 2011.)

Schlessman lab research projects

Research Focus: Protein structure-function studies, primarily using x-ray crystallography

crystal

diffraction image electron density map

Harms, Schlessman, Sue, and García-Moreno, PNAS(USA), 2011.

.

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

crystallographic model

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): olocal unfoldingowater penetrationointernal ion-pair formationodomain-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

V66R

T62R

Schlessman et al., in preparation

Protein engineering studies include:oshort- & intermediate-range interactions between internal & surface groupsodesign & 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 siteocreation & removal protein cavities to probe the effects on protein stabilityo"switches" that trigger protein unfolding (useful in drug delivery) or dimerization (potential model for neurodegenerative disease initiation)oWhat could you engineer?

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

Applications of Protein Electrostatics Studies

K36

E23 E23 / K36

Other projects: 1. Collaboration with Prof. Isaac (Spy structure/function studies) 2. Development and structural studies of novel protein

crystallization agents with Prof. Lin

CAVEAT: Is SNase unique? Robinson et al., submitted

Biological roles and regulation of A to I RNA editing

Associate Professor Daniel Morse

Adenosine Deaminases that Act on RNA (ADARs) catalyze hydrolytic

deamination of adenosines within double-stranded RNA

Regulation ofTranslation

Codon Changes IncreaseProtein Diversity

Regulation ofSplicing

Regulation of Translation,Localization, and StabilityRegulation of

TranslationRegulation ofTranslation

Codon Changes IncreaseProtein Diversity

Codon Changes IncreaseProtein Diversity

Regulation ofSplicing

Regulation ofSplicing

Regulation of Translation,Localization, and StabilityRegulation of Translation,Localization, and Stability

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.

Adenosine (A) Inosine (I)Adenosine (A) Inosine (I)

In vitro selection of structure-switching RNA aptamers that canbe used as very sensitive and specific molecular probes

No ligand, fluorescence

is quenchedFluorophore moves away

from quencher,

fluorescence increases

fluorophore

complex structure

fluorophore

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.

Smith Lab Research ProjectsHow do small molecules and nanoparticles interact with biological membranes?

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

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.

high

How does the structure of a bifunctional metalloprotein change as switches between roles?

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.

Iron-responsive element binding protein-1

Low intracellular [Fe2+ ]

Cytoplasmic aconitase

High intracellular [Fe2+ ]

What are the biochemical changes that occur in a deciduous leaf over the course of a season?

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

Gingko biloba – a “living fossil”

Abbie Sigman (‘12) developed an anti-oxidant assay based on the oscillating reaction from Integrated Lab 3.

We use a variety of spectroscopic, chemical, and biochemical techniques to analyze the leaf samples.

There are no students currently working on this project.

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SSweet Lab: Structural characterization of psychrophilic endotoxin

1611.21penta-acyl

1793.45hexa-acyl

1976.01hepta-acyl1384.78

tetra-acyl

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Sweet, et al., submitted to BBA MCB Lipids

Sweet Lab: Isolation and study of new arctic psychrophiles

Nghiem, S.V., et al., Proc. Bionature 2013

http://usnapsp.blogspot.com/2012/04/bromex-2012-wrap-up.html

image credit: Charles Sweet

Sweet Lab: Bioprospecting for algal biofuel feedstocks

Image credit: Solix Biosystems

Next-gen processing and distillation (Bio-SPK) of drop-in biodistillate for boats and jets

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).

Estuarine algae isolated from the Severn in winter (cold-tolerant algae may have beneficial fuel oils)

Image credit: Charles Sweet

Umbach, B. E. Trident Scholar

Project 2014

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

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!