The Bio Bunch - USNA

Biology and Biochemistry

2020-2021 Research Projects

The Bio

Bunch

Profs O’Carroll and Sweet not taking students next year.*

*

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Basta Lab Research ProjectsCombating antibiotic resistance


Problem: Antibiotic resistance (AMR) is an emerging

global public health concern. Deaths attributable to AMR

are expected to surpass other major causes of death by

2050.





2050.

Identify essential protein





2050.


Learn as much as you can about

what it does





2050.



what it does

Develop drug to inhibit protein





2050.



what it doesKill bacterium






2050.

Virtually all bacteria possess a peptidoglycan (PG) layer that is essential for their growth and

viability.



what it doesKill bacterium


PG



what it does


Kill bacterium

Project 1: Investigating the reactions catalyzed by mycobacterial LD-transpeptidase enzymes.

Project 2: Evaluating newly patented drugs against LD-transpeptidase enzymes.


LD-transpeptidases

(Ldts)·PG biosynthesis·PG modification

LD-transpeptidases

(Ldts)

Inhibited by carbapenem antibiotics

Evaluate atypical

carbapenems


With the risk of biological warfare on the rise, strategies are needed to combat emerging biological threats!

The development of new antimicrobials can contribute to warfighter protection.



what it does


Kill bacterium

Project 1: Investigating the reactions catalyzed by mycobacterial LD-transpeptidase enzymes.

Project 2: Evaluating newly patented drugs against LD-transpeptidase enzymes.

LD-transpeptidases

(Ldts)·PG biosynthesis·PG modification

LD-transpeptidases

(Ldts)

Inhibited by carbapenem antibiotics

Evaluate atypical

carbapenems


So what will YOU be doing?

I want to study ALL aspects of Ldt enzymes• Molecular cloning• Protein overexpression and purification• Interpreting crystallographic data• Various microbiological techniques• Biochemical assay development

50

37

Msm LdtD

Zandi, T., Marshburn, R., Stateler, P., Brammer Basta, L. ACS Infect. Dis. 2019, 5, 12, 2047-2054.

Leaves, Leaf Eaters, and The

Chemistry That Connects Them

Research in the Rehill Lab AY 2021

Gypsy Moth-Red Oak Projects 1. Isolation and Identification of Red Oak Tannins (in

conjunction w/ Dr. Dillner) {liquid chromatography, mass spectroscopy and NMR spectroscopy} Previously: Jacob Cole (Research Award Winner 2012), Josh Kotler, Ian Eisenhauer, Awad Mohamed , Aidan Lang, Breanna Akins, Ixel Ochoa, Andrew Mitchell

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} Previously: Amanda Lau, Andrew Almonte, Adam Hammer, Amy Krick, Lianne Marquez & Erin Kusuda, Hazel Acosta, Caitlyn thompson

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 , leaf collection?}

• Previously: Harold Hickey, Jenn Underhill, Nia

Chandler, Eleanor Pratt, Nii Quaye

Using in vitro selection to produce RNAs that act as sensors

Associate Professor Daniel Morse

How the Guanine Riboswitch Works In Vivo

Adapting the Guanine Riboswitch for use In Vitro

Adapting the Guanine Riboswitch for use In Vitro

In Vitro Selection Strategy for Changing the Specificity of the Guanine Riboswitch

(Hypoxanthine; HX)

Research Focus: Protein structure-function studies usingX-ray crystallography

crystal diffraction image electron density mapHarms, Schlessman, Sue, and García-Moreno, (2011) PNAS(USA).

.

Primary project: Structural studies of Staphylococcal nuclease variantsdetermine protein X-ray crystal structures

to identify molecular determinants of

protein electrostatics behavior and support protein engineering projects

Techniques:• protein crystal growth (pipetting!)

• collect X-ray diffraction data

• run computer software to determine

and analyze structures

crystallographic

model

Schlessman lab research projects

USNA Chemistry X-ray diffractometer

(MI-131)

Sample project 1: Optimization of data collection strategies for crystallographic studies of domain-swapped Staphylococcal nuclease variants

.Domain swapping occurs when secondary structural elements of a protein exchange position with those of identical neighboring molecule

• Initially proposed as model for protein misfolding events

• Strategy for protein engineering (e.g. pH-dependent protein switch)

• Library of candidate proteins from JHU ready for structural study

Project significance:

• Domain-swapped Staphylococcal nuclease crystasl grew with large unit cells requiring data collection at synchrotron facilities

• Multiple-sweep data collection strategies may enable in-house data collection

• Increases pace for candidate screening


Model for proposed pH-dependent switch protein

C-terminal domain swapping in Staph nuclease

PDB ID: 1SND

Adapted from Green et al., (1995) Nature.

Sample project 2: What is the structural effect of metal ion substitution in the staphylococcal nuclease active site?

.

Protein Data Bank contains >200 staphylococcal nuclease variant structures

• Most consist of ternary complex of protein, Ca2+ and thymidine 3’,5’ diphosphate

(Ki ~ 10-7 M)

• Improves conformational homogeneity

• Enzymatic activity is stimulated by Mg2+ and inhibited by Ca2+, Hg2+, Zn2+, Cd2+, Ba2+

and Sr2+ (Cuatrecases, et al, (1967) J Biol Chem)

• Bio-safe targets: Mg2+, Zn2+ and Sr2+

Project significance:

• Heavier metals provide more scattering in X-ray diffraction and may provide tools for improving crystal quality or initial phasing

• May provide details about active site

geometric flexibility for future protein

engineering studies


PDB ID: 3BDC

I use a variety of biochemical and biophysical techniques -

spectroscopy, calorimetry, activity assays - to address

fundamental questions of protein structure and function.

For example, how can a single protein have two different

conformations and two different functions?

How can an enzyme detect and repair a single misplaced

oxygen on a large protein?

The study of

methionine

sulfoxide reductase

A from E.coli is a

joint project with

Prof Jamie

Schlessman.

HERV-K associated with:

- autoimmunity/neurological disease (ALS) - various cancers (melanoma, testicular, ovarian,

prostate, and breast cancers).

O’Carroll Lab Research:

Biochemistry and molecular biology of human endogenous retroviruses.

We have HIV-like sequences in our own

genome!

Expression of HERV-K depends on the RcRE signal on the RNA genome and the protein Rec.

The Rec protein facilitates export of the RNA genome from the nucleus to the cytoplasm.

Specific Aim 1: Mutational analysis of the Rec multimerization domain.

- Multimerization is an essential step in successful Rec-RcRE interactions.

- Use molecular biology techniques to engineer specific amino acid mutations in the putative multimerization domain.

- Bogerd et al. found three trios involved in oligomerization.

But which ones specifically?

Specific Aim 2: Investigation of thepromoter activity of the antisense strand.

- In collaboration with Dr. Sergey Iordanskiy at USUHS, we found the antisense strand has a potential promoter.

- Antisense RNA can pair with sense RNA making dsRNA and trigger an immune response.

- What is the molecular basis of antisense promoter activity?

1) How? Which transcription factors? 2) Under what conditions? Irradiation? Stress?

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

Can projects performed with “regular” chemistry

professors fulfill the requirement?

Yes, if it:

– 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!

Sweet CR, Watson RE, Landis CA, and Smith JP, Mar Drugs 2015, 13(8)

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Sweet Lab: Characterization of psychrophilic endotoxin structure

Psychrobacter cryohalolentis endotoxin

Sweet Lab: Study of Chesapeake and Arctic psychrophiles

Sweet Lab: Planktonic microbiome, June 2019 Field Expedition

The Bio Bunch - USNA

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