Determining the binding constants of xeno-estrogens using fluorescence methods

1. Determining the binding constants of xeno-estrogens using fluorescence methods

2. Outline • Introduction • Estrogen Receptor & Xenobiotic Estrogens • Current Determination Methods • Chemical & Biological • Fluorescence Method • Protein Production & Purification • Detection • Analysis • Conclusions

3. What is Estrogen? • Female Hormone • Regulation of the Menstrual Cycle and related female childbearing organs (such as the uterus and ovaries) • Maintain healthy bones and heart • Required for development of the breast

4. Hormone Action Figure 1. The mechanism by which estrogen acts to regulate gene expression.

5. The Human Estrogen Receptor • Belongs to the nuclear receptor superfamily • Regulates hormone action • Can be divided into three separate functional domains • Transactivation • DNA Binding • Ligand Binding

6. Ligand Binding Domain Figure 2. Three dimensional representation of the ligand binding domain of the human estrogen receptor.

7. Xenobiotic Estrogens • Environmental estrogens • What are they? • Naturally occurring or synthetic chemicals that can act like human estrogen made by the ovary • Mimic the effect of estrogen • What are some examples? • Several Pesticides(including DDT) • Food Preservatives (BHT and BHA) • Industrial Detergent by-products (nonylphenol) • Red Dye #3

8. Xenobiotic Estrogens • Environmental Estrogens • What are they? • Naturally occurring or synthetic chemicals that can act like human estrogen made by the ovary • Mimic the effect of estrogen • What are some examples? • Dioxins • Several Pesticides(including DDT) • Food Preservatives (BHT and BHA) • Industrial Detergent by-products (nonylphenol) • Diethylstilbestrol (DES) From structural classes such as: • Pyrazole • Stilbestrol • Isoflavones • benzofurans

9. Xenobiotic Estrogens • What is their link to altered fertility and breast cancer? • Some xenoestrogens increase cell division and thus may contribute to breast cancer Nonylphenol BHT o,p’-DDT

10. Outline • Introduction • Estrogen Receptor & Xenobiotic Estrogens • Current Determination Methods • Chemical & Biological • Fluorescence Method • Protein Production & Purification • Detection • Analysis • Conclusions

11. Chemical EPA’s MRM (multiple residue method) GC-MS Method accurate Difficult to identify Novel Estrogen Mimics Biological MCF7 Cells Sensitive to estrogen Growth occurs at increased rates in the presence of estrogen mimics Time consuming Current Determination Methods

12. Outline • Introduction • Estrogen Receptor & Xenobiotic Estrogens • Current Determination Methods • Chemical & Biological • Fluorescence Method - Protein Production & Purification • Detection • Analysis • Questions

13. Introduction to Proteins • Proteins play a vital role in all living systems • Biological function of a protein is determined by its three dimensional structure • Ability to interact with molecules is dependent on structure and conformational flexibility

14. The 20 Amino Acids

15. Fundamentals of protein structure Some Residues buried other are not

16. Aromatic Amino Acids

17. Production of Hormone Binding Domain of the Estrogen Receptor Inoculate Large Liquid Culture and grow to OD600 = 0.8 Start with Bacterial Plate transformed with pMAL-HBD Induce via IPTG and Grow Overnight @ 25oC Grow Overnight @ 37oC Inoculate Liquid culture with a single colony

18. Purification of Hormone Binding Domain of the Estrogen Receptor Purify ER-MBP from bacterial proteins using affinity column chromatography Use Lysozyme to rupture cells and release proteins Spin to separate bacterial debris from HBD-MBP Collect Fractions & identify purified product

19. Purification of Hormone Binding Domain of the Estrogen Receptor Separate the HBD from MBP via column chromatography (either size exclusion or ion-exchange) Hydroxylamine Fraction containing purified fusion protein MBP HBD The purified fusion protein is cleaved with hydroxylamine Analyze and save purified HBD

20. Results of SDS-Page Purified Protein

21. Outline • Introduction • Estrogen Receptor & Xenobiotic Estrogens • Current Determination Methods • Chemical & Biological • Fluorescence Method - Protein Production & Purification • Detection • Analysis • Questions

22. Shine UV light Shine UV light Fluoresce Blue light

23. Large Conformational change takes place Conformational change

24. Without Estrogen With Estrogen

25. Can get binding data!

26. Data Obtained • Can answer the questions: • “How tightly does a certain xenoestrogen bind”? • “How fast does it bind”? • Can estrogen compete ‘off’ the xenoestrogen? • Other chemically important information

27. Conclusions • The Fluorescence Method for xeno-estrogen detection • Quick & Highly sensitive • Detect novel estrogen mimics in samples • Obtain important biophysical data

28. Questions

29. SAR Questions 12 17 11 16 13 1 10 6 2 14 15 9 8 5 3 4 7

30. Detection limit question (QCM) • The binding of a small ligand • MW =< 500 • closely packed protein • protein diameter (~5-7 nm) • 1:1 stoichiometry Yield a Change of 1-2 ng cm-2 or less Calculation with the Sauerbrey equation for a 10 MHz crystal yields an observed frequency change no greater than 0.4 Hz