Determining the binding constants of xeno-estrogens using fluorescence methods

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

What is Estrogen? u 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

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

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

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

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

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

Xenobiotic Estrogens u What is their link to altered fertility and breast cancer? –Some xenoestrogens increase cell division and thus may contribute to breast cancer Nonylphenol BHTo,p’-DDT

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

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

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

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

The 20 Amino Acids

Fundamentals of protein structure Some Residues buried other are not

Aromatic Amino Acids

Production of Hormone Binding Domain of the Estrogen Receptor Start with Bacterial Plate transformed with pMAL-HBD Inoculate Liquid culture with a single colony Grow 37 o C Inoculate Large Liquid Culture and grow to OD 600 = 0.8 Induce via IPTG and Grow 25 o C

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

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

Results of SDS-Page Purified Protein

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

Shine UV light Fluoresce Blue light Shine UV light

Large Conformational change takes place Conformational change

Without Estrogen With Estrogen

Can get binding data!

Data Obtained u 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

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

Questions

SAR Questions

Detection limit question (QCM) u 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