Title: Determining the binding constants of xeno-estrogens using fluorescence methods
1Determining the binding constants of
xeno-estrogens using fluorescence methods
2Outline
- Introduction
- Estrogen Receptor Xenobiotic Estrogens
- Current Determination Methods
- Chemical Biological
- Fluorescence Method
- Protein Production Purification
- Detection
- Analysis
- Conclusions
3What 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
4Hormone Action
Figure 1. The mechanism by which estrogen acts
to regulate gene expression.
5The 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
6Ligand Binding Domain
Figure 2. Three dimensional representation of
the ligand binding domain of the human estrogen
receptor.
7Xenobiotic 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
8Xenobiotic 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
9Xenobiotic 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
10Outline
- Introduction
- Estrogen Receptor Xenobiotic Estrogens
- Current Determination Methods
- Chemical Biological
- Fluorescence Method
- Protein Production Purification
- Detection
- Analysis
- Conclusions
11Current Determination Methods
- Chemical
- EPAs 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
12Outline
- Introduction
- Estrogen Receptor Xenobiotic Estrogens
- Current Determination Methods
- Chemical Biological
- Fluorescence Method
- - Protein Production Purification
- Detection
- Analysis
- Questions
13Introduction 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
14The 20 Amino Acids
15Fundamentals of protein structure
Some Residues buried other are not
16Aromatic Amino Acids
17Production 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 _at_ 25oC
Grow Overnight _at_ 37oC
Inoculate Liquid culture with a single colony
18Purification 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
19Purification 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
20Results of SDS-Page
Purified Protein
21Outline
- Introduction
- Estrogen Receptor Xenobiotic Estrogens
- Current Determination Methods
- Chemical Biological
- Fluorescence Method
- - Protein Production Purification
- Detection
- Analysis
- Questions
22Shine UV light
Shine UV light
Fluoresce Blue light
23Large Conformational change takes place
Conformational change
24Without Estrogen
With Estrogen
25Can get binding data!
26Data 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
27Conclusions
- The Fluorescence Method for xeno-estrogen
detection - Quick Highly sensitive
- Detect novel estrogen mimics in samples
- Obtain important biophysical data
28Questions
29SAR Questions
12
17
11
16
13
1
10
6
2
14
15
9
8
5
3
4
7
30Detection limit question (QCM)
- The binding of a small ligand
- MW lt 500
- closely packed protein
- protein diameter (5-7 nm)
- 11 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