Determining the binding constants of xeno-estrogens using fluorescence methods

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Determining the binding constants of
xeno-estrogens using fluorescence methods
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Outline

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

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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

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

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

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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

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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
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Outline

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

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Current 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

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Outline

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

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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

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The 20 Amino Acids
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Fundamentals of protein structure
Some Residues buried other are not
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Aromatic Amino Acids
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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 _at_ 25oC
Grow Overnight _at_ 37oC
Inoculate Liquid culture with a single colony
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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
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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
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Results of SDS-Page
Purified Protein
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Outline

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

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Shine UV light
Shine UV light
Fluoresce Blue light
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Large Conformational change takes place
Conformational change
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Without Estrogen
With Estrogen
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Can get binding data!
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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

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Conclusions

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

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Questions
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SAR Questions
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17
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16
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1
10
6
2
14
15
9
8
5
3
4
7
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Detection 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