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Determining the binding constants of xeno-estrogens using
fluorescence methods
Outline Introduction
– Estrogen Receptor & Xenobiotic Estrogens Current Determination Methods
– Chemical & Biological Fluorescence Method
– Protein Production & Purification– Detection– Analysis
Conclusions
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
HO
OH
Hormone Action
Figure 1. The mechanism by which estrogen acts to regulate gene expression.
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
Ligand Binding Domain
Figure 2. Three dimensional representation of the ligand binding domain of the human estrogen receptor.
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
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
Xenobiotic Estrogens
What is their link to altered fertility and breast cancer?– Some xenoestrogens increase cell division
and thus may contribute to breast cancer
HO
Nonylphenol BHT
OHC Cl
Cl
Cl
Cl
Cl
o,p’-DDT
Outline Introduction
– Estrogen Receptor & Xenobiotic Estrogens Current Determination Methods
– Chemical & Biological Fluorescence Method
– Protein Production & Purification– Detection– Analysis
Conclusions
Current Determination Methods
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
Outline Introduction
– Estrogen Receptor & Xenobiotic Estrogens Current Determination Methods
– Chemical & Biological Fluorescence Method
- Protein Production & Purification– Detection– Analysis
Questions
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
NH2
OOH
alanine
NH2
OHO
valineNH2
O
HO
leucine
NH2
OOH
isoleucine
NH2
NH
O
OH
tryptophan
NH2
OHO
HO
tyrosine
H2N
O
OHphenylalanine
NH2HN
N
O
OH
histidine
H2NH2N O
OHlysine
NH2HN
NH
H2N
O
OH
arginine
NH2 O
OH
O
-O
aspartate
NH2
O
HO
O
O-
glutamate
H2NOH
OOH
serine
NH2
OH
OHO
threonineNH2 O
NH2
O
HO
asparagine
NH2
O
H2N
O
OH
glutamine
H2NSH
OOH
cysteineNH2
S
O
HO
methionine
H2N
O
OHglycine
HN O
OHproline
The 20 Amino Acids
Fundamentals of protein structure
Some Residues buried other are not
Aromatic Amino Acids
NH2
NH
O
OH
tryptophan
NH2
OHO
HO
tyrosine
H2N
O
OHphenylalanine
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 Overnight @ 37oC
Inoculate Large Liquid Culture and grow to OD600 = 0.8
Induce via IPTG and Grow Overnight @ 25oC
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
MBP HBD
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 Introduction
– Estrogen Receptor & Xenobiotic Estrogens Current Determination Methods
– Chemical & Biological Fluorescence Method
- Protein Production & Purification– Detection– Analysis
Questions
Shine UV light
Fluoresce Blue light
Shine UV light
Large Conformational change takes place
Conformational change
300 310 320 330 340 350 360 370 380
100
150
200
250
300
350
400
450
500
550
Flo
ure
sce
nce
Inte
nsi
ty
Wavelength
Without Estrogen
With Estrogen
Can get binding data!
-2 0 2 4 6 8 10 12 14 16-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
Data: Data1_BModel: XenoEstrogen Chi^2/DoF = 0.04609R^2 = 0.69397 Kd 0.17725 ±0.06854
Fra
ctio
n B
ou
nd
Free XenoEstrogen (uM)
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
Conclusions
The Fluorescence Method for xeno-estrogen detection– Quick & Highly sensitive – Detect novel estrogen mimics in samples– Obtain important biophysical data
Questions
SAR Questions
HO
OH
1
2
34
5
6
7
8
9
10
1112
13
14 15
16
17
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 lessCalculation with the Sauerbrey equation for a
10 MHz crystal yields an observed frequency change no greater than 0.4 Hz
