Transcript Determination of Xeno-Estrogens Using a Modified

Determining the binding
constants of xeno-estrogens using
fluorescence methods
Outline
Introduction
– Estrogen Receptor & Xenobiotic Estrogens
Current Determination
– Chemical & Biological
Methods
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
OH
HO
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
Cl
OH
Cl
C
Cl Cl
HO
Cl
Nonylphenol
BHT
o,p’-DDT
Outline
Introduction
– Estrogen Receptor & Xenobiotic Estrogens
Current Determination
– Chemical & Biological
Methods
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
– Chemical & Biological
Methods
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
OH
O
O
OH
NH2
The 20
Amino
Acids
NH2
HO
HO
NH2
alanine
O
NH2
leucine
O
valine
O
H2N
O
glycine H
isoleucine
O
HO
OH
NH2
NH2
O
O
OH
N
H
tryptophan
H2N
OH
phenylalanine
tyrosine
O
H2N
N
HN
H2N
O
H2N
OH
NH2
histidine
O
-
OH
arginine
O
NH2
OH
O
O-
HO
NH2 O
aspartate
O
glutamate
O
OH
O
NH2
O
OH
H2N
serine
NH
OH
lysine
NH2
H
N
OH
NH2
NH2
HO
HO
O
O
OH
cysteine
S
HO
H2N
NH2
methionine
SH
H
N
proline
O
OH
OH
O
NH2 O
asparagine
O
threonine
H2N
glutamine
O
Fundamentals of protein structure
Some Residues buried other are
not
Aromatic Amino Acids
O
OH
NH2
N
H
tryptophan
HO
NH2
O
O
OH
tyrosine
H2N
OH
phenylalanine
Production of Hormone Binding
Domain of the Estrogen Receptor
Start with Bacterial Plate
transformed with pMAL-HBD
Inoculate Large Liquid Culture
and grow to OD600 = 0.8
Induce via IPTG and
Grow Overnight @ 25oC
Grow Overnight @ 37oC
Inoculate Liquid culture with a
single colony
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
Purification of Hormone Binding
Domain of the Estrogen Receptor
Hydroxylamine
Fraction
containing
purified fusion
protein
Separate the HBD
from MBP via column
chromatography
(either size exclusion
or ion-exchange)
MBP HBD
The purified
fusion protein is
cleaved with
hydroxylamine
Analyze and
save purified
HBD
Results of SDS-Page
Purified Protein
Outline
Introduction
– Estrogen Receptor & Xenobiotic Estrogens
Current Determination
– Chemical & Biological
Methods
Fluorescence Method
- Protein Production & Purification
– Detection
– Analysis
Questions
Shine
Shine UV
UV light
light
Fluoresce Blue light
Large Conformational change takes
place
Conformational change
550
500
Without Estrogen
Flourescence Intensity
450
400
350
300
250
With Estrogen
200
150
100
300
310
320
330
340
350
Wavelength
360
370
380
Can get binding data!
1.0
0.8
Fraction Bound
0.6
0.4
0.2
Data: Data1_B
Model: XenoEstrogen
0.0
Chi^2/DoF
= 0.04609
R^2
= 0.69397
-0.2
Kd
0.17725
±0.06854
-0.4
-2
0
2
4
6
8
10
Free XenoEstrogen (uM)
12
14
16
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
OH
12
17
11
13
1
6
2
10
14
9
HO
3
8
5
4
16
7
15
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