1. Investigation of Ligand Binding to G-protein Coupled Receptors with the
Use of Ellipsometry
A thesis submitted on the 25th of February, 2016
To the Department of Biology and Biomedical Engineering of
Rose-Hulman Institute of Technology
In partial fulfillment of the requirements for the degree of
Bachelor of Science in Biology, May 2016
By
__________________________________________________
Anna Mary Weber
Approved: ____________________________________________
Dr. Jennifer O’Connor, Ph.D., Advisor

2. 2

3. ABSTRACT
Weber, Anna Mary
B.S. Biology, Rose-Hulman Institute of Technology
May 2016
Investigation of Ligand Binding to G-protein Coupled Receptors with the Use of Ellipsometry
Jennifer O’Connor Ph.D.
G-protein coupled receptors (GPCRs) are integral membrane proteins associated with the
signaling of many biological processes. There has been links identified between GPCRs and
various diseases. The pharmaceutical industry is targeting these proteins, but are unable to track
the efficiency, primarily due to limitations with current technologies. Ellipsometry, an optical
technique, can be used to quantify and explain the binding of ligands to receptors of the cell
surface. A549 cell cultures, which express the GPCR, CXCR4, and its associated ligand,
CXCL12, were used for experimentation. Optimal ligand and antibody concentrations were
determined using the well-establish method, ELISA. Baseline and biological testing was
performed on the ellipsometer. Psi-Delta analysis was performed on the collected data where
trends were visually inspected.

4. ii
TABLE OF CONTENTS
LIST OF FIGURES………….…………………………………………………………………...iii
INTRODUCITON…………….…………………………………………………………..………1
LITERATURE REVIEW….…………………………………………………………..………….3
MATERIALS AND METHODS…………………………………………………………….....…5
Cell Culture…………………………………………………………….....……………….5
Enzyme Linked Immunosorbent Assay…………………………………………………...5
Slide Preparation …………………………………………………………….....…………6
Spectroscopic Ellipsometry……………………………………………………………….6
Ellipsometric Experimentation……………………………………………………………6
Psi-Delta Data Analysis……………………………………………………………...........7
RESULTS ……………………………………………………………………………..…….……8
DISCUSSION……………………………………………………………………………………11
CONCLUSIONS………………………………………………………………………………...12
BIBLIOGRAPHY………………………………………………………………..………………13

5. iii
LIST OF FIGURES
Figure 1: Cell Culture………………………………………………….………………………...14
Figure 2: Schematic of Enzyme Linked Immunosorbent Assay…….…………………………..15
Figure 3: Ellipsometry Apparatus……………………………………………………………….16
Figure 4: Example Psi Delta Output From Ellipsometer….…………………………………….17
Figure 5: Glass Slide Characterization Data…………………………………………………….18
Figure 6: Glass Slide with Ligand Data…...…………………………………………………….19
Figure 7: Glass Slide with Antibody Data…...…………………………………………………..20
Figure 8: Glass Slide with Ligand and Antibody Data…...………………………….…………..21

6. 1
INTRODUCTION
G-protein coupled receptors (GPCRs) are integral cell surface proteins that are comprised of
seven trans-membrane helices, responsible for driving many biological processes through the use
of several signal transduction pathways (Kriechbaumer et al., 2012). When a ligand binds to its
specific receptor, a conformational change occurs resulting in heterotrimeric G proteins to
become activated. These proteins are responsible for the transmission of signal molecules to the
interior of the cell (Oldham et al., 2007). Both cellular signaling and mutation of the proteins
can potentially result in detrimental physiological function.
Various diseases including schizophrenia, cancer, and diabetes have all been directly related to
GPCRs (Kriechbaumer et al., 2012). Because GPCRs are both present in high frequencies in the
human genome, with about 800 locations being recognized to date, and play a crucial role in cell
signaling, they are a prime target for drugs and treatment procedures (Giraldo et al., 2011).
Presently, upwards of 60 percent of pharmaceuticals target GPCRs that have been designed to
treat ailments of many physiological systems (Schoneberg et al., 2004). Well known
pharmaceutical companies like GlaxoSmithKline, Eli Lilly, and Pfizer are all investigating in
treatment options targeting these receptors (Kriechbaumer et al., 2012).
Drug discovery is a lengthy process today taking anywhere from 10-12 years from the initial
research and development phase to the creation of a commercially recognized drug (Zang et al.,
2012). The development phase is time consuming due to the extensive testing procedures and
inefficient screening processes currently on the market. More relevant systems of analysis and
high throughput screening are desired in order to expedite the drug discovery process.

7. 2
Currently, the main method of ligand and receptor binding analysis is done through the use of
cell based screening assays. This method is the most common likely due to the ease of the
process making use of simple components like fluorescence and cell markers (Zang et al., 2012).
Also associated with cell based screening assays, however, are critical assumptions that represent
limitations with this method. It is assumed cell viability is stable throughout and at a
continuously, relatively high level, resulting in potential anomalies during the data analysis
portion (Azouz, 2014). The compromise in cell viability can be attributed to the lengthy
processing time associated with these assays. Due to the limitations in accurately representing
cell receptor and ligand binding, a more efficient method of categorization must be determined.
I tested the use of ellipsometry to analyze the binding of cell and ligands. Ellipsometry is an
optical technique in which the efficacy of ligand bind can be measured through the analysis of a
polarized light spectrum that this refracted off of a specimen. This method has been utilized by
some other researchers and is able to successfully quantify the interaction of membranes and
ligand binding to a GPCR (Kriechbaumer et al., 2012). Ellipsometry is able to track minute
changes in the environment, specifically those at the surface of a membrane making this method
appropriate to investigate interactions.

8. 3
LITERATURE REVIEW
Though GPCRs represent significant physiological importance, both intensive study and
investigation of therapeutic treatment have prevailed as a challenging task. Structural and
functional analysis of some of the proteins are restricted by the inability to create and maintain
situations comparable to that of nature. The membrane lacks stability when introduced into
certain buffers resulting in fluctuations in data analysis (Kriechbaumer et al., 2012).
Because the stability of the membranes alone is questionable, the use of the GPCR in a cellular
system is desirable. For a model system, we used of an epithelial cell line, A549, said to express
a specific GPCR, CXCR4, based upon the findings of previous researchers.
Chemokines are a group of cytokines that act as signaling proteins, functioning primarily as part
of the immune response (Zlotnik et al., 2000). Regulation associated with these proteins is
mainly driven by the interaction of the molecules with GPCRs. In humans, upwards of 40
chemokines have been identified ranging in location from the lymphatic system to the nervous
system (Zlotnik et al., 2000). Chemokine receptors, though representing a small family of
proteins currently, are further subdivided into four subfamilies: CXC, CC, CX3C, and XC. The
nomenclature of the subfamilies is attributed to the arrangement of residues on the side chains of
the proteins (Zlotnik et al., 2000). CXC chemokine receptors (CXCRs) are named for the
separation of the two cysteine residues separated by a one different amino acid.
CXCRs are found on a variety of cells, most notably hematopoietic cells and vascular endothelial
cells. CXCR4 has been characterized and is functionally active said having the ability pair to

9. 4
certain G-proteins. The A549 cell line expresses mRNA for CXCR4 which makes it a good
target for experimentation (Murdoch et al., 1999). CXCR4 binds naturally with its ligand
CXCL12 (Kriechbaumer et al., 2012). The selected cell line, A549 expressing this receptor, was
used in combination with the appropriate chemokine ligand and associated antibody.

10. 5
MATERIALS and METHODS
Cell Culture
A549, human epithelial, cell culture was grown in a Corning cell culture flasks in F-12K
Medium (Kaighn's Modification of Ham's F-12 Medium) supplemented with 10% Fetal Bovine
Serum (FBS). Cultures were incubated at 37°C with media renewal 2 to 3 times per week.
Cultures were grown to confluency and then passaged and either subcultured or frozen down. To
passage cells, growth medium was removed and remaining cell layer was treated with 1 mL 10%
concentrated trypsin and incubated until cells became suspended. For subculture, half of the
trypsin was removed and placed in another culture vessel With F-12K media supplemented with
10% FBS. To freeze cells, confluent cells were treated with trypsin, then centrifuged to
concentrate, and resuspended in F-12K media with 10% DMSO. After acclimating in an ethanol
bath overnight at -80°C, cells were stored in liquid nitrogen.
Enzyme Linked Immunosorbent Assay (ELISA)
A double antibody sandwich ELISA was performed (Current, 2008).. Lyophilized Recombinant
Human/Feline CXCL12/SDF-1 beta and lyophilized Human CXCL12/SDF-1 Antibody were
both reconstituted in molecular grade water. Dilutions were made to 200 ng/mL and 10 μg/mL
of ligand and antibody, respectively. A 96 well ELISA plate was collected. 10 μg/mL of the
antibody was added and 1:2 serially diluted across the wells. The antibody was blocked using a
5% Bovine Serum Albumin solution and incubated for 2 hours. After incubation, the wells were
washed with water and shaken dry. This wash and drying process was repeated two more times.
Blocking buffer was added again and incubated for an additional 10 minutes. The wells were
washed and dried five more times. Any additional liquids were removed with a pipette. 200

11. 6
ng/mL of ligand was added and 1:2 serially diluted across the wells. The plate was incubated for
2 hours. The wash process was repeated at this step 3 times and a blocking buffer was added.
Wells were washed again. Specific antibodies were added and incubated for 2 hours and room
temperature. The wells were washed and horse radish peroxidase conjugated antibodies were
added. Plate was incubated at room temperature for 1 hour (Current, 2008).. Absorbance was
measured using the BioTek ELISA Reader.
Slide Preparation
Standard microscope slides were used for experimentation. Opaque tape pieces were affixed to
entire bottom surface of the slide.
Spectroscopic Ellipsometry
A J.A. Woollam Co., Inc. α-SE spectroscopic ellipsometer apparatus was used for
experimentation in conjunction with Version 4.72 of CompleteEASE Data Acquisition and
Analysis Software for Spectroscopic Ellipsometers.
The measurements were collected at a standard angle of incidence of 70°. Measurement controls
were maintained at a constant collection method through all experiments with standard mode and
sample alignment settings.
Ellipsometric Experimentation
All microscope slides were coated with tape on one side. Aliquots of 100 ng/mL ligand and
5μg/mL antibody were made.

12. 7
For a single component analysis, 10 μL of desired biological sample were added to the non-
coated face of the slide and incubated at 37°C. The slide was washed and shaken dry.
Measurements were taken at above parameters.
For a two component analysis, 10 μL of ligand were added to the non-coated face of the slide
and incubated at 37°C. The slide was washed and shaken dry. 10 μL of antibody were
subsequently added to the same portion of the slide and incubated for 2 hours at 37°C. The slide
was washed and shaken dry again. Measurements were taken at noted parameters.
Psi-Delta Data Analysis
Data from CompleteEASE software was compiled. Numerical data was extracted into Excel and
averages were taken for respective subsets. Psi and Delta values were plotted in Excel and
trends were analyzed visually. Data of each of the biological specimens was compared to that of
the baseline characterization slide data.

13. 8
RESULTS
Cell Culture
During the cell culturing process, we encountered some issues. The incubator was
malfunctioning resulting in the death of some of the cultures. Once repaired, however, cells
were able to be successfully grown and passaged. During passaging, it was determined that 10%
concentrated trypsin must be used suspend the cells. They bind to the culture vessel very well
and were unable to be removed with any lower concentrations.
Six aliquots were frozen down stored in the liquid nitrogen dewer for future use.
ELISA
The ELISA is a well-established method used to characterize binding. The double antibody
sandwich method was utilized to confirm the binding of the antibody to the ligand. A literature
protocol was slightly adapted for the procedure. The absorbance values were measured using an
reader and indicated that the optimal concentrations were somewhere between 100-200 ng/mL
for the ligand and about 10 μg/mL for the antibody.
Slide Preparation
Initial baseline characterization was taken of both plastic petri dishes and glass slides. Petri
dishes were deemed incompatible with the ellipsometer system rather quickly as the sides of the
dish are too tall resulting in unnecessary interactions with the incoming polarized light. The
slides as well, initially resulted in difficulties as the ellipsometer was unable to obtain an accurate
reading as the light was penetrating the glass completely.

14. 9
We researched several different methods to counteract this penetration: the use of either a
frosted slide, a roughened slide, or a coated slide. After further analysis, we were unsure how
the frosted slide would affect the system biologically so decided this method was not
appropriate. With testing both the mechanically roughened and coated slide, data was very
similar. Because mechanical roughing is difficult to control and create consistency, the coated
slides were chose for experimentation. 3M Scotch Matte Finish Magic Tape was used to coat
one side of the microscope slide. In theory, the tape material should be homogenous so each
slide should reflect similar data.
Ellipsometric Experimentation
Due to complications with the ellipsometer, data was unable to be collected for the cell line or
for any of the subsequent measurements involving cells.
For other samples, each set of measurements was taken with 3 replicates and 5 pseudo-replicates.
Data collection for the slides, slides with ligand, and slide with antibody were all quite simple.
Some difficulties arose when taking measurements of the slide and both biologics. This is was
corrected for by changing the orientation of the slide on the table of the ellipsometer.
Optimal concentrations of ligand for ellipsometry was found to be 100ng/mL whereas antibody
concentration was 5μg/mL.

15. 10
Psi-Delta Analysis
Baseline characterization of the slide presented the smoothest data. The psi value represented a
positive correlation with wavelength ranging in values from around 20 – 20.5 (See Figure 5).
The delta value peaked with a value of approximately 2 and then had a fairly steep and steady
decline to values around 0.3.
Values of the biological samples were much more variable compared to those of baseline. Each
of the subsequent measurements were evaluated compared to the baseline to track changes in
both psi and delta values.
Aggregate data of slide and ligand represents a steady upward trend in the psi portion. Values at
lower wavelength are at approximately 24.19 and reach maximum values of 24.55. At the upper
limits of wavelength, it can be seen that there is a slight drop off, however. Delta values are
primarily in the negative region climbing towards 0 at higher wavelengths (See Figure 6). Psi
values were substantially higher compared to that of the baseline, but delta values decreased.
Comparatively, aggregate data of the slide and antibody are very similar. Trends are consistent.
Psi values are marginally higher, only about 0.1 in most cases (See Figure 7). The differentials
from the baseline are on the same scale and the slide and ligand.
When ligand and antibody were both bound introduced, the both psi and delta values decreased
compared to the baseline. The data had a much more linear appearance compared to any of the
other figures (See Figure 8).

16. 11
DISCUSSION
Due to the complications associated with incubator, the cells were unable to be tested as part of
the model system. Implementation of this portion is critical for understanding and analyzing the
full biological system and relating significance. Thus it is necessary to complete further and
more in-depth research to categorize the efficiency of the system for this propose.
However, based upon obtained collected data and preliminary results, the spectroscopic
ellipsometer shows promise for system evaluation. Several criterion support this assertion.
Compared to other systems, ellipsometry utilizes both smaller samples sizes and is able to track
changes in lower concentrations. The most substantial benefit is associated with the acquisition
time. As mentioned, cell based screening assay may take up to several hours which results in a
decrease in cell viability. With ellipsometry, data acquisition is completed on a scale of seconds.
The quick data collection provides the Psi-Delta output which can be further analyzed.
Though psi and delta alone, do not provide much significance other than the potential for visual
inspection of trends, these parameters are useful in the creation of an optical model. Optical
constants and biological factors are implemented to devise a model that will explain changes in
the optical surface properties of the cellular system. Derivation of this model is crucial in
relating the numerical values to the biological significance. Thus explaining what is physically
occurring in the system. This process is necessary in the end goal of tracking efficiency of
ligand binding the GPCR.

17. 12
CONCLUSION
The small sample sizes and quick acquisition time associated with spectroscopic ellipsometry
presents benefits over other analytical techniques presently used in characterization of ligand
binding to receptors. Though this method shows promise, it must be further evaluated with the
implementation of a complete model system. Characterization of the system by utilizing the Psi-
Delta data to develop a model with optical constants and further relating that to biological
properties and significance must be completed in order to fully and accurately describe
interactions.

18. 13
BIBLIOGRAPHY
Azouz, H. 2014. Q&A: Brian Kobilka. Nature. 514: S12-S13. [Online].
Current Protocols in Molecular Biology. 2008. Unit 11. Wiley Interscience, Hoboken, NY.
Giraldo, J and J-P. Pin. 2011. G Protein-Coupled Receptors: From Structure to Function.
[Online.] The Royal Society of Chemistry, Cambridge, UK.
<https://books.google.com/books?id=KMpvQwj1YEQC&pg=PR3&lpg=PR3&dq=ISBN+978-1-
84973-183-6&source=bl&ots=WCWGzxSR3s&sig=jYaF7O-aADoEmwKx6Hp-
V0C7qRk&hl=en&sa=X&ei=fT3YVPv8FYi0yQTqzIC4BA&ved=0CDUQ6AEwBA#v=onepag
e&q=ISBN%20978-1-84973-183-6&f=false>
Kriechbaumer, V., A. Nabok, R. Widdowson, D. P. Smith, and B. M. Abell. 2012. Quantification
of Ligand Binding to G-Protein Coupled Receptors on Cell Membranes by Ellipsometry. PLoS
One. 7(9): 1-9. [Online.]
Murdoch, C., P. Monk, and A. Finn. 1999. Functional expression of chemokine receptor
CXCR4 on human epithelial cells. Immunology. 98(1): 36-41. [Online].
Oldham, W. M., and H. Hamm. 2007. How do Receptors Activate G Proteins?. Advances in
Protein Chemistry. 74: 67-93. [Online].
Schoneberg, T., A. Schultz, H. Biebermann, T. Hermsdorf, H. Rompler, and K. Sangkul. 2004.
Mutant G-protein-coupled receptors as a cause of human diseases. Pharmacology &
Therapeutics. 104(3): 173-206. [Online].
Zang. R., L. Ding, I-C., Tang, J. Wang, and S-T. Yang. 2012. Cell-Based Assays in High-
Throughput Screening for Drug Discovery. International Journal of Biotechnology for Wellness
Industries. 1(1): 31-51. [Online].
Zlotnik, A. and O. Yoshie. 2000. Chemokines: A New Classification System and Their Role in
Immunity. Cell. 12(2): 121-127. [Online].

19. 14
FIGURES
Figure 1: A549 cells growing in F-12K growth medium supplemented with 10% FBS.

20. 15
Figure 2: Schematic representation of double antibody sandwich ELISA protocol (Current,
2008).

21. 16
Figure 3: J.A. Woollam Co., Inc. α-SE spectroscopic ellipsometer apparatus.

22. 17
Figure 4: Example Psi-Delta data output from J.A. Woollam Co., Inc. α-SE ellipsometer.

23. 18
Figure 5: Aggregate Psi-Delta data for characterization of glass slide coated with tape.
0
0.5
1
1.5
2
2.5
19.8
19.9
20
20.1
20.2
20.3
20.4
20.5
20.6
350 450 550 650 750 850
Delta
Psi
Wavelength (nm)
Spectroscopic Ellipsometric Data
Psi
Delta

24. 19
Figure 6: Aggregate Psi-Delta data for glass slide with 100ng/mL ligand.
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
24
24.1
24.2
24.3
24.4
24.5
24.6
350 450 550 650 750 850
Delta
Psi
Wavelength (nm)
Spectroscopic Ellipsometric Data
Psi
Delta

25. 20
Figure 7: Aggregate Psi-Delta data for glass slide with 5μg/mL antibody.
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
24.25
24.3
24.35
24.4
24.45
24.5
24.55
24.6
24.65
24.7
24.75
350 450 550 650 750 850
Delta
Psi
Wavelength (nm)
Spectroscopic Ellipsometric Data
Psi
Delta

26. 21
Figure 8: Aggregate Psi-Delta data for characterization of glass with ligand and antibody.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
19.7
19.8
19.9
20
20.1
20.2
20.3
20.4
350 450 550 650 750 850
Delta
Psi
Wavelength (nm)
Spectroscopic Ellipsometric Data
Psi
Delta