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DePaul Discoveries
Volume 1 | Issue 1
Article 11
2012
Single-Molecule Fluorescence Studies of
Glycosylated and Aglycosylated Antibodies
Irina Timoshevskaya
DePaul University, [email protected]
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Acknowledgements
Faculty Advisor: Dr. Cathrine Southern, Department of Chemistry
Recommended Citation
Timoshevskaya, Irina (2012) "Single-Molecule Fluorescence Studies of Glycosylated and Aglycosylated Antibodies," DePaul
Discoveries: Vol. 1: Iss. 1, Article 11.
Available at: http://via.library.depaul.edu/depaul-disc/vol1/iss1/11
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Timoshevskaya: Single-Molecule Fluorescence Studies of Glycosylated and Aglycosylated Antibodies
D E PA U L D I S C O V E R I E S ( 2 O 1 2 )
Single-Molecule Fluorescence Studies of
Glycosylated and Aglycosylated Antibodies
Irina Timoshevskaya*
Department of Chemistry
ABSTRACT
Antibodies are Y-shaped, flexible proteins whose structures can be studied using Förster Resonance
Energy Transfer (FRET) at the single-molecule level. Dye molecules must be attached to these proteins so as to carry
out FRET studies of antibodies. In order to label the binding sites of an antibody, dye molecules were attached to a
small molecule, or hapten, which the antibody binds to. Evidence for this binding was provided by ultraviolet-visible
(UV-Vis) spectroscopy. To label the stem region of a humanized immunoglobulin G (IgG) antibody, the DNA for this
antibody was mutated to introduce a cysteine residue to which dyes can be attached. In this research, the DNA was
sequenced and checked to provide the desired sequence for protein production.
INTRODUCTION
to study whether different conformations are present,
Immunoglobulin G (IgG) antibodies have structural
what happens to conformations once the carbohydrates
conformations that can be studied using Förster
are removed, and if there is a preferred conformation.
Resonance Energy Transfer (FRET) at the single-
The IgG antibody used in this research is the 39C2
molecule level. An antibody is a very flexible protein
catalytic aldolase antibody.
composed of four chains (two heavy and two light) and
has a Y-shaped structure as shown in Figure 1. The
FRET was the chosen technique to determine the
lower portion contains a constant region (Fc fragment)
distances between areas of interest such as between
that is composed of similar amino acids found in all
the two portions of the Fc region near the carbohydrate
antibody molecules whereas the upper portion is the
groups on the antibody and between the antigen-binding
“variable region” (two Fab fragments) with varying
sites. FRET requires the use of two dye molecules,
amino acids that results in antibodies that specifically
a donor and an acceptor. These dye molecules can
bind different antigen molecules. The stem region
covalently bond to macromolecules and are used for
contains carbohydrates, or sugar molecules, that
fluorescence spectroscopy, as they absorb and fluoresce
provide shape to the Fc region. Previous studies have
visible light. Once the donor molecule is excited, it can
shown that if the sugars are removed, the antibody is
transfer energy to the acceptor molecule which proceeds
unable to elicit an immune response2. The purpose of
to fluoresce. The closer the acceptor is to the donor, the
this research is to examine the distribution of antibody
more likely it is that energy transfer will take place.
structures with and without the sugars present in order
Therefore, the relative amounts of donor and acceptor
fluorescence can be used to calculate the distance
* Faculty Advisor: Dr. Cathrine Southern, Department of Chemistry
Summer 2011
between the donor and acceptor molecules, resulting
Author contact: [email protected]
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Published by Via Sapientiae, 2012
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DePaul Discoveries, Vol. 1 [2012], Iss. 1, Art. 11
S I N G L E - M O L E C U L E F LU O R E S C E N C E S T U D I E S O F G LY C O SY L AT E D A N D A G LY C O SY L AT E D A N T I B O D I E S
in the ability to determine structural information about
ester form of the hapten. The product was reacted with
the protein2 (Figure 2). Single molecule FRET can be
the donor and acceptor dye molecules (AlexaFluor®
used to obtain a histogram of the distances between
568 and Cy 5.5®). Thin layer chromatography (TLC)
the donor and acceptor sites on the antibody3,4. Single
was used to establish a desired solvent system, found
molecule spectroscopy was the method chosen as it
to be a 60:40 mixture of methanol and ethyl acetate.
examines one molecule at a time rather than multiple
A
molecules at once.
Spectrometer was used to study the concentration of
ThermoScientific
NanoDrop
1000
UV-Visible
the samples and to determine the percent of antibody
In order to study the distance between the antigen-
binding sites labeled with the dye-hapten. The UV-
binding sites of an antibody, dye molecules were
Visible Spectrometer displayed a peak at 318 nm which
attached to a hapten, or a small molecule that specifically
indicates an enaminone, the product of the hapten
adheres to the binding site of an antibody. This dye-
binding to the antibody, is present.
hapten conjugate was then reacted with the antibody to
observe the degree of binding. FRET experiments were
R E S U LTS
performed on this system, but no significant energy
The protein sequences were analyzed and the desired
transfer was observed. So as to attach dye molecules to
DNA and amino acid sequences for the light (plasmid
the Fc region, manipulation of the DNA of a humanized
5.3) and heavy (plasmid 6.4) chains of the humanized
(part mouse, part human) IgG antibody was necessary.
IgG antibody were obtained and sent to Creative
The DNA sequences for the light and heavy chains of
BioLabs for protein production. There were no unwanted
the humanized IgG antibody were checked for unwanted
mutations in the light chain. However, the heavy chain
mutations. A satisfactory sequence has been obtained
is the area in which the DNA mutation was executed and
and submitted to a company for the production of the
the new sequence includes the cysteine point mutation
antibody. The labeling of the antigen binding sites and
into the CH2 region of the Fc region. Figure 4 presents
the examination of the humanized IgG DNA sequence
the desired sequence for the heavy chain. There were
are both described below.
sequence errors found in the variable heavy chain that
will be fixed by Creative BioLabs.
METHODS
P R OT E I N S EQ U E N C E A N A LYS I S
Once the protein sequences were analyzed, UV-Vis
From previous research, the Stratagene QuikChange
spectroscopy was used to determine if there is evidence
Lightning Site Directed Mutagenesis Kit was used to
for dye labeling. It was found that approximately
introduce a cysteine point mutation into the CH2 region
85% of the binding sites of the antibody were labeled
of the Fc region. The cysteine point mutation allows for
with a dye molecule. As evidenced by Figure 5, there
dye attachment, crucial for FRET spectroscopy analysis.
is a substantial peak around 568 nm signifying the
The sequences were checked using the CLC Sequence
AlexaFluor® 568 donor dye fluorophore and a peak at
Viewer (Figure 3). Literature references for the plasmids
about 680nm, characteristic of the Cy 5.5® acceptor
were provided so as to determine the desired amino
dye fluorophore. There is also an evident peak at
acid sequences for the light and heavy chains of the IgG
318nm, denoting the enaminone formed which is the
antibody by comparison .
product of the hapten binding to the antibody. FRET
5-7
experiments were performed on this sample; however,
DY E AT TAC H M E N T
substantial energy transfer was not observed and further
The carboxylic acid form of the hapten was previously
experiments are necessary.
reacted with reagents to generate the succinimidyl
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2
Figure 2. Illu
FRET, in wh
molecule (D
distance-de
transfer to a
molecule (A
fluoresce. T
fluorescenc
D and A can
determine th
between the
The antibod
from the pro
Harris, L. J.
Hasel, K. W
A. Biochemis
1581.
Timoshevskaya: Single-Molecule Fluorescence Studies of Glycosylated and Aglycosylated Antibodies
D E PA U L D I S C O V E R I E S ( 2 O 1 2 )
C O N C LU S I O N
The results demonstrate it was possible to optimize
dye attachment to the 38C2 antibody and UV-Visible
D
spectroscopy provides evidence that the antibody was
A
labeled with the AlexaFluor® 568 donor dye fluorophore
and the Cy 5.5® acceptor dye fluorophore. Furthermore,
D
A
the light and heavy DNA sequences were checked for
any unwanted mutations and the desired sequences
From previous research, the Stratagene QuikChange
were found and sent to create the protein sequence.
Based on this and previous research, it will be possible
to label the purified protein with the dye molecules and
to perform single-molecule FRET spectroscopy. FRET
will examine any possible antibody conformations.
FIGURE 2
In order to study the distance between the antigen-binding sites of an a
Kit
was used
toinintroduce
cysteine
point mutation into the C
Illustrations
of FRET,
which a donoramolecule
(D) undergoes
molecules
were attached
to a hapten,
or a small
molecule
distance-dependent
energy transfer
to an acceptor
molecule
(A), that specifically adhe
causing A topoint
fluoresce.
The amountallows
of fluorescence
observed
from
cysteine
mutation
for dye
attachment,
crucial fo
site of an antibody. This dye-hapten conjugate was then reacted with the antib
D and A can be used to determine the distance between the two
molecules. The antibodies shown are from the protein data bank,
degree of binding.
experiments
on this system,
but no
sequences
wereFRET
checked
usingwere
the performed
CLC Sequence
Viewer
(F
Harris, L. J.; Larson, S. B.; Hasel, K. W.; McPherson, A. Biochemistry,
1997, 36, 1581.
transfer
was observed. So as to attach dye molecules to the Fc region, manipu
the
plasmids were provided so as to determine the desired am
.
of a humanized (part mouse, part human) IgG antibody was necessary. The D
heavy chains of the IgG antibody by comparison5-7.
the light and heavy chains of the humanized IgG antibody were checked for un
A satisfactory sequence has been obtained and submitted to a company for the
antibody. The labeling of the antigen binding sites and the examination
of the
Figure
3.
DNA sequence are both described below.
Methods
Antigen-binding
Sites
sequence.
mutation i
guanine.
Protein Sequence Analysis:
Fab
Fragment
Hinge
Region
Fc
Fragment
Sugars
FIGURE 1
The structure of an immunoglobulin G (IgG) molecule.
The regions of the antibody that correspond to the Fab (upper,
antigen-binding fragment), the Fc (lower, crystallizable, constant
fragment), and the hinge region are also shown. The location of the
sugars, which are bound to the Fc fragment, is indicated.
.
Published by Via Sapientiae, 2012
FIGURE 3
Dye
Attachment:
Example of a section of the protein sequence. Evidence of a cysteine
point mutation in which cysteine was changed to guanine.
The carboxylic acid form of the hapten was previousl
the succinimidyl ester form of the hapten. The product was r
dye molecules (AlexaFluor® 568 and Cy 5.5®). Thin layer ch
— 134 establish
—
a desired solvent system, found to be a 60:40 mixtu
3
ThermoScientific NanoDrop 1000 UV-Visible Spectrometer
Fc region. Figure 4 presents the desired sequence for the heavy chain. There were sequence
FRET
were performed on this sample; however, s
DePaul Discoveries, Vol.antibody.
1 [2012], Iss.
1, Art.experiments
11
N G L E - M O L E C U L E F LU O R E S C E N C E S T U D I E S O F G LY C O SY L AT E D A N D A G LY C O SY L AT E D A N T I B O D I E S
errors foundS Iin
the variable heavy chain that will be fixed by Creative BioLabs.
transfer was not observed and further experiments are necessary.
CH2 sequence:
Absorbance
GCACCTGAACTCCTGGGGGGACCGTCA
Figure 4. The desired DNA sequence of 6.4
GTCTTCCTCTTCCCCCCAAAACCCAAG
plasmid for the CH2 region of the heavy
GACACCCTCATGATCTGCCGGACCCCT
chain which contains the cysteine point
GAGGTCACATGCGTGGTGGTGGACGTG
mutation.
AGCCACGAAGACCCTGAGGTCAAGTTC
AACTGGTACGTGGACGGCGTGGAGGTG
CATAATGCCAAGACAAAGCCGCGGGAG
Wavelength (nm)
GAGCAGTACAACAGCACGTACCGTGTG
Conclusion
FIGURE 4
FIGURE 5
GTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAATGGCAAGGAGTACAAG
The desired DNA sequence of 6.4 plasmid for the C 2 region of the
Illustration
of
the UV-Vis
sprectroscopy
the possible
dye-haptento
to optimize
determine dye attachme
The
results
demonstrate
it of
was
Once the protein sequences were analyzed,
UV-Vis
spectroscopy
was
used
to determine
heavy chain which contains the cysteine point mutation.
percent of binding sites occupied on the 38C2 antibody. Peaks for the
TGCAAGGTCTCCAACAAAGCCCTCCCA
enaminone,
donorspectroscopy
and acceptor dyeprovides
fluorophoesevidence
are evident.
and
UV-Visible
that the antibody was la
if GCCCCCATCGAGAAAACCATCTCCAAA
there is evidence for dye labeling. It was found that approximately 85% of the binding sites of
AlexaFluor® 568 donor dye fluorophore and the Cy 5.5® acceptor dye flu
GCCAAA
the antibody were labeled with a dye molecule.
bysequences
Figure 5,
there
is aforsubstantial
the As
lightevidenced
and heavy DNA
were
checked
any unwanted mut
H
®
sequences
were found
sent to create the
protein
sequence.
Based on
peak around 568 nm signifying the AlexaFluor
568 donor
dyeandfluorophore
and
a peak
at about
research, it will be possible to label the purified protein with the dye mol
680nm, characteristic of the Cy 5.5® acceptor dye fluorophore. There is also an evident peak at
single-molecule FRET spectroscopy. FRET will examine any possible an
318nm, denoting the enaminone formed which is the product of the hapten binding to the
References:
1. Krapp, S.; Mimura, Y.; Jefferis, R.; Huber, R.; Sondermann, P. J. Mol. Biol.
2. Roy, R.; Hohng, S.; Ha, T. Nature Methods. 2008, 5, 507.
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Acad Sci U S A. 2008, 105, 11176.
4. Gansen, A.; Tóth, K.; Schwarz, N.; Langowski, J. J. Phys. Chem. B. 2009, 1
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Krapp, S.; Mimura, Y.; Jefferis, R.; Huber, R.; Sondermann, P. J. Mol. Biol.
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