Transcript Emission Spectra of Prodan Bound to the Mature

Abstract
A library of related monoclonal antibodies isolated from various points in the
murine immune response has been developed. The antibodies were elicited
using the same diketone hapten used to elicit the catalytic aldolase 38C2
antibody (Wagner, J., Lerner, R. A., Barbas, C. F., (1995) Science 270, 1797).
The binding properties of two commercially available fully mature aldolase
antibodies, four members of the 38C2 family from our lab, and a non-specific
IgG were studied by fluorescence spectroscopy. Prodan (6-propionyl-2(dimethylamino)naphthalene) is a fluorescent hapten analogue that binds
reversibly to the 38C2 family of antibodies with micromolar affinity. Steady
state fluorescence spectra, polarization, and lifetime distributions were measured
for the hapten analogue in solution and bound to the various antibodies. Prodan
exhibits a marked blue shift in its fluorescence emission and an increase in
intensity and polarization upon binding. These data are analyzed to determine
binding constants, and scale with fluorescence studies of hapten binding from
tryptophan quenching. Prodan lifetime distributions as measured by
multifrequency phase fluorometry show two types of antibody binding
environments. These results are interpreted in terms of the model of antibody
evolution in which higher affinity is achieved through binding site rigidification.
Antibody Structure
• Antibodies, or immunoglobulin (Ig)
molecules, are composed of four
chains, two light and two heavy
• The structure of Ig molecules can
be divided into a constant region, in
which the amino acid sequence is
largely conserved, and a variable
region, where the amino acid
sequence for different Ig molecules
has considerably more variation
• Within the variable region, there are
three hypervariable regions,
referred to as complementarity
determining regions (CDR1-3), as
they are located at the binding sites
of the antibody molecule
RasMol image of IgG molecule, PDB code 1IGT,
Harris, L. J., Skaletsky, E., and McPherson, A.
(1998) J. Mol. Biol. 275, 861-872.
Antigen-binding
Sites
Variable
Regions
CDR1
CDR2
CDR3
Constant
Regions
Carbohydrates
1
Affinity Maturation
• Upon exposure to an unknown antigen, the immune system
begins a process called affinity maturation
• The germline antibody that binds the antigen most tightly
undergoes somatic hypermutation in the variable region to
form antibodies that each have a new active site and,
therefore, modified affinity for the antigen
• The immune system selects for the modified antibody that
most tightly binds the antigen and somatic hypermutation
occurs again to produce a more mature antibody with an even
more specialized binding site
• Therefore, more mature antibodies should show both more
rigid binding sites and also smaller dissociation constants
2
Aldolase Family of Antibodies
• 38C2 is a catalytic antibody, first developed at the Scripps Research
Institute - Wagner, J., Lerner, R. A., Barbas, C. F., Science 270, 1797 (1995)
• This antibody was raised against a diketone hapten by the process of
reactive immunization, and catalyzes the aldol reaction
• Reactive immunization involves an actual chemical reaction between
the hapten and the antibody, rather than the typical non-covalent
interaction between the hapten and the antibody
• The evidence for catalytic activity is the observation of an absorption
band at 316 nm due to the vinylogous amide formed in the reaction of
the diketone hapten with a lysine residue in the binding site of the
antibody
• The antibodies in this study do not show an absorption peak at 316
nm, indicating that the interaction between the hapten and these
antibodies is not covalent
3
Aldolase Family of Antibodies
• The antibodies used in this study were raised against the
same diketone hapten used to generate the mature 38C2
antibody
• Antibodies were collected at different time points of the
murine immune response
Antibodies Examined:
Primary (A3.1.1): collected 12 days after initial exposure to the hapten
Secondary (2c26.1): collected 5 days after the first boost
Tertiary (3.22): collected 5 days after the second boost
Mature 38C2: collected 14 days after the second boost
Mature 84G3: collected 14 days after the second boost
4
Ligand Molecules
• 1,3-Diketone
• Hapten all
antibodies studied
were raised against
O
• Prodan, or 6-propionyl-2(dimethylamino)naphthalene
• Fluorescent molecule
• Structural similarities to the
1,3-diketone hapten
indicated that prodan could
bind to the antibodies studied
O
O
O
OH
N
H
O
N CH3
CH3
5
Questions Addressed
• Can Prodan, a highly fluorescent and
environmentally sensitive small molecule,
mimic hapten binding?
• What does Prodan tell us about the relative
hydrophobicities of the antibody binding
sites?
• Does Prodan fluorescence provide evidence
for an increase in the rigidity of the antibody
binding sites as a function of antibody
maturity?
6
Experimental Techniques
Antibody dissociation constants were determined
using techniques in which the experimental
observable is proportional to the fraction of ligand
bound
• Antibody was titrated into a solution of Prodan in PBS
• Prodan fluorescence experiments done using a Perkin
Elmer LS50B fluorimeter
• Prodan fluorescence anisotropy measured with a
Panvera Beacon 2000 using an excitation filter
centered at 360 nm and a transmission filter centered at
490 nm
7
Experimental Techniques
• Multifrequency phase fluorometry was used to measure the
fluorescence lifetimes and lifetime distributions of the Trp residues
in the antibodies and Prodan
• The phase and modulation data were fit to a combination of discrete
lifetimes and Gaussian distributions of lifetimes
Discrete:
Intensity decay:
I (t )    i exp( t /  i )
i
Gaussian Distribution:

I (t )    ( ) exp( t /  )d
0
 1    2 
1
 (t ) 
 exp   
 
2

 2

 


Fractional contribution
 i i
f

to fluorescence intensity: i
  i i
i
fi 
 fi ( )d

0
 i fi ( )d
0
8
O
Prodan Emission and Anisotropy
N CH3
CH3
Filter Transmission
Methanol
450
500
Wavelength (nm)
Phosphate
Buffer
Intensity (Arbitrary Units)
Acetone
550
• The emission spectrum of
Prodan blue-shifts with
decreasing solvent polarity
• The emission spectrum of
Prodan in acetone resembles
that of Prodan bound to the
antibodies studied
• Both free (low anisotropy)
and bound (high anisotropy)
Prodan fluoresce in the range
of the emission filter used in
the anisotropy experiments,
therefore, the anisotropy in
this wavelength range can be
used to determine the
600 fraction of Prodan bound
9
Emission Spectra of Prodan Bound
to the Mature 38C2 Antibody
• Excitation is at 361 nm
• Prodan emission shifts to
445 nm upon binding to
the 38C2 antibody
• The emission intensity at
445 nm increases with
increasing antibody
concentration
• At 490 nm, both free and
bound Prodan contribute
to the fluorescence signal
Intensity (arbitrary units)
Bound Prodan
Free Prodan
400
450
500
Wavelength (nm)
550
600
10
Intensity (arbitrary units)
Prodan Binds Specifically in the
Antibody Binding Site
400
20 nM Prodan
20 nM Prodan, 400 nM 38C2
20 nM Prodan, 800 nM 38C2
20 nM Prodan, 800 nM 38C2, 800 nM Hapten
440
480
520
Wavelength (nm)
560
• Excitation is at 361 nm
• Prodan emission shifts to
445 nm upon binding to
the 38C2 antibody
• The emission intensity at
445 nm increases with
increasing antibody
concentration
• At 490 nm, both free and
bound Prodan contribute
to the fluorescence signal
• Addition of hapten to a
solution of Prodan bound
to an antibody resulted in
the displacement of
600
Prodan
11
Determination of Antibody Dissociation Constants
• The fluorescence anisotropy of Prodan is related to the fraction
of Prodan bound to antibody
• A plot of the anisotropy vs. the concentration of free antibody
binding sites, [S], allows the dissociation constant, Kd, of the
antibodies to be determined according to the following
relationship:
( Fmax  Fmin )[ S ]
F
 Fmin
K d  [S ]
• The concentration of free antibody was approximated by the
total antibody concentration, which is valid as the total Prodan
concentration used (20 nM-100 nM) was at least ten-fold lower
than the observed Kd values
12
Determination of Antibody Dissociation
Constants From Anisotropy Data
350
84G3
38C2
Tertiary
Anisotropy (mA)
300
Kd= 0.122 ± 0.005 
Kd= 0.48 ± 0.06 
Kd= 1.8 ± 0.4 
Secondary Kd= 2.1 ± 0.5 
Primary Kd= 1.1 ± 0.2 
250
200
150
100
50
0
500
1000
1500
Total Concentration of Ab Binding Sites (nM)
2000
13
Summary of Antibody Binding Affinity Data
Antibody
Heavy Chain CDR3
Sequence
Kd’/Kdq(84G3)
% Trp
Kd /Kd(84G3)
residues in Trp quenching
binding site by hapten Prodan anisotropy
84G3
Unknown
42%
(10/24)
1
1
38C2
CKIYKYSFSYW
42%
(10/24)
1
4
CIRGGTAYNRYDGAYW
38%
(10/26)
3
15
CATAHYVNPGRFTKTLDYW
38%
(10/26)
54
17
CTRWGYAYW
43%
(12/28)
Non-specific
binding
9
Tertiary
Secondary
(2c26.1)
Primary
14
10
0
-10
1.0
-3
0.5
0.0
-0.5
x10
Resid.
Multifrequency Phase Fluorometry
0.8
Unligated Phase
Unligated Modulation
Ligated Phase
Ligated Modulation
60
40
0.6
0.4
20
0.2
0
0.0
2
3
4 5 6 78
2
10
3
4 5 6 78
100
Modulation
Phase Angle (Degrees)
80
• The best fit to the
phase and modulation
data for Prodan bound
to 38C2 gives two
lifetime components
• One is a discrete
lifetime of <1 ns
(below the resolution
of the instrument) and
one is a distribution
centered at 4.0 ns with
a full-width at halfmaximum of 0.8 ns
Modulation Frequency (MHz)
15
Steady-State and Multifrequency Phase Fluorometry
lEx Max. lEm Max. Stokes Shift
(cm-1)
(nm)
(nm)
System
L (ns) fwL(ns) f L*
c2
Prodan
1.81 (0.06) discrete 0.42
1.44
375
528
7700
+84G3
5.03 (0.12)
0.96
0.78 1.63
375
473
5500
+38C2
4.16 (0.03)
0.77
0.80 1.24
360
452
5700
+Tertiary
4.03 (0.05)
1.00
0.46 1.39
379
461
4700
+Secondary 2.90 (0.02)
1.86
0.38 1.30
380
494
6000
0.91
0.40 1.66
389
461
4000
375
531
7800
Primary
IgG
4.72 (0.03)
1.66 (0.07) discrete 0.40
1.83
The remainder of the fluorescence intensity is due to a discrete lifetime component
with a lifetime <1 ns, below the resolution of our instrument
16
Conclusions
• Both the blue-shifted emission and increased anisotropy of Prodan can be
used as a probe of binding affinity of antibodies.
• Though raised against a diketone hapten, the antibodies studied exhibit Kd
values ranging from 0.122 – 2.1 M.
• The 50-fold range of Prodan binding constants determined for the antibodies
studied are consistent with the binding behavior of the antibodies for the
hapten as measured by tryptophan quenching (more mature antibodies bind
more strongly).
• The emission spectrum of Prodan bound to antibodies is blue-shifted relative
to that observed for Prodan in solution or in the presence of a non-specific
antibody, indicating the hydrophobic nature of the antibody binding sites.
• The lifetime data exhibit a marked increase (factor of three) in the lifetime of
Prodan when bound to an antibody.
• Lifetime distribution widths decrease with increasing antibody maturity,
suggesting that affinity maturation produces antibodies with increasingly
rigid binding sites.
17
Acknowledgements
• NSF C-RUI collaborators
– Prof. Richard Goldsby, creation of antibody family
– Prof. David Hansen, synthesis of hapten
– Prof. David Ratner and Nalini Sha-Mahoney,
genetic characterization of antibodies
– Phil Chiu ‘02, Trp quenching data
• Camille and Henry Dreyfus Scholar/Fellow
Program for Undergraduate Institutions
• Faculty Research Awards Progam, Amherst
College
18
Emission Spectra of Prodan Bound
to the Tertiary Antibody
Free Prodan
Intensity (Arbitrary Units)
Bound Prodan
400
450
500
Wavelength (nm)
550
• Excitation is at 361 nm
• As seen with the 38C2
antibody, the prodan emission
shifts to 445 nm upon binding
to the tertiary antibody, and
the intensity at 445 nm
increases with increasing
antibody concentration
• Again, both bound and free
prodan contribute to the
fluorescence signal at 510 nm
600
14
Quenching of Intrinsic Tryptophan
Fluorescence on Hapten Binding
3
3
80
40
340
360
380
Wavelength (nm)
320
340
360
380
Wavelength (nm)
400
340
360
380
Wavelength (nm)
400
3
Intensity (a.u.)
Intensity (a.u.)
320
150x10
120x10
100
80
60
40
20
400
Intensity (a. u.)
Intensity (a.u.)
120x10
120x10
3
80
100
50
40
320
340
360
380
Wavelength (nm)
400
320
Emission Spectra of Prodan Bound to
the Mature 38C2 Antibody
Free Prodan
Intensity (Arbitrary Units)
60
50
40
Bound Prodan
30
20
• Excitation is at 361
nm
• Emission intensity
at 445 nm is due to
bound prodan, and
is observed to
increase with
increasing antibody
concentration
10
0
400
450
500
Wavelength (nm)
550
600
Emission Spectra of Prodan Bound to
the Mature 38C2 Antibody
Bound Prodan
Intensity in Arbitrary Units
120
100
Free Prodan
80
60
40
• Excitation is at 361
nm
• Emission intensity
at 445 nm is due to
bound prodan, and
is observed to
increase with
increasing antibody
concentration
20
400
450
500
Wavelength (nm)
550
600
Summary of Antibody Binding Affinity Data
Antibody
Heavy Chain CDR3
Sequence
Kd /Kd38C2
% Trp
Trp
residues in
binding site quenching
by hapten
Kd from prodan
anisotropy
(fluorescence)
CKIYKYSFSYW
42%
(10/24)
1
0.51 ± 0.06 M
(0.75 ± 0.06 M)
CIRGGTAYNRYDGAYW
38%
(10/26)
3
0.8 ± 0.3 M
(0.73 ± 0.04 M)
Secondary
(A2c26.1)
CATAHYVNPGRFTKTLDYW
38%
(10/26)
54
1.8 ± 0.7 M
Secondary
(A2c22.1)
CTRGNYGYVGAYW
38%
(10/26)
180*
N/A
CTRWGYAYW
43%
(12/28)
Non-specific
binding
1.1 ± 1.0 M
38C2
Tertiary
Primary
*Ligand
used was acetyl acetone, not the hapten
18
Determination of Antibody Dissociation
Constants From Anisotropy Data
Primary
Kd = 2.5 ± 0.4 M
Secondary Kd = 1.3 ± 0.8 M
Tertiary
Kd = 410 ± 20 nM
Mature 38C2 Kd = 250 ± 40 nM
Anisotropy at 518 nm (mP)
300
250
200
150
100
50
0
200
400
600
Total Antibody Concentration (nM)
17
Determination of Antibody Binding Constants
From Tryptophan Fluorescence Quenching
F0-F (Arbitrary Units)
100x10
Non-Specific Antibody
Primary Antibody
Secondary Antibody
Tertiary Antibody
Mature 38C2 Antibody
3
80
60
40
20
0
0
1
2
3
4
Hapten Concentration (M)
5x10
-6
11
Determination of Antibody Dissociation Constants
• Quenching of the intrinsic tryptophan fluorescence of the antibodies
is related to the fraction of antibody binding sites filled (Fb)
according to the following relationship:
F0  F
Fb 
F0  Fm q
Fb: Fraction of binding sites filled
F0: Trp fluorescence in the absence of ligand
F: Trp fluorescence in the presence of ligand
Fmq: Trp fluorescence with maximum quenching
• A plot of F0-F vs. the total ligand concentration (Lt) can be used to
determine the dissociation constants (Kd) of the antibodies according
to the following relationship:
( K d  St  Lt )  K d2  2 K d St  2 K d Lt  St2  2St Lt  L2t
F0  F  ( F0  Fm q )
2St
St: Total concentration of antibody binding sites (all other parameters defined above)
8
Quenching of Intrinsic Tryptophan
Fluorescence on Hapten Binding
38C2
Increasing
hapten
Intensity (a.u.)
120x10
3
80
40
320
IgG
400
Increasing
hapten
Intensity (a. u.)
160x10
340
360
380
Wavelength (nm)
3
120
80
40
320
340
360
380
Wavelength (nm)
400
• Excitation at 295 nm for
38C2, 284 nm for IgG
• The fluorescence
intensity due to intrinsic
tryptophan residues in
the antibodies decreases
with increasing hapten
concentration
• The fluorescence
quenching observed for
the mature 38C2
antibody (specific
binding) is greater than
that observed for IgG
(non-specific binding)
9
Quenching of Intrinsic Tryptophan
Fluorescence on Hapten Binding
Tertiary
Secondary (A2c26.1)
Increasing
hapten
Intensity (a. u.)
Increasing 120x103
hapten
100
Intensity (a.u.)
0x10
lexc=295 nm
in all cases
3
80
60
40
20
100
50
340
360
380
Wavelength (nm)
Intensity (a.u.)
Secondary (A2c22.1)
600
500
400
300
200
100
320
320
400
340
360
380
Wavelength (nm)
400
Primary
Increasing
hapten
Increasing
3
120x10
hapten
340
360
380
Wavelength (nm)
Intensity (a.u.)
320
80
40
400
320
340
360
380
Wavelength (nm)
400
10
Determination of Antibody Dissociation Constants
From Tryptophan Fluorescence Quenching
10
Mature 38C2
Kdq= 0.05 ± 0.10 M
Tertiary
Kd= 0.15 ± 0.09 M
Secondary (A2c26.1) Kd= 2.7 ± 1.6 M
Secondary (A2c22.1) Kd= 9.0 ± 0.9 M
3
F0-F (Arbitrary Units)
100
80
60
40
20
0
0
1
2
3
4
Hapten Concentration (M)
5
6x10
-6
• Ab concentrations  3M
• Primary antibody shows
non-specific binding
similar to IgG (data not
shown)
• Other antibodies show
binding behavior more
similar to 38C2
• Because 38C2 forms a
covalent bond with the
hapten, the value reported
is an apparent Kd, Kdq
• Non-specific binding
occurs at high hapten
concentrations
• Use of fluorescent ligand
would allow for lower
ligand concentrations
11