EVOLUTION OF CATALYTIC ANTIBODY FLEXIBILITY AS PROBED BY MULTIFREQUENCY PHASE FLUOROMETRY AS PROBED...
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EVOLUTION OF CATALYTIC ANTIBODY FLEXIBILITY EVOLUTION OF CATALYTIC ANTIBODY FLEXIBILITY AS PROBED BY MULTIFREQUENCY PHASE FLUOROMETRY AS PROBED BY MULTIFREQUENCY PHASE FLUOROMETRY Patricia B. O’Hara, Philip Chiu Department of Chemistry Amherst College Amherst, MA, 01002 Abstract This research focuses on the family of catalytic antibodies, which includes the mature aldolase antibody 38C2. Shown to catalyze an aldol condensation by means of a protein bound enamine intermediate (1995 Science 270, 1797-1800), aldolase antibody 38C2 is now commercially available (Aldrich catalogue #47,995-0). We have created, purified, and characterized a closely related family of catalytic antibodies in mice at various points in the immune response. These antibodies have been harvested after reactive immunization with the same diketone hapten used to elicit 38C2. In order to maximize the diversity of the antibodies available, our isolations have focused on the population available in the germinal center, the center of hypervariability. Here we make use of the natural fluorescence of the tryptophan residues, five of which cluster at each of the antigen’s binding site in the antibody. An ISS K-2 multifrequency phase fluorometer was used to measure the lifetime distribution for the tryptophans in the protein. Two sets of distributions can be resolved for the tryptophan’s in the 38C2 family: one narrow gaussian distribution centered at approximately 1 ns, and a wider distribution centered at 4 ns. We will show how the width of these distributions vary according to the different stages of evolution and test the assumption that germ line antibodies are flexible and able to bind similar but not identical antigens. Conversely that antibodies having undergone affinity maturation are more rigid, and able to bind only one specific antigen with high affinity. Introduction It has been hypothesized that the active site of an immature or germ line antibody displays enormous flexibility, a necessary feature of this generic and relatively indiscriminate first line defender. Over the course of an infection, antibody producing cells undergo affinity maturation in lymph tissue. This poorly understood process results in a population of cells which produce antibody molecules with much higher specificity and higher affinity. The high specificity and affinity are thought to be accompanied by a loss of conformational entropy at the active site. Analysis of crystal structures of germ line and mature antibodies supports this hypothesis (Wedemeyer et al, 1997, Science 276 1665-1669). Since it has been known for many years that the binding of antigen to antibodies results in a large quench in the intensity of the antibodies’ natural tryptophan fluorescence, we set out to explore whether we could use the native tryptophan fluorescence of antibodies to explore this hypothesis in greater detail. X-ray crystal structure of catalytic aldolase antibody 38C2 reveals the presence of a lysine, H93, in the active site. This lysine is thought to play a direct role in catalysis of aldol condensation by covalent bond formation with a diketone substrate analogous to classic mechanisms of other class I aldolases (Shulz et al 1995 Science 270 1797-1800). We used the same diketone hapten to produce a family of antibodies with the purpose of studying the evolution of binding site flexibility over the course of affinity maturation. A panel of primary, secondary, and tertiary monoclonal antibodies have been isolated from mice using standard hybridoma technology in the lab of Richard Goldsby in the biology department at Amherst College. Here we have spectroscopically characterized changes in fluorescence of 38C2, and a family of catalytic antibodies related to 38C2 upon ligand binding. The ability of each antibody to form a covalent adduct with the substrate analogue can first be evaluated by UV difference spectrum. These series of experiments focus on characterizing the steady state and time resolved native Trp fluorescence of this family and examining changes induced by binding to ligand. Results UV difference spectra for 38C2, which contains an active site lysine, K H93, display a UV signal at 316 nm when titrated with ligand. This absorption is characteristic of a vinylogous amide and demonstrates the covalent nature of this ligand/protein interaction. None of our primary, secondary, or tertiary antibodies displayed this characteristic peak, and therefore bind the ligand by alternative non-covalent mechanisms. The native tryptophan fluorescence intensity of 38C2 quenches 35% upon binding of antigen. This can be interpreted as a total quenching of the 10 tryptophan at the two active sites out of the 27 tryptophan in the entire antibody molecule and is consistent with quenching patterns seen for other antibodies. Modeling of 38 C2 with Rasmol show that there is nothing unique about active site cluster of tryptophan residues. The five tryptophan at each active site are all within 10 A of Lys 93 and presumable the ligand. Alignment of the sequence of our antibodies with germ line sequences confirm that we are observing antibodies undergoing affinity maturation. Upon excitation with 284 nm light and emission collected without filters, two lifetimes are for all antibody samples, observed, a short lifetime centered at about 1.0 ns and a longer lifetime centered at about 4.0 ns. The short lifetime value varies from 0.88 ns for 38C2, to 1.21 ns, 1.07 ns and 0.82 ns for primary, secondary and tertiary antibodies we have made. Addition of ligand changes the short lifetime by 18% for 38C2; and 66%, 14% and 104% for primary, secondary and tertiary respectively. The long lifetime value varies from 3.71 ns for 38C2 to 3.96 ns, 4.03 ns, and 4.29 ns for the primary, secondary, and tertiary antibodies we have made. Addition of ligand changes the long lifetime by 18% for 38C2; and 2%, 3% and 8% for primary, secondary and tertiary respectively. Fit of the data to a two component gaussian distribution is better than or comparable to a two component discrete fit. χ 2 values for all of these are less that 2. The lifetime distributions for the short lifetime value is 0.35 ns for 38C2, and 0.51 ns, 0.47 ns, and 0.71 ns for primary, secondary and tertiary respectively. Upon addition of ligand, these distributions narrow 15% for 38C2, and 18%, 98%, and 99% primary, secondary and tertiary respectively. •The lifetime distributions for the long lifetime value is 0.75 ns for 38C2, and 1.11 ns, 0.93 ns, and 1.07 ns for primary, secondary and tertiary respectively. Upon addition of ligand, these distributions narrow 40% for 38C2, and 41%, 3%, and 0% primary, secondary and tertiary respectively. Conclusions Lifetime centers for the tryptophan fluorescence in the four antibodies studied were similar to those observed in other proteins (RNAase 0.98 ns,3.57 ns; and glucagon 1.1ns, 3.6 ns; Lakowicz, p493) and to tryptophan in aqueous solution (0.81 ns, 3.33 ns Lakowicz, p488). The two lifetime components are both from the same emissive state, and represent two different rotational isomers of the tryptophan. This suggests that the antibodies that we studied did not contain any tryptophan in unusual environments. The longer lifetime component shows a slight increase from primary to secondary to tertiary, but we are not confident of its significance, and 38C2 is right in line with all of the antibodies we isolated. The shorter lifetime component appears to decreases as the antibody matures; here mature 38C2 is most similar to our tertiary antibody. • Ligand Binding in almost all cases resulted in a decrease in the lifetime for both long and short lifetimes. In almost all cases, the drop is not significant. An exception is the 66% quench in the shorter lifetime of the only primary antibody studied. Sequence analysis of the CDR3 loop of this antibody (the loop that makes the most contact with the ligand) reveals an additional tryptophan for this antibody. Nearby positive charges can quench this fluorescence. Perhaps, in the transition from ligand free to bound, the positively charged arginine adjacent to this tryptophan is recruited to assist in making favorable enthalpic interactions with the negatively charged ligand. This ligand induced change brings the tryptophan in close proximity to the positive charge for only one conformer corresponding to the short lifetime. The lifetime distributions for both long and short lifetimes narrowed upon ligand binding (in all but one case). For the secondary and tertiary antibodies, the long lifetime distribution was unaffected, and the short lifetime distribution was dramatically reduced. For these two antibodies, the ligand bound state is best described by a discrete lifetime (i.e no distribution). For primary antibody and 38C2 mature antibody ligand binding resulted in a narrowing of both long and short lifetimes, with the longer lifetime component showing the greatest narrowing. This suggest that ligand binding results in conformational annealing, which is preferential to one conformer for secondary and tertiary but distributed across both conformers for primary and 38C2. C hange in long lifetim e value upon addition ofligand 0 1 2 3 4 5 A 3.1.1 2c 26.1 3.22 aldolase Antibody Lifetim e (ns) withoutligand w ith ligand C hange in long lifetim e fraction upon addition ofligand 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 A 3.1.1 2c 26.1 3.22 aldolase Antibody Fraction withoutligand w ith ligand C hange in long lifetim e w idth upon addition ofligand 0.0 0.2 0.4 0.6 0.8 1.0 1.2 A 3.1.1 2c 26.1 3.22 aldolase Antibody W idth (ns) withoutligand w ith ligand C hange in shortlifetim e value upon addition ofligand 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 A 3.1.1 2c 26.1 3.22 aldolase 38C 2 Antibody Lifetim e (ns) withoutligand w ith ligand C hange in shortlifetim e fraction upon addition ofligand 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 A 3.1.1 2c 26.1 3.22 aldolase 38C 2 Antibody Fraction withoutligand w ith ligand C hange in shortlifetim e w idth upon addition ofligand 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 A 3.1.1 2c 26.1 3.22 aldolase 38C2 Antibody W idth(ns) withoutligand w ith ligand Antibody Name Response Stage Harvest Time From Mice A 3.1.1 Primary 12 days after initial immunization 2c 26.1 Secondary 5 days after the first boost 3.22 Tertiary 5 days after the second boost Aldolase Catalytic Antibody 38C2 Mature 14 days after the second boost Antibody Name # Nucleotide Mutations Relative to the Germline Gene Sequence in V region Amino Acid Sequence in CDR 3 (location of key lysine residue in 38 C2) A 3.1.1 Light Chain- 0 Heavy Chain- 11 CTRWGYAYWGQGTLVTVS 2c 26.1 Light Chain- 2 Heavy Chain- 14 CATAHYVNPGRFTK TLDWG 3.22 Light Chain – 2 Heavy Chain – 15 CIRGGTAYNRYDGAYWGQG Aldolase Catalytic Antibody 38C2 Light Chain – 13 Heavy Chain – 25 CK IYKYSFSYWGQGTLVTVS Acknowledgments This work was supported by an NSF collaborative research at undergraduate colleges grant. Amherst College colleagues contributing to the catalytic antibody project include Professor Richard Goldsby, immunology; Professor David Ratner, molecular biology and genetics; and Professor David Hansen, bioorganic chemist. We are especially grateful to Nalini Shah-Mahoni from Professor Ratner’s lab who performed the gene sequencing studies and Elise Nguyen ‘04 who assisted with the antibody purifications. M ax fluorescence intensity vs.fraction ofsites bound w ith acetylacetone 0 10000 20000 30000 40000 50000 60000 70000 80000 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 fraction ofactive sites bound w ith ligand intensity at334 nm (a.u.) UV/Vis subtraction Assay of Aldolase 38C2 UV/Vis substraction Assay of Asc. 26.1
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Transcript of EVOLUTION OF CATALYTIC ANTIBODY FLEXIBILITY AS PROBED BY MULTIFREQUENCY PHASE FLUOROMETRY AS PROBED...
EVOLUTION OF CATALYTIC ANTIBODY FLEXIBILITY EVOLUTION OF CATALYTIC ANTIBODY FLEXIBILITY
AS PROBED BY MULTIFREQUENCY PHASE FLUOROMETRYAS PROBED BY MULTIFREQUENCY PHASE FLUOROMETRY
Patricia B. O’Hara, Philip ChiuDepartment of Chemistry
Amherst CollegeAmherst, MA, 01002
Abstract
This research focuses on the family of catalytic antibodies, which includes the mature aldolase antibody 38C2. Shown to catalyze an aldol condensation by means of a protein bound enamine intermediate (1995 Science 270, 1797-1800), aldolase antibody 38C2 is now commercially available (Aldrich catalogue #47,995-0). We have created, purified, and characterized a closely related family of catalytic antibodies in mice at various points in the immune response. These antibodies have been harvested after reactive immunization with the same diketone hapten used to elicit 38C2. In order to maximize the diversity of the antibodies available, our isolations have focused on the population available in the germinal center, the center of hypervariability. Here we make use of the natural fluorescence of the tryptophan residues, five of which cluster at each of the antigen’s binding site in the antibody. An ISS K-2 multifrequency phase fluorometer was used to measure the lifetime distribution for the tryptophans in the protein. Two sets of distributions can be resolved for the tryptophan’s in the 38C2 family: one narrow gaussian distribution centered at approximately 1 ns, and a wider distribution centered at 4 ns. We will show how the width of these distributions vary according to the different stages of evolution and test the assumption that germ line antibodies are flexible and able to bind similar but not identical antigens. Conversely that antibodies having undergone affinity maturation are more rigid, and able to bind only one specific antigen with high affinity.
Introduction It has been hypothesized that the active site of an immature or germ line antibody displays enormous flexibility, a necessary feature of this generic and relatively indiscriminate first line defender. Over the course of an infection, antibody producing cells undergo affinity maturation in lymph tissue. This poorly understood process results in a population of cells which produce antibody molecules with much higher specificity and higher affinity. The high specificity and affinity are thought to be accompanied by a loss of conformational entropy at the active site. Analysis of crystal structures of germ line and mature antibodies supports this hypothesis (Wedemeyer et al, 1997, Science 276 1665-1669). Since it has been known for many years that the binding of antigen to antibodies results in a large quench in the intensity of the antibodies’ natural tryptophan fluorescence, we set out to explore whether we could use the native tryptophan fluorescence of antibodies to explore this hypothesis in greater detail.
X-ray crystal structure of catalytic aldolase antibody 38C2 reveals the presence of a lysine, H93, in the active site. This lysine is thought to play a direct role in catalysis of aldol condensation by covalent bond formation with a diketone substrate analogous to classic mechanisms of other class I aldolases (Shulz et al 1995 Science 270 1797-1800). We used the same diketone hapten to produce a family of antibodies with the purpose of studying the evolution of binding site flexibility over the course of affinity maturation. A panel of primary, secondary, and tertiary monoclonal antibodies have been isolated from mice using standard hybridoma technology in the lab of Richard Goldsby in the biology department at Amherst College. Here we have spectroscopically characterized changes in fluorescence of 38C2, and a family of catalytic antibodies related to 38C2 upon ligand binding. The ability of each antibody to form a covalent adduct with the substrate analogue can first be evaluated by UV difference spectrum. These series of experiments focus on characterizing the steady state and time resolved native Trp fluorescence of this family and examining changes induced by binding to ligand.
Results UV difference spectra for 38C2, which contains an active site lysine, K H93, display a UV signal at 316 nm when titrated with ligand. This absorption is characteristic of a vinylogous amide and demonstrates the covalent nature of this ligand/protein interaction. None of our primary, secondary, or tertiary antibodies displayed this characteristic peak, and therefore bind the ligand by alternative non-covalent mechanisms.
The native tryptophan fluorescence intensity of 38C2 quenches 35% upon binding of antigen. This can be interpreted as a total quenching of the 10 tryptophan at the two active sites out of the 27 tryptophan in the entire antibody molecule and is consistent with quenching patterns seen for other antibodies.
Modeling of 38 C2 with Rasmol show that there is nothing unique about active site cluster of tryptophan residues. The five tryptophan at each active site are all within 10 A of Lys 93 and presumable the ligand.
Alignment of the sequence of our antibodies with germ line sequences confirm that we are observing antibodies undergoing affinity maturation.
Upon excitation with 284 nm light and emission collected without filters, two lifetimes are for all antibody samples, observed, a short lifetime centered at about 1.0 ns and a longer lifetime centered at about 4.0 ns.
The short lifetime value varies from 0.88 ns for 38C2, to 1.21 ns, 1.07 ns and 0.82 ns for primary, secondary and tertiary antibodies we have made. Addition of ligand changes the short lifetime by 18% for 38C2; and 66%, 14% and 104% for primary, secondary and tertiary respectively.
The long lifetime value varies from 3.71 ns for 38C2 to 3.96 ns, 4.03 ns, and 4.29 ns for the primary, secondary, and tertiary antibodies we have made. Addition of ligand changes the long lifetime by 18% for 38C2; and 2%, 3% and 8% for primary, secondary and tertiary respectively.
Fit of the data to a two component gaussian distribution is better than or comparable to a two component discrete fit. χ 2 values for all of these are less that 2.
The lifetime distributions for the short lifetime value is 0.35 ns for 38C2, and 0.51 ns, 0.47 ns, and 0.71 ns for primary, secondary and tertiary respectively. Upon addition of ligand, these distributions narrow 15% for 38C2, and 18%, 98%, and 99% primary, secondary and tertiary respectively. •The lifetime distributions for the long lifetime value is 0.75 ns for 38C2, and 1.11 ns, 0.93 ns, and 1.07 ns for primary, secondary and tertiary respectively. Upon addition of ligand, these distributions narrow 40% for 38C2, and 41%, 3%, and 0% primary, secondary and tertiary respectively.
Conclusions Lifetime centers for the tryptophan fluorescence in the four antibodies studied were similar to those observed in other proteins (RNAase 0.98 ns,3.57 ns; and glucagon 1.1ns, 3.6 ns; Lakowicz, p493) and to tryptophan in aqueous solution (0.81 ns, 3.33 ns Lakowicz, p488). The two lifetime components are both from the same emissive state, and represent two different rotational isomers of the tryptophan. This suggests that the antibodies that we studied did not contain any tryptophan in unusual environments.
The longer lifetime component shows a slight increase from primary to secondary to tertiary, but we are not confident of its significance, and 38C2 is right in line with all of the antibodies we isolated. The shorter lifetime component appears to decreases as the antibody matures; here mature 38C2 is most similar to our tertiary antibody. • Ligand Binding in almost all cases resulted in a decrease in the lifetime for both long and short lifetimes. In almost all cases, the drop is not significant. An exception is the 66% quench in the shorter lifetime of the only primary antibody studied. Sequence analysis of the CDR3 loop of this antibody (the loop that makes the most contact with the ligand) reveals an additional tryptophan for this antibody. Nearby positive charges can quench this fluorescence. Perhaps, in the transition from ligand free to bound, the positively charged arginine adjacent to this tryptophan is recruited to assist in making favorable enthalpic interactions with the negatively charged ligand. This ligand induced change brings the tryptophan in close proximity to the positive charge for only one conformer corresponding to the short lifetime.
The lifetime distributions for both long and short lifetimes narrowed upon ligand binding (in all but one case). For the secondary and tertiary antibodies, the long lifetime distribution was unaffected, and the short lifetime distribution was dramatically reduced. For these two antibodies, the ligand bound state is best described by a discrete lifetime (i.e no distribution). For primary antibody and 38C2 mature antibody ligand binding resulted in a narrowing of both long and short lifetimes, with the longer lifetime component showing the greatest narrowing. This suggest that ligand binding results in conformational annealing, which is preferential to one conformer for secondary and tertiary but distributed across both conformers for primary and 38C2.
Change in long lifetime value upon addition of ligand
0
1
2
3
4
5
A 3.1.1 2c 26.1 3.22 aldolase
Antibody
Life
time
(ns)
w ithout ligand
w ith ligand
Change in long lifetime fraction upon addition of ligand
00.10.20.30.40.50.60.70.8
A 3.1.1 2c 26.1 3.22 aldolase
Antibody
Fra
ctio
n
w ithout ligand
w ith ligand
Change in long lifetime width upon addition of ligand
0.0
0.2
0.4
0.6
0.8
1.0
1.2
A 3.1.1 2c 26.1 3.22 aldolase
Antibody
Wid
th (
ns)
w ithout ligand
w ith ligand
Change in short lifetime value upon addition of ligand
0.00.20.40.60.81.01.21.4
A 3.1.1 2c 26.1 3.22 aldolase38C2
Antibody
Life
time
(ns)
w ithout ligand
w ith ligand
Change in short lifetime fraction upon addition of ligand
0.00
0.050.10
0.15
0.20
0.250.30
0.35
A 3.1.1 2c 26.1 3.22 aldolase38C2
Antibody
Fra
ctio
n
w ithout ligand
w ith ligand
Change in short lifetime width upon addition of ligand
00.10.20.30.40.50.60.70.8
A 3.1.1 2c 26.1 3.22 aldolase 38C2
Antibody
Wid
th (n
s) w ithout ligand
w ith ligand
Antibody Name Response Stage Harvest Time From Mice
A 3.1.1
Primary
12 days after initial immunization
2c 26.1
Secondary
5 days after the first boost
3.22
Tertiary
5 days after the second boost
Aldolase Catalytic Antibody 38C2
Mature
14 days after the second boost
Antibody Name
# Nucleotide Mutations Relative to the Germline Gene Sequence in V region
Amino Acid Sequence in CDR 3 (location of key lysine residue in 38 C2)
A 3.1.1
Light Chain- 0Heavy Chain- 11
CTRWGYAYWGQGTLVTVS
2c 26.1
Light Chain- 2Heavy Chain- 14
CATAHYVNPGRFTKTLDWG
3.22
Light Chain – 2Heavy Chain – 15
CIRGGTAYNRYDGAYWGQG
Aldolase Catalytic Antibody 38C2
Light Chain – 13Heavy Chain – 25
CKIYKYSFSYWGQGTLVTVS Acknowledgments
This work was supported by an NSF collaborative research at undergraduate colleges grant. Amherst College colleagues contributing to the catalytic antibody project include Professor Richard Goldsby, immunology; Professor David Ratner, molecular biology and genetics; and Professor David Hansen, bioorganic chemist. We are especially grateful to Nalini Shah-Mahoni from Professor Ratner’s lab who performed the gene sequencing studies and Elise Nguyen ‘04 who assisted with the antibody purifications.
Max fluorescence intensity vs. fraction of sites bound with acetylacetone
01000020000300004000050000600007000080000
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
fraction of active sites bound with ligand
inte
nsity
at
334
nm (
a.u.
)
UV/Vis subtraction Assay of Aldolase 38C2
UV/Vis substraction Assay of Asc. 26.1
