©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Temperature and pH Dependence of Fluorescein Binding within the Monoclonal Antibody 9-40 Active Site as Monitored by Hydrostatic Pressure (*)

(Received for publication, November 27, 1995; and in revised form, December 18, 1995)

Jenny Carrero Edward W. Voss Jr. (§)

From the Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801-3797

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In a comparative study, the thermodynamic parameter, DeltaV, was obtained using hydrostatic pressure-induced dissociation of fluorescein (Fl) from the active site of monoclonal antibody (mAb) 9-40 and its mutant and native derivatives equilibrated at six pH values (8.0, 7.5, 7.0, 6.5, 6.0, and 5.5) and four temperatures (35, 25, 15, and 5 °C). mAb 9-40 and its Fab and single-chain Fv (scFv) derivatives at pH 8.0 were found to have identical Fl dissociation behavior under pressure as a function of temperature. The pressure dissociation at 25 °C as a function of pH showed a sigmoidal dependence of DeltaV with a midpoint value at pH 7.4 for mAb 9-40. Comparison of experimental results for scFv 9-40/212 with its mutant scFv 9-40/212 indicated that the pH dependence of mAb 9-40 was due to the titration of His-34L in the active site. Iodide quenching of bound Fl showed that the hapten in this active site was solvent accessible. Imperfect packing, which leads to increased conformational dynamics, was determined as a possible cause of the low affinity for mAb 9-40.


INTRODUCTION

The understanding of antibody (Ab) (^1)structure-function has been enhanced during the past two decades through the advent of hybridoma methodology(1, 2) , the increase in resolved atomic structures due primarily to x-ray crystallography (3, 4, 5, 6) , generation of Ab structural derivatives such as single-chain Fv (scFv) fragments(7, 8) , and site-specific mutagenesis of well characterized Abs(9, 10) . However, despite these significant advances, a comprehensive understanding of all the factors that directly impact the binding of antigen remain ill defined. Such factors as domain-domain interactions, binding affinity (K) as it correlates with primary and secondary interactions(11) , the total effect of somatic mutations, and the structure-function role of the first constant domains in both the heavy and light chains remain unresolved. The role and magnitude of these parameters have been inferred from a spectrum of studies, but direct measurements have not been made that correlate the results with variable domain or protein dynamics(12) .

To experimentally isolate these parameters for comparative and direct quantitative measurements, model Ab reagents must be carefully selected so that the results can be integrated into meaningful principles. The conclusions from this study were based on two anti-fluorescein (anti-Fl) mAbs, mAb 4-4-20 and mAb 9-40(13, 14, 15) , which are highly related at the idiotypic or unliganded level, reflecting nearly identical primary structures in both the heavy and light chains(14) . However, these Abs were only marginally related at the metatype or liganded level, suggesting significantly different conformations of the liganded state(16, 17) . Primary structural studies have also shown that 4-4-20 is a high affinity somatic mutant of the lower affinity germ line 9-40 molecule(15) . The affinities of these two Abs differ by 1000-fold for the Fl ligand with the value of 2.8 times 10^7M(10) and 2 times 10M(14) for 9-40 and 4-4-20, respectively.

Comparatively, in mAb 9-40 the heavy chain variable (V(H)) region of the amino acid sequence differs by nine amino acid residues from 4-4-20 with most of the differences localized in the HCDR3 hypervariable region, whereas the variable light chain (V(L)) differed by only two amino acid residues(14, 15) . Based on the crystal structure of 4-4-20, it was shown that only one 4-4-20 contact residue (Arg-34L) was not present in 9-40 (His-34L). In summary, 9-40 provided an opportunity to determine a set of rules that define affinity maturation by making a comparison of a low affinity Ab to a high affinity Ab that were structurally and sequentially similar.

In an effort to help establish a set of rules specifying structure-function relationships that may be eventually used for Ab engineering, hydrostatic pressure-dependent behavior of mAb 9-40 and its structural derivative scFv 9-40, a genetically engineered derivative of the anti-Fl mAb 9-40 (9, 10) comprised of the V(L) and V(H) domains of the corresponding mAb joined by a short polypeptide linker (7, 8) was studied. From crystallographic studies, it was known that when Fl was bound, Arg-34L in 4-4-20 was salt linked to the enolic oxygen(18, 19) . The crystal structure of 9-40 was not yet available, and therefore the role of His at the 34L position in connection to Fl was not known. However, the influence of the contact residue His-34L was monitored by comparing scFv9-40/212 with scFv9-40/212. Specifically, hydrostatic pressure was used to induce dissociation of Fl as a function of pH from the active site of mAb 9-40 and its native and mutant derivatives, scFv9-40/212 and scFv9-40/212, respectively. The thermodynamic parameter, change in partial molar volume (DeltaV), was found to be pH-dependent for mAb 9-40.

Because the affinities of mAb 9-40 and its derivatives, Fab 9-40 and scFv 9-40/212, for Fl were identical(10) , the influence of IgG constant domains on Ab stability were also investigated. Hydrostatic pressure was used to study the effect of IgG constant domains on 9-40 binding without the additional complication of affinity as a variable. In this study, pressure induced dissociation behavior as a function of temperature of mAb and its Fab and scFv derivatives were identical, within error. Finally, compared with 4-4-20, iodide quenching of bound Fl in 9-40 and 4-4-20 also showed Fl was more solvent-accessible in 9-40.


MATERIALS AND METHODS

scFv Ab 9-40

scFv 9-40/212 and scFv9-40/212 were constructed and expressed as described previously(7, 9) .

Fab 9-40

Fab 9-40 was prepared from IgG 9-40 by papain digestion using immobilized papain (Pierce) as per product protocol. Fab fragments were purified from the undigested IgG and Fc fragments using protein A-Sepharose and Fl-Sepharose 4B chromatography(20, 21) .

mAb 9-40

mAb 9-40 was obtained from ascites fluid by affinity purification using Fl-Sepharose 4B as described previously (20, 21) .

Iodide Quenching

All fluorescence experiments were performed on a photon counter spectrofluorometer (Gregg-PC, ISS Instruments, Champaign, IL). Aliquots of potassium iodide were taken from a 4 M potassium iodide stock solution to prepare two sets of six protein samples, all of which contained 0.02 µM Fl, 2 µM mAb 9-40, and 0, 0.25, 0.50, 0.75, 1.00 or 1.25 M potassium iodide, respectively, in the first set or 1 µM mAb 4-4-20 in the second set. In both cases, anisotropy at 0.0 M potassium iodide indicated that the fluorophore was fully bound. Emission spectra were recorded in the wavelength region spanning 500-600 nm with a slit width of 8 nm. Fluorescence intensity at each pressure was acquired by integrating the area under the emission spectrum. The Stern-Volmer equation was used to calculate the degree of intensity fluorescence quenching as a function of iodide as follows:

where I(0) was the intensity at zero quencher concentration, I was the intensity at quencher concentration [Q], k was the bimolecular constant, and (0) was the lifetime of the fluorophore.

Hydrostatic Pressure Fluorescence Measurements

Hydrostatic pressure in the range of 1 bar to 2.4 Kbar was achieved using the pressure cell described by Paladini and Weber(22, 23) . For all pressures, the protein samples were allowed to equilibrate for 4 min before measurements were made. The protein-ligand sample was excited at 480 nm with a slit width of 8 nm. For fluorescence polarization measurements, the emission wavelength was 525 nm with a slit width of 16 nm. Polarization of each sample in the pressure cell at 0 bar was identical to the polarization obtained without the cell. Fluorescence polarization values measured in the pressure cell were corrected for birefringence of the quartz windows as described by Paladini and Weber (22) . The dissociation parameter, alpha, was calculated using as follows:

where R is the ratio of the fluorescence quantum yields of free and bound forms, r is the anisotropy at each pressure, and r(F) and r(B) are the temperature- and pH-dependent anisotropies for free and bound states, respectively(22) , which are calculated from polarization as r = 2P/(3 - P), where P is the polarization. The pH dependence of the Fl quantum yield was corrected by incorporation of pH-dependent R values in . Dissociation constants (K(d)) at each pressure were calculated using the following relation:

where [Abs](0) and [Fl](0) were the total concentrations of Ab active sites for scFv, Fab, or mAb and Fl, respectively(24, 25, 26) . The dissociation constant related to the free energy of dissociation as follows:

where R was the gas constant and T was the temperature in Kelvin. The linear regions of the dissociation curves were used to calculate values for K(d) and DeltaG, for IgG, Fab, scFv 9-40/212, and scFv 9-40/212 at all temperatures and pH values. DeltaV was obtained from the following relation:

Hydrostatic Protein Samples

All stock solutions prepared in 40 mM Tris contained 0.2 µM Ab active sites and 0.02 M Fl except scFv 9-40/212, which contained 3 µM active sites. The polarization of each sample at 25 °C was taken immediately prior to each pressure measurement to ensure integrity of the sample. The temperature measurements were performed at pH 8.0 and at 5, 15, 25, and 35 °C. The temperature was regulated with a circulating temperature bath and monitored to ±0.1 °C with the thermocouple in direct contact with the stainless steel pressure cell. For the pH measurements, the experiments were performed at 25 °C, the protein was dialyzed overnight against pH 8.0, 7.5, 7.0, 6.5, 6.0, or 5.5 ± 0.1 pH unit for mAb and scFv 9-40/212. For scFv 9-40/212, only three samples at pH 6.5, 7.2, and 8.0 were prepared because of the relatively large amount of protein necessary.


RESULTS

Iodide Quenching

Results of the iodide quenching studies are shown in Fig. 1. The bimolecular collision constant of 5.1 times 10^8 sM, which was calculated using for mAb 9-40, was equal to that expected for the collision in solution of free iodide with Fl bound by a protein. Therefore, when Fl was bound by mAb 9-40 it was solvent accessible. The iodide quenching of mAb 4-4-20 was previously examined by Coelho-Sampaio and Voss(26) . The increased iodide quenching of Fl in the mAb 9-40 active site as compared with 4-4-20 indicates that Fl was more accessible in 9-40.


Figure 1: Iodide quenching of fluorescence intensity of Fl bound to mAb 9-40 (box) and mAb 4-4-20 ().



Hydrostatic Pressure

A pseudo-first-order process such as conformational rearrangements at the Fl binding site as a function of pressure can result in a pressure-induced polarization dependence(27) . To ensure that the change in polarization induced by pressure observed in this study was due to Fl dissociation from the Ab active site and not conformational rearrangements, hydrostatic pressure dissociation measurements were made on each Ab fragment as a function of protein and Fl concentration, as performed by Coelho-Sampaio and Voss (26) for scFv 4-4-20/212. The resulting dependence of the dissociation parameters on the protein and Fl concentration verified the bimolecular nature of the process being monitored (data not shown)(27) .

Temperature Perturbation Measurements

The raw data for the application of pressure on IgG, Fab, and scFv 9-40/212 is shown in Fig. 2. The value of K(d) at atmospheric pressure calculated using and are tabulated in Table 1. For each protein sample, at atmospheric pressure the degree of dissociation of the Fl from the active site varied with higher temperatures resulting in greater dissociation. DeltaV and its variation with temperature for mAb, Fab and scFv 9-40/212 decreased as temperature increased (Fig. 3). The DeltaV values indicated that Fl dissociation was identical for scFv 9-40/212, Fab 9-40, and mAb 9-40 at all temperatures. For each Ab, the pressure dissociation curves, at all the temperatures examined, reached the same plateau value. Fl fluorescence was quenched 86% upon binding to the IgG Ab 9-40 and its derivatives at pH 8.0. The quenching of Fl fluorescence bound by the anti-Fl Ab allowed the intensity recovery of Fl fluorescence upon application of hydrostatic pressure to serve as an assay for Fl dissociation. Fig. 4shows the fluorescence intensity recovery for mAb 9-40, Fab, and scFv 9-40/212 at 25 °C plotted as a function of hydrostatic pressure. The similarity in these three curves further corroborated the similar dissociation patterns for mAb, Fab, and scFv.


Figure 2: Raw polarization data for dissociation of Fl from scFv at 35 (), 25 (), 15 (+), and 5 °C (times) and IgG at 35 (box), 25 (up triangle), 15 (), and 5 °C ().






Figure 3: DeltaV (ml/mol) as a function of temperature for mAb 9-40 (box), Fab 9-40 (), and scFv 9-40 ().




Figure 4: Intensity recovery for Fl fluorescence bound by mAb 9-40 (box), Fab 9-40 (), and scFv 9-40 () as a function of pressure.



pH Perturbation Measurements

The raw data for the application of pressure on mAb, scFv and scFv as a function of pH is shown in Fig. 5. The value of K(d) for 9-40 at atmospheric pressure over the pH range 8.0-6.5 for mAb 9-40, scFv 9-40/212 are shown in Table 2. In the whole molecule the dissociation rate constant was found increased as the pH was lowered, in the pH range studied. The native scFv 9-40/212 dissociation was found to be identical to the IgG from pH 8.0 to pH 6.5, where scFv appeared to have become unstable (Fig. 6). The DeltaV decreased dramatically below pH 6.5 in agreement with scFv instability. Hydrostatic pressure measurements were performed on the mutant scFv to ascertain the effect of that contact residue on the pH dependence of DeltaV. Because of its low K(a), large quantities scFv were needed to be able obtain reasonably high polarization values. Hydrostatic pressure induced dissociation of Fl for scFv yielded values of DeltaV as a function of pH that were relatively constant (Fig. 6).


Figure 5: Raw polarization data for dissociation of Fl from IgG at pH 8.0 (box), 7.5 (), 7.0 (), 6.5 (), 6.0 (times), and 5.5 (+).






Figure 6: DeltaV (ml/mol) as a function of temperature for mAb 9-40 (box), scFv 9-40 (), and scFv 9-40 ().




DISCUSSION

Hydrostatic pressure has already been shown to be a valuable tool in the study of protein subunit interactions because thermodynamic information can be obtained under isothermal conditions(22, 23) . The thermodynamic parameter of interest in this study was DeltaV. DeltaV and its temperature dependence for mAb, Fab, and scFv 9-40/212 were found to be identical within error (Fig. 3). Identity of the temperature dependence for mAb 9-40 and its derivatives indicated that the change in volume upon Fl dissociation occurred specifically in the variable domains; in other words, no influence by constant domains was observed under hydrostatic pressure. Values of DeltaV obtained as a function of pH are shown in Fig. 6. The pH dependence of DeltaV demonstrated that an ionizable group specifically in the active site was titrated in the pH range observed (Fig. 6). A lack of pH dependence of DeltaV for scFv implied that the His in the native 9-40 active site was the titratable group.

In order to fully understand the behavior of DeltaV observed in this study, it is desirable to know which amino acids are the ligand contact residues within the 9-40 active site. The topographical structure of the 9-40 active site was not available, but knowledge of the 4-4-20 active site provided important information about the mAb 9-40 active site. Overall, the 4-4-20 active site is an electropositive environment that complements the electronegative influence of Fl, which is a dianion, at neutral pH, in aqueous media. Fl is composed of two substructures: the xanthenone ring and the phenyl carboxyl moiety, and it is known from the crystal structure to be a site-filling antigen (18, 19) . In 4-4-20, the xanthenone ring occupies the deepest part of the Ab active site and resides in an aromatic slot flanked by Tyr-32L, Trp-96L, and Trp-33H. This aromatic-aromatic stacking stabilizes the xanthenone ring in the active site by van der Waals' forces. The electrostatic interactions with Fl in the 4-4-20 active site include the first enolic oxygen salt bridged to the Arg-34L, the second enolic oxygen hydrogen bonded to the His-27L, and the phenyl carboxylate oxygen hydrogen bonded to Tyr-32L. In the 4-4-20 crystal structure, the charged Arg-34L coordinates two water molecules as it salt links to the enolic oxygen group.

Although the crystal structure of mAb 9-40 had not been solved, the amino acid residues that comprised its active site were inferred from the crystal structure of mAb 4-4-20. Wang et al.(28) have suggested three types of combining sites: cavity, groove, and planar. This topographic classification is based on the analysis of 20 Ab x-ray structures. Presumably the shape of the active site of mAb 9-40, whose amino acid sequences of the variable domains differ only by 10 residues (15) , should not change significantly from mAb 4-4-20 pocket shape nor should the relative positions of the residues. Based on this premise, Omelyanenko et al.(29) used molecular modeling to show that the distance of His-34L was 3.5 Å from the Fl enolic group so that sterically no salt link could exist. The His-34L or the Fl enolic group was the most likely source of this pH dependence of DeltaV, observed in this study, because both His (30) and the Fl enolic oxygen (31) were found to be titrated in the pH range observed. However, the hydrostatic pressure behavior of scFv lended credence to the hypothesis that the pH dependence was due to the ionization of the His-34L.

To discuss the temperature and pH dependence of DeltaV in relation to the 9-40 active site, the origin of DeltaV should be understood. Several factors have been proposed to contribute to the change in the partial molar volume that accompanies ligand dissociation from a protein(22) : replacement of interaction between nonpolar groups at two sides of the apolar boundary by water dipoles, imperfect packing of the liganded state of the Ab, and electrostriction of water by newly formed charged groups. In the first such contribution to DeltaV, replacement of interactions between nonpolar groups at the two sides of the apolar boundary by water dipoles, the apolar boundary of interest in our system was that between Fl and the active site. Studies have shown that water molecules form a three-dimensional network that bridges the antigen with the Ab active site increasing the complementarity between the interacting surfaces(32) . Iodide quenching of Fl bound by mAb 9-40 demonstrated that Fl was solvent-accessible. The fact that Fl was solvent accessible and that water molecules have been shown to be present in Ab active sites, in general, make greater interactions of nonpolar groups at the apolar boundary upon dissociation unlikely as a major contribution to DeltaV for mAb 9-40. A second contribution to DeltaV may have been the existence of free volume due to imperfect packing. mAb 9-40 is the germ line clone of the 4-4-20 idiotype family. The mutated residues in mAb 4-4-20 active site varied in volume significantly from the original germ line residues(33) . Imperfect organization of volume in the original germ line residues can cause the packing in mAb 9-40 to include cavities or voids such as that observed by Omelyanenko et al.(29) at the His-34L site in 9-40 using molecular modeling. The existence of cavities or voids would have provided additional flexibility at the site of the void, which may have eventually translated to an unstable environment for the Fl hapten and thus low affinity. Accordingly, higher affinity would then result from somatic mutations, which improved active site packing as previously suggested by Herron et al.(34) . Higher temperatures could have increased thermal energy of the solvent bath, which provided energy for fluctuations in the active site that allowed migration of solvent to fill these cavities. The temperature dependence would have been from subsequent decrease of voids where the temperature was increased, which would have diminished DeltaV. The last determinant of DeltaV was electrostriction of water by newly exposed charged groups. The decrease in DeltaV, which can arise from electrostriction of water of 20 ml/mol or 30 Å^3/molecule, is not negligible. Newly exposed charged groups should not have occurred in the exterior of the IgG molecule upon Fl dissociation because the degree of solvent exposure of the electrostatic surface charge was not expected to change. Therefore, newly exposed charge groups from Fl dissociation, if present, would have occurred in the active site. The identity of the dissociation behavior observed for the IgG, Fab, and scFv 9-40/212 provided experimental evidence that localized DeltaV to the variable domains, probably in the active site.

In the 9-40 active site, the 34L position was occupied by a His. The salt link between His-34L and the enolic oxygen was not expected to exist because the geometry of His restricts its radial charge range. All else being equal, a void would have existed where the salt link and the coordinated water molecules appeared in the 4-4-20 crystal structure(18, 19) . The titration of His-34L in the 9-40 active site may then have given rise to pH dependence of DeltaV from the ionization of His in the active site and coordination of water by charged His similar to those observed in the crystal structure of 4-4-20. The initial premise was that a cavity or void existed at the site of the His because of its geometry with respect to Fl. Although it had been shown that water molecules may line the active site and Fl was solvent-accessible, this did not preclude the presence of voids deep in the active site. The crystal structure of 4-4-20 showed that the 34L position lay deep in the hydrophobic pocket of the active site and the residue at that position in 9-40 should have followed suit. At high pH, the His should have been neutral and could not have coordinated any water molecules at that position thus leaving a void. The dissociation of Fl from the active site would have contributed to DeltaV if upon dissociation the void was then filled by a water molecule. Protonation of the His at low pH would have then imparted a positive charge on that residue that allowed His to coordinate a water molecule at that position. Occupation of the void by a coordinated water molecule would have decreased DeltaV upon Fl dissociation at lower pH because of the lack of a void due to occupation by a coordinated water molecule. The difference in DeltaV observed as the pH decreased from pH 8.0 to pH 5.5 was approximately 16 ml/mol or 24 Å^3/molecule, which is comparable with the volume of a water molecule. Accordingly, the curve in Fig. 6would then suggest that the pK(a) of His is pH 7.4 or 1.2 pH units greater than its aqueous value. It is well known that pK(a) of individual residues vary significantly when in a protein environment(35) . The other ionizable group was that of the Fl enolic oxygen. The pK(a) of the enolic oxygen of Fl in mAb 9-40 was found to be 6.7 by Omelyanenko et al.(29) , which was higher than the pK(a) for the enolic oxygen of free Fl at 6.2(31) . However, both the bound and free pK(a) values of the enolic oxygen of Fl were lower than the midpoint observed in Fig. 6. Therefore, the pH dependence of DeltaV was attributed to the ionization of His-34L and the possible coordination of water molecules at that site.

Denzin and Voss (10) found that mutation of His-34L to Arg decreased the affinity of scFv 9-40/212, although it was not possible to obtain a value. In this study using hydrostatic pressure, the K(a) for scFv was found to be 2.2 times 10^6M, which was lower than the K(a) of the native molecule. The pressure dependence of scFv 9-40/212 did not show the same type of titration behavior as a function of pH in this range. The value of DeltaV remained constant at the pH range observed, indicating that the site specific mutation mitigated the titration of the ionizable group (Fig. 6). According to the above hypothesis, this was an expected result because the Arg was ionized throughout the pH range used in this study. The ionization of Arg allowed the coordination of water molecules such as those seen in the 4-4-20 crystal structure throughout the pH range observed.

Denzin and Voss (10) constructed scFv to test the hypothesis that the salt link between Arg-34L and the enolic oxygen was responsible for the high affinity of 4-4-20 as previously had been suggested by the lower affinity of scFv 4-4-20/212(9) . Investigators using x-ray crystallography have previously shown that in certain Ab-antigen systems(32) , a site-directed mutation only affected the area where the mutation occurred. In that study, with the crystal structure of Fv D1.3, anti hen lysozyme Ab, and its site directed mutant Fv D1.3, where the affinity of this Ab for its antigen decreased 3-fold, it was found that all the detectable changes in conformation occurred around the site of mutation. The rest of the antigen-Ab interface remained as in the complex with the wild type Fv. The fact that the mutation of His-34L to Arg caused a decrease in affinity where an increase was expected indicated that the Arg salt link was not the only determinant of 4-4-20 high affinity.

In an effort to study the effect of constant domains on binding, Müller et al.(36) investigated the relative dynamics of the variable domains of mAb 4-4-20 with its Fab 4-4-20 and scFv 4-4-20/212 derivatives. In that study, the variable domains were identical, but the Abs differed in the amount of constant domains. Müller et al.(36) found that scFv 4-4-20/212 was substantially less stable than the Fab 4-4-20 and mAb 4-4-20. These results were obtained for mAb 4-4-20 and its derivatives specifically as a glycerol-water mixture. Coelho-Sampiao and Voss (26) studied scFv 4-4-20/212 with hydrostatic pressure and found that DeltaV of dissociation for Fl-scFv 4-4-20/212 was 10 times higher than that found for Fl dissociation from intact mAb 4-4-20. The greater dissociation of Fl from scFv 4-4-20/212 was explained in terms of a higher overall flexibility of unliganded scFv 4-4-20/212 and of a less stable binding site in scFv 4-4-20/212 relative to mAb 4-4-20. Although informative, these studies compared derivatives that contained identical variable domains but possessed slightly different affinities (mAb 4-4-20 K(a) = 1.3 times 10M; scFv 4-4-20/212 K(a) = 2.9 times 10^9M). In this study, mAb 9-40, and its derivatives were used because the affinities are identical for all. It was found that no difference in ligand dissociation behavior between scFv 9-40/212 and mAb 9-40 was observed, which indicated that the variable domains dynamics were not coupled to the constant domains for mAb 9-40 and its derivatives in contrast to that observed for 4-4-20. This result was originally unexpected given the lack of the C(H)1 and the C(L)1 constant domains, which were previously found to stabilize mAb 4-4-20(26, 36) . However, this finding may be explained in terms of increased conformational domain dynamics for 9-40 and may have been predicted by the identical affinities for mAb 9-40 and its derivatives.

The relationship of Ab structure and dynamics to affinity can be visualized by a model originally proposed to predict different rates of association for an Ab with cross-reactive ligands that has been modified to include metatype dynamics (Fig. 7). The nonliganded conformers correspond to a dynamic distribution of idiotypic states similar to distribution of states discussed in Frauenfelder and Wolynes(12) . A number of unliganded conformers in this distribution can bind the homologous ligand. Once bound by the ligand the width of the Ab-homologous-ligand-conformer distribution is expected to decrease because the ligand stabilizes the Ab dynamics. Cross-reactivity occurs when a conformer from the nonliganded conformer distribution binds to a nonhomologous ligand and restricts the liganded conformer distribution to an Ab-nonhomologous-ligand distribution (represented by metatype distribution x or y) with average set of structural determinants that probably differs from those held by the Ab-homologous-ligand complex distribution (represented by metatype distribution w). For 4-4-20, no cross-reactivity existed. It bound with high affinity to Fl and a conformational change was known to occur once the ligand was bound(16, 17) . For 9-40, the value for DeltaV at pH 8.0, 25 °C was greater than that observed for mAb 4-4-20. It has been shown in this study that a possible determinant of DeltaV for mAb 9-40 was the presence of voids or cavities. Although the exact fractional contribution from the three causes of DeltaV previously mentioned at present cannot be quantitated, the more likely contribution to the difference in DeltaV between mAb 9-40 and mAb 4-4-20 may have been imperfect packing from the presence of voids. The presence of voids can contribute to the distribution of states of 9-40 by migration throughout the Ab variable domain matrix(37) . An ensemble of Ab molecules each with a void in a slightly different position would be a distribution of conformations (12) . As a result, the distribution of nonliganded conformations for 9-40 may be greater than that for 4-4-20. The presence of an unliganded distribution of states for mAb 9-40 was corroborated by the ability of 9-40 to cross-react with Fl analogs (^2)in contrast to 4-4-20. Moreover, the bound state of 9-40 was believed to have a wider distribution of conformations than 4-4-20, as well. The overall distribution of ligand states for 9-40 would be greater than 4-4-20 because the structural characteristics of the observed cross-reactive species would not be expected to be the same. This hypothesis was in agreement with the iodide quenching of bound Fl. Increased dynamics of the 9-40 variable domains compared with 4-4-20 would have allowed Fl to be more accessible to the quencher. The higher affinity of 4-4-20 can be viewed as a consequence of, among other factors, a high degree of conformational rigidity, partly stabilized by the constant domains, which translated to a smaller distribution of nonliganded cross-reactive conformers. In summary, the dynamic fluctuations of the 9-40 variable domains were believed to be below the limit of flexibility that could be stabilized by constant domains.


Figure 7: Model depicting relationship between Ab stucture and dynamics.




FOOTNOTES

*
This work was supported by a grant from the Biotechnology Research Development Corp., Peoria, IL. Fluorescence measurements were performed at the Laboratory for Fluorescence Dynamics at the University of Illinois at Urbana-Champaign, which is supported jointly by Division of Research Resources of the National Institutes of Health Grant RR03155-01 and by the University of Illinois. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Microbiology, University of Illinois at Urbana-Champaign, 407 S. Goodwin Ave., Urbana, IL 61801-3797. Tel.: 217-333-0299; Fax: 217-244-6697; e-voss1{at}uiuc.edu.

(^1)
The abbreviations used are: Ab, antibody; scFv, single-chain Fv; mAb, monoclonal antibody; Fl, fluorescein.

(^2)
J. Carrero and E. W. Voss, Jr., unpublished results.


ACKNOWLEDGEMENTS

We thank the Laboratory for Fluorescence Dynamics for the use of instrumentation and technical support. We are grateful to William D. Mallender and Mark Mummert for stimulating discussion in reference to this work.


REFERENCES

  1. Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W., and Howard, J. C. (1997) Nature 266, 550-552
  2. James, K., and Bell, G. T. (1987) J. Immunol. Methods 100, 5-40 [Medline] [Order article via Infotrieve]
  3. Davies, D. R., Padlan, E. A., and Sheriff, S. (1990) Annu. Rev. Biochem. 59, 439-473 [CrossRef][Medline] [Order article via Infotrieve]
  4. Wilson, I. A., Rini, J. M., Fremont, D. H., Fieser, G. G., and Stura, E. A. (1991) Methods Enzymol. 203, 153-176 [Medline] [Order article via Infotrieve]
  5. Wilson, I. A., and Steinfield, R. L. (1993) Curr. Opin. Struct. Biol. 3, 113-123 [CrossRef]
  6. Padlan, E. A. (1994) Mol. Immunol. 31, 169-217 [CrossRef][Medline] [Order article via Infotrieve]
  7. Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S., Lee, T. Pope, S. H., Riorden, G. S., and Whitlow, M. (1988) Science 242, 423-426 [Medline] [Order article via Infotrieve]
  8. Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M.-S., Novotny, J., Margolies, M. N., Ridge, R. J., Bruccoleri, R., Haber, E., Crea, R., and Opperman, H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5879-5883 [Abstract]
  9. Denzin, L. K., Whitlow, M., and Voss, E. W., Jr. (1991) J. Biol. Chem. 266, 14095-14103 [Abstract/Free Full Text]
  10. Denzin, L. K., and Voss, E. W., Jr. (1992) J. Biol. Chem. 267, 8925-8931 [Abstract/Free Full Text]
  11. Mummert, M., and Voss, E. W., Jr. (1995) Mol. Immunol. 32, 1225-1233 [CrossRef][Medline] [Order article via Infotrieve]
  12. Frauenfelder, H., and Wolynes, P. G. (1985) Science 229, 337-345 [Medline] [Order article via Infotrieve]
  13. Kranz, D. M., Ballard, D. W., and Voss, E. W., Jr. (1983) Mol. Immunol. 20, 1313-1322 [Medline] [Order article via Infotrieve]
  14. Bedzyk, W. D., Reinitz, D. M., and Voss, E. W., Jr. (1986) Mol. Immunol. 23, 1319-1328 [Medline] [Order article via Infotrieve]
  15. Bedzyk, W. D., Herron, J. N., Edmundson, A. B., and Voss, E. W., Jr. (1990) J. Biol. Chem. 265, 133-138 [Abstract/Free Full Text]
  16. Voss, E. W., Jr., Miklasz, S., Petrossian, A., and Dombrink-Kurtzman, M. A. (1988) Mol. Immunol. 25, 751-759 [Medline] [Order article via Infotrieve]
  17. Voss, E. W., Jr., Dombrink-Kurtzman, M. A., and Ballard, D. W. (1989) Mol. Immunol. 26, 971-977 [Medline] [Order article via Infotrieve]
  18. Herron, J. N., He, X.-M., Mason, M. L., and Voss, E. W., Jr. (1989) Proteins 5, 271-280 [Medline] [Order article via Infotrieve]
  19. Herron, J. N., Terry, A. H., Johnson, S., He, X.-M., Guddat, L. W., Voss, E. W., Jr., and Edmundson, A. B. (1994) Biophys. J. 67, 2167-2183 [Abstract]
  20. Kranz, D. M., and Voss, E. W., Jr. (1981) Mol. Immunol. 18, 889-898 [Medline] [Order article via Infotrieve]
  21. Reinitz, D. M., and Voss, E. W., Jr. (1985) J. Immunol. 135, 3365-3371 [Abstract/Free Full Text]
  22. Paladini, A. A., and Weber, G. (1981) Rev. Sci. Instrum. 52, 419-427
  23. Paladini, A. A., and Weber, G. (1981) Biochemistry 20, 2587-2593 [Medline] [Order article via Infotrieve]
  24. Li, T. M., Hook, J. W., III, Drickamer, H. G., and Weber, G. (1976) Biochemistry 15, 3205-3211 [Medline] [Order article via Infotrieve]
  25. Li, T. M., Hook, J. W., III, Drickamer, H. G., and Weber, G. (1976) Biochemistry 15, 5571-5580 [Medline] [Order article via Infotrieve]
  26. Coelho-Sampaio, T., and Voss, E. W., Jr. (1993) Biochemistry 32, 10929-10935 [Medline] [Order article via Infotrieve]
  27. Weber, G. (1987) High Pressure Chemistry and Biochemistry (van Eldik, R., and Jonas, J., eds) p. 401, D. Reidel Publishing Co., Dordrecht, The Netherlands
  28. Wang, D., Ligo, J., Mitra, D., Akolkar, P., Gruezo, F., and Kabat, B. (1991) Mol. Immunol. 28, 1387-1397 [Medline] [Order article via Infotrieve]
  29. Omelyanenko, V. G., Jiskoot, W., and Herron, J. N. (1993) Biochemistry 32, 10423-10429 [Medline] [Order article via Infotrieve]
  30. Tanford, C. (1962) Adv. Protein. Chem. 17, 69-98
  31. Martin, M. M., and Lindvist, L. (1975) J. Lumin. 10, 381-390
  32. Ysern, X., Fields, B. A., Bhat, T. N., Goldbaum, F. A., Acqua, W. D., Schwarz, F. P., Poljak, and Mariuzza (1994) J. Mol. Biol. 238, 496-500 [CrossRef][Medline] [Order article via Infotrieve]
  33. Creighton, T. E. (1984) Proteins , p. 7, W. H. Freeman and Co., New York
  34. Herron, J. N., Kranz, D. M., Jameson, D. M., and Voss, E. W., Jr. (1986) Biochemistry 25, 4602-4609 [Medline] [Order article via Infotrieve]
  35. Creighton, T. E. (1984) Proteins , p. 135, W. H. Freeman and Co., New York
  36. Müller, J. D., Nienhaus, G. U., Tetin, S., and Voss, E. W. (1994) Biochemistry 33, 6221-6227 [Medline] [Order article via Infotrieve]
  37. Rashin, A. A., Iofin, M., and Honig, B. (1986) Biochemistry 25, 3619-3625 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.