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• W2040996863 abstract "In a comparative study, the thermodynamic parameter, ΔV, 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 ΔV 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 9-40/212Arg-34L 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. In a comparative study, the thermodynamic parameter, ΔV, 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 ΔV 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 9-40/212Arg-34L 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. The understanding of antibody (Ab) ( 1The abbreviations used are: AbantibodyscFvsingle-chain FvmAbmonoclonal antibodyFlfluorescein.) structure-function has been enhanced during the past two decades through the advent of hybridoma methodology(1.Galfre G. Howe S.C. Milstein C. Butcher G.W. Howard J.C. Nature. 1997; 266: 550-552Crossref Scopus (1182) Google Scholar, 2.James K. Bell G.T. J. Immunol. Methods. 1987; 100: 5-40Crossref PubMed Scopus (191) Google Scholar), the increase in resolved atomic structures due primarily to x-ray crystallography (3.Davies D.R. Padlan E.A. Sheriff S. Annu. Rev. Biochem. 1990; 59: 439-473Crossref PubMed Scopus (693) Google Scholar, 4.Wilson I.A. Rini J.M. Fremont D.H. Fieser G.G. Stura E.A. Methods Enzymol. 1991; 203: 153-176Crossref PubMed Scopus (43) Google Scholar, 5.Wilson I.A. Steinfield R.L. Curr. Opin. Struct. Biol. 1993; 3: 113-123Crossref Scopus (264) Google Scholar, 6.Padlan E.A. Mol. Immunol. 1994; 31: 169-217Crossref PubMed Scopus (794) Google Scholar), generation of Ab structural derivatives such as single-chain Fv (scFv) fragments(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. Whitlow M. Science. 1988; 242: 423-426Crossref PubMed Scopus (1323) Google Scholar, 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. Opperman H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5879-5883Crossref PubMed Scopus (1411) Google Scholar), and site-specific mutagenesis of well characterized Abs(9.Denzin L.K. Whitlow M. Voss Jr., E.W. J. Biol. Chem. 1991; 266: 14095-14103Abstract Full Text PDF PubMed Google Scholar, 10.Denzin L.K. Voss Jr., E.W. J. Biol. Chem. 1992; 267: 8925-8931Abstract Full Text PDF PubMed Google Scholar). 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 (Ka) as it correlates with primary and secondary interactions(11.Mummert M. Voss Jr., E.W. Mol. Immunol. 1995; 32: 1225-1233Crossref PubMed Scopus (20) Google Scholar), 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.Frauenfelder H. Wolynes P.G. Science. 1985; 229: 337-345Crossref PubMed Scopus (425) Google Scholar). antibody single-chain Fv monoclonal antibody fluorescein. 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.Kranz D.M. Ballard D.W. Voss Jr., E.W. Mol. Immunol. 1983; 20: 1313-1322Crossref PubMed Scopus (14) Google Scholar, 14.Bedzyk W.D. Reinitz D.M. Voss Jr., E.W. Mol. Immunol. 1986; 23: 1319-1328Crossref PubMed Scopus (11) Google Scholar, 15.Bedzyk W.D. Herron J.N. Edmundson A.B. Voss Jr., E.W. J. Biol. Chem. 1990; 265: 133-138Abstract Full Text PDF PubMed Google Scholar), which are highly related at the idiotypic or unliganded level, reflecting nearly identical primary structures in both the heavy and light chains(14.Bedzyk W.D. Reinitz D.M. Voss Jr., E.W. Mol. Immunol. 1986; 23: 1319-1328Crossref PubMed Scopus (11) Google Scholar). However, these Abs were only marginally related at the metatype or liganded level, suggesting significantly different conformations of the liganded state(16.Voss Jr., E.W. Miklasz S. Petrossian A. Dombrink-Kurtzman M.A. Mol. Immunol. 1988; 25: 751-759Crossref PubMed Scopus (45) Google Scholar, 17.Voss Jr., E.W. Dombrink-Kurtzman M.A. Ballard D.W. Mol. Immunol. 1989; 26: 971-977Crossref PubMed Scopus (22) Google Scholar). 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.Bedzyk W.D. Herron J.N. Edmundson A.B. Voss Jr., E.W. J. Biol. Chem. 1990; 265: 133-138Abstract Full Text PDF PubMed Google Scholar). The affinities of these two Abs differ by 1000-fold for the Fl ligand with the value of 2.8 × 107M-1(10.Denzin L.K. Voss Jr., E.W. J. Biol. Chem. 1992; 267: 8925-8931Abstract Full Text PDF PubMed Google Scholar) and 2 × 1010M-1(14.Bedzyk W.D. Reinitz D.M. Voss Jr., E.W. Mol. Immunol. 1986; 23: 1319-1328Crossref PubMed Scopus (11) Google Scholar) for 9-40 and 4-4-20, respectively. Comparatively, in mAb 9-40 the heavy chain variable (VH) 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 (VL) differed by only two amino acid residues(14.Bedzyk W.D. Reinitz D.M. Voss Jr., E.W. Mol. Immunol. 1986; 23: 1319-1328Crossref PubMed Scopus (11) Google Scholar, 15.Bedzyk W.D. Herron J.N. Edmundson A.B. Voss Jr., E.W. J. Biol. Chem. 1990; 265: 133-138Abstract Full Text PDF PubMed Google Scholar). 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.Denzin L.K. Whitlow M. Voss Jr., E.W. J. Biol. Chem. 1991; 266: 14095-14103Abstract Full Text PDF PubMed Google Scholar, 10.Denzin L.K. Voss Jr., E.W. J. Biol. Chem. 1992; 267: 8925-8931Abstract Full Text PDF PubMed Google Scholar) comprised of the VL and VH domains of the corresponding mAb joined by a short polypeptide linker (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. Whitlow M. Science. 1988; 242: 423-426Crossref PubMed Scopus (1323) Google Scholar, 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. Opperman H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5879-5883Crossref PubMed Scopus (1411) Google Scholar) 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.Herron J.N. He X.-M. Mason M.L. Voss Jr., E.W. Proteins. 1989; 5: 271-280Crossref PubMed Scopus (213) Google Scholar, 19.Herron J.N. Terry A.H. Johnson S. He X.-M. Guddat L.W. Voss Jr., E.W. Edmundson A.B. Biophys. J. 1994; 67: 2167-2183Abstract Full Text PDF PubMed Scopus (70) Google Scholar). 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 scFv9-40/212Arg-34L. 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 scFv9-40/212Arg-34L, respectively. The thermodynamic parameter, change in partial molar volume (ΔV), 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.Denzin L.K. Voss Jr., E.W. J. Biol. Chem. 1992; 267: 8925-8931Abstract Full Text PDF PubMed Google Scholar), 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. scFv 9-40/212 and scFv9-40/212Arg-34L were constructed and expressed as described previously(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. Whitlow M. Science. 1988; 242: 423-426Crossref PubMed Scopus (1323) Google Scholar, 9.Denzin L.K. Whitlow M. Voss Jr., E.W. J. Biol. Chem. 1991; 266: 14095-14103Abstract Full Text PDF PubMed Google Scholar). 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.Kranz D.M. Voss Jr., E.W. Mol. Immunol. 1981; 18: 889-898Crossref PubMed Scopus (76) Google Scholar, 21.Reinitz D.M. Voss Jr., E.W. J. Immunol. 1985; 135: 3365-3371PubMed Google Scholar). mAb 9-40 was obtained from ascites fluid by affinity purification using Fl-Sepharose 4B as described previously (20.Kranz D.M. Voss Jr., E.W. Mol. Immunol. 1981; 18: 889-898Crossref PubMed Scopus (76) Google Scholar, 21.Reinitz D.M. Voss Jr., E.W. J. Immunol. 1985; 135: 3365-3371PubMed Google Scholar). 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: I0/I=1+k+τ0[Q](Eq. 1) where I0 was the intensity at zero quencher concentration, I was the intensity at quencher concentration [Q], k+ was the bimolecular constant, and τ0 was the lifetime of the fluorophore. Hydrostatic pressure in the range of 1 bar to 2.4 Kbar was achieved using the pressure cell described by Paladini and Weber(22.Paladini A.A. Weber G. Rev. Sci. Instrum. 1981; 52: 419-427Crossref Scopus (146) Google Scholar, 23.Paladini A.A. Weber G. Biochemistry. 1981; 20: 2587-2593Crossref PubMed Scopus (208) Google Scholar). 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.Paladini A.A. Weber G. Rev. Sci. Instrum. 1981; 52: 419-427Crossref Scopus (146) Google Scholar). The dissociation parameter, α, was calculated using as follows: αp=[1+R(rrF)(rBr)]1(Eq. 2) where R is the ratio of the fluorescence quantum yields of free and bound forms, r is the anisotropy at each pressure, and rF and rB are the temperature- and pH-dependent anisotropies for free and bound states, respectively(22.Paladini A.A. Weber G. Rev. Sci. Instrum. 1981; 52: 419-427Crossref Scopus (146) Google Scholar), 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 (K3) at each pressure were calculated using the following relation: Kd=αp{[Abs]0(1αp)[F1]0}(1αp)(Eq. 3) where [Abs]0 and [Fl]0 were the total concentrations of Ab active sites for scFv, Fab, or mAb and Fl, respectively(24.Li T.M. Hook III, J.W. Drickamer H.G. Weber G. Biochemistry. 1976; 15: 3205-3211Crossref PubMed Scopus (64) Google Scholar, 25.Li T.M. Hook III, J.W. Drickamer H.G. Weber G. Biochemistry. 1976; 15: 5571-5580Crossref PubMed Scopus (129) Google Scholar, 26.Coelho-Sampaio T. Voss Jr., E.W. Biochemistry. 1993; 32: 10929-10935Crossref PubMed Scopus (21) Google Scholar). The dissociation constant related to the free energy of dissociation as follows: ΔGdiss=RTlnKd(Eq. 4) 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 Kd and ΔGdiss, for IgG, Fab, scFv 9-40/212, and scFv 9-40/212Arg-34L at all temperatures and pH values. ΔV was obtained from the following relation: ΔV=(δG/δp)τ(Eq. 5) All stock solutions prepared in 40 mM Tris contained 0.2 μM Ab active sites and 0.02 M Fl except scFv 9-40/212Arg-34L, 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 9-40/212Arg-34L, only three samples at pH 6.5, 7.2, and 8.0 were prepared because of the relatively large amount of protein necessary. Results of the iodide quenching studies are shown in Fig. 1. The bimolecular collision constant of 5.1 × 108 s-1M-1, 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.Coelho-Sampaio T. Voss Jr., E.W. Biochemistry. 1993; 32: 10929-10935Crossref PubMed Scopus (21) Google Scholar). 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. 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.Weber G. van Eldik R. Jonas J. High Pressure Chemistry and Biochemistry. D. Reidel Publishing Co., Dordrecht, The Netherlands1987: 401Crossref Google Scholar). 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.Coelho-Sampaio T. Voss Jr., E.W. Biochemistry. 1993; 32: 10929-10935Crossref PubMed Scopus (21) Google Scholar) 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.Weber G. van Eldik R. Jonas J. High Pressure Chemistry and Biochemistry. D. Reidel Publishing Co., Dordrecht, The Netherlands1987: 401Crossref Google Scholar). The raw data for the application of pressure on IgG, Fab, and scFv 9-40/212 is shown in Fig. 2. The value of Kd 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. ΔV and its variation with temperature for mAb, Fab and scFv 9-40/212 decreased as temperature increased (Fig. 3). The ΔV 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. 4 shows 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.Tabled 1 Open table in a new tab Figure 3:ΔV (ml/mol) as a function of temperature for mAb 9-40 (□), Fab 9-40 (■), and scFv 9-40 (◊).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4:Intensity recovery for Fl fluorescence bound by mAb 9-40 (□), Fab 9-40 (□), and scFv 9-40 (■) as a function of pressure.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The raw data for the application of pressure on mAb, scFv and scFvArg-34L as a function of pH is shown in Fig. 5. The value of Kd 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 ΔV decreased dramatically below pH 6.5 in agreement with scFv instability. Hydrostatic pressure measurements were performed on the mutant scFvArg-34L to ascertain the effect of that contact residue on the pH dependence of ΔV. Because of its low Ka, large quantities scFvArg-34L were needed to be able obtain reasonably high polarization values. Hydrostatic pressure induced dissociation of Fl for scFvArg-34L yielded values of ΔV as a function of pH that were relatively constant (Fig. 6).Tabled 1 Open table in a new tab Figure 6:ΔV (ml/mol) as a function of temperature for mAb 9-40 (□), scFv 9-40 (■), and scFv 9-40Arg-34L (◊).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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.Paladini A.A. Weber G. Rev. Sci. Instrum. 1981; 52: 419-427Crossref Scopus (146) Google Scholar, 23.Paladini A.A. Weber G. Biochemistry. 1981; 20: 2587-2593Crossref PubMed Scopus (208) Google Scholar). The thermodynamic parameter of interest in this study was ΔV. ΔV 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 ΔV obtained as a function of pH are shown in Fig. 6. The pH dependence of ΔV 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 ΔV for scFvArg-34L implied that the His in the native 9-40 active site was the titratable group. In order to fully understand the behavior of ΔV 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.Herron J.N. He X.-M. Mason M.L. Voss Jr., E.W. Proteins. 1989; 5: 271-280Crossref PubMed Scopus (213) Google Scholar, 19.Herron J.N. Terry A.H. Johnson S. He X.-M. Guddat L.W. Voss Jr., E.W. Edmundson A.B. Biophys. J. 1994; 67: 2167-2183Abstract Full Text PDF PubMed Scopus (70) Google Scholar). 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.Wang D. Ligo J. Mitra D. Akolkar P. Gruezo F. Kabat B. Mol. Immunol. 1991; 28: 1387-1397Crossref PubMed Scopus (29) Google Scholar) 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.Bedzyk W.D. Herron J.N. Edmundson A.B. Voss Jr., E.W. J. Biol. Chem. 1990; 265: 133-138Abstract Full Text PDF PubMed Google Scholar), 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.Omelyanenko V.G. Jiskoot W. Herron J.N. Biochemistry. 1993; 32: 10423-10429Crossref PubMed Scopus (34) Google Scholar) 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 ΔV, observed in this study, because both His (30.Tanford C. Adv. Protein. Chem. 1962; 17: 69-98Crossref Scopus (419) Google Scholar) and the Fl enolic oxygen (31.Martin M.M. Lindvist L. J. Lumin. 1975; 10: 381-390Crossref Scopus (449) Google Scholar) were found to be titrated in the pH range observed. However, the hydrostatic pressure behavior of scFvArg-34L 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 ΔV in relation to the 9-40 active site, the origin of ΔV 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.Paladini A.A. Weber G. Rev. Sci. Instrum. 1981; 52: 419-427Crossref Scopus (146) Google Scholar): 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 ΔV, 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.Ysern X. Fields B.A. Bhat T.N. Goldbaum F.A. Acqua W.D. Schwarz F.P. Poljak Mariuzza J. Mol. Biol. 1994; 238: 496-500Crossref PubMed Scopus (63) Google Scholar). 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 ΔV for mAb 9-40. A second contribution to ΔV 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.Creighton T.E. Proteins. W. H. Freeman and Co., New York1984: 7Google Scholar). 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.Omelyanenko V.G. Jiskoot W. Herron J.N. Biochemistry. 1993; 32: 10423-10429Crossref PubMed Scopus (34) Google Scholar) 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.Herron J.N. Kranz D.M. Jameson D.M. Voss Jr., E.W. Biochemistry. 1986; 25: 4602-4609Crossref PubMed Scopus (78) Google Scholar). 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 ΔV. The last determinant of ΔV was electrostriction of water by newly exposed charged groups. The decrease in ΔV, which can arise from electrostriction of water of ~20 ml/mol or 30 Å3/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 ΔV 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.Herron J.N. He X.-M. Mason M.L. Voss Jr., E.W. Proteins. 1989; 5: 271-280Crossref PubMed Scopus (213) Google Scholar, 19.Herron J.N. Terry A.H. Johnson S. He X.-M. Guddat L.W. Voss Jr., E.W. Edmundson A.B. Biophys. J. 1994; 67: 2167-2183Abstract Full Text PDF PubMed Scopus (70) Google Scholar). The titration of His-34L in the 9-40 active site may then have given rise to pH dependence of ΔV 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 ΔV 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 ΔV upon Fl dissociation at lower pH because of the lack of a void due to occupation by a coordinated water molecule. The difference in ΔV observed as the pH decreased from pH 8.0 to pH 5.5 was approximately 16 ml/mol or 24 Å3/molecule, which is comparable with the volume of a water molecule. Accordingly, the curve in Fig. 6 would then suggest that the pKa of His is pH 7.4 or 1.2 pH units greater than its aqueous value. It is well known that pKa of individual residues vary significantly when in a protein environment(35.Creighton T.E. Proteins. W. H. Freeman and Co., New York1984: 135Google Scholar). The other ionizable group was that of the Fl enolic oxygen. The pKa of the enolic oxygen of Fl in mAb 9-40 was found to be 6.7 by Omelyanenko et al.(29.Omelyanenko V.G. Jiskoot W. Herron J.N. Biochemistry. 1993; 32: 10423-10429Crossref PubMed Scopus (34) Google Scholar), which was higher than the pKa for the enolic oxygen of free Fl at 6.2(31.Martin M.M. Lindvist L. J. Lumin. 1975; 10: 381-390Crossref Scopus (449) Google Scholar). However, both the bound and free pKa values of the enolic oxygen of Fl were lower than the midpoint observed in Fig. 6. Therefore, the pH dependence of ΔV was attributed to the ionization of His-34L and the possible coordination of water molecules at that site. Denzin and Voss (10.Denzin L.K. Voss Jr., E.W. J. Biol. Chem. 1992; 267: 8925-8931Abstract Full Text PDF PubMed Google Scholar) 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 Ka for scFvArg-34L was found to be 2.2 × 106M-1, which was lower than the Ka of the native molecule. The pressure dependence of scFv 9-40/212Arg-34L did not show the same type of titration behavior as a function of pH in this range. The value of ΔV 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.Denzin L.K. Voss Jr., E.W. J. Biol. Chem. 1992; 267: 8925-8931Abstract Full Text PDF PubMed Google Scholar) constructed scFvArg-34L 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 4-4-20/212His-34L(9.Denzin L.K. Whitlow M. Voss Jr., E.W. J. Biol. Chem. 1991; 266: 14095-14103Abstract Full Text PDF PubMed Google Scholar). Investigators using x-ray crystallography have previously shown that in certain Ab-antigen systems(32.Ysern X. Fields B.A. Bhat T.N. Goldbaum F.A. Acqua W.D. Schwarz F.P. Poljak Mariuzza J. Mol. Biol. 1994; 238: 496-500Crossref PubMed Scopus (63) Google Scholar), 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 D1.3Asp-92L, 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.Müller J.D. Nienhaus G.U. Tetin S. Voss E.W. Biochemistry. 1994; 33: 6221-6227Crossref PubMed Scopus (17) Google Scholar) 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.Müller J.D. Nienhaus G.U. Tetin S. Voss E.W. Biochemistry. 1994; 33: 6221-6227Crossref PubMed Scopus (17) Google Scholar) 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.Coelho-Sampaio T. Voss Jr., E.W. Biochemistry. 1993; 32: 10929-10935Crossref PubMed Scopus (21) Google Scholar) studied scFv 4-4-20/212 with hydrostatic pressure and found that ΔV 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 Ka = 1.3 × 1010M-1; scFv 4-4-20/212 Ka = 2.9 × 109M-1). 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 CH1 and the CL1 constant domains, which were previously found to stabilize mAb 4-4-20(26.Coelho-Sampaio T. Voss Jr., E.W. Biochemistry. 1993; 32: 10929-10935Crossref PubMed Scopus (21) Google Scholar, 36.Müller J.D. Nienhaus G.U. Tetin S. Voss E.W. Biochemistry. 1994; 33: 6221-6227Crossref PubMed Scopus (17) Google Scholar). 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.Frauenfelder H. Wolynes P.G. Science. 1985; 229: 337-345Crossref PubMed Scopus (425) Google Scholar). 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 × 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.Voss Jr., E.W. Miklasz S. Petrossian A. Dombrink-Kurtzman M.A. Mol. Immunol. 1988; 25: 751-759Crossref PubMed Scopus (45) Google Scholar, 17.Voss Jr., E.W. Dombrink-Kurtzman M.A. Ballard D.W. Mol. Immunol. 1989; 26: 971-977Crossref PubMed Scopus (22) Google Scholar). For 9-40, the value for ΔV 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 ΔV for mAb 9-40 was the presence of voids or cavities. Although the exact fractional contribution from the three causes of ΔV previously mentioned at present cannot be quantitated, the more likely contribution to the difference in ΔV 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.Rashin A.A. Iofin M. Honig B. Biochemistry. 1986; 25: 3619-3625Crossref PubMed Scopus (253) Google Scholar). An ensemble of Ab molecules each with a void in a slightly different position would be a distribution of conformations (12.Frauenfelder H. Wolynes P.G. Science. 1985; 229: 337-345Crossref PubMed Scopus (425) Google Scholar). 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 ( 2J. Carrero and E. W. Voss, Jr., unpublished results.) 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. 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." @default.
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