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• W2043532306 abstract "We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala49 and Ile70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 °C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala49 and Ile70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects. We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala49 and Ile70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 °C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala49 and Ile70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects. One of the major objectives of antibody engineering is to stabilize the three-dimensional structure of the antibody. Disulfide bonds often significantly stabilize the structure of native proteins. Thus, the introduction of artificial disulfide bonds is recognized as a useful protein engineering technique to increase conformational stability. Although this technique has been applied to many proteins, there are no reports of engineered disulfide bonds in an immunoglobulin fold framework.Cystine is hydrophobic, and thus, most of naturally occurring disulfide bonds are buried in the protein (1Thornton J.M. J. Mol. Biol. 1981; 151: 261-287Crossref PubMed Scopus (668) Google Scholar, 2Nagano N. Ota M. Nishikawa K. FEBS Lett. 1999; 458: 69-71Crossref PubMed Scopus (114) Google Scholar, 3Rose G.D. Geselowitz A.R. Lesser G.J. Lee R.H. Zehfus M.H. Science. 1985; 229: 834-838Crossref PubMed Scopus (960) Google Scholar, 4Fauchère J.L. Pliska V. Eur. J. Med. Chem. 1983; 18: 369-375Google Scholar). Therefore, the introduction of an engineered disulfide bond into the hydrophobic core better maintains the biophysical properties of the target protein. There are several examples of artificial disulfide bonds that can replace a pair of buried hydrophobic residues, the accessible surface areas of which were <20% (5Dani V.S. Ramakrishnan C. Varadarajan R. Protein Eng. 2003; 16: 187-193Crossref PubMed Scopus (91) Google Scholar, 6Mårtensson L.-G. Karlsson M. Carlsson U. Biochemistry. 2002; 41: 15867-15875Crossref PubMed Scopus (34) Google Scholar, 7Wakarchuk W.W. Sung W.L. Campbell R.L. Cunningham A. Watson D.C. Yaguchi M. Protein Eng. 1994; 7: 1379-1386Crossref PubMed Scopus (150) Google Scholar, 8Mitchinson C. Wells J.A. Biochemistry. 1989; 28: 4807-4815Crossref PubMed Scopus (145) Google Scholar, 9Burton R.E. Hunt J.A. Fierke C.A. Oas T.G. Protein Sci. 2000; 9: 776-785Crossref PubMed Scopus (22) Google Scholar, 10Reading N.S. Aust S.D. Biotechnol. Prog. 2000; 16: 326-333Crossref PubMed Scopus (45) Google Scholar, 11Ko J.H. Jang W.H. Kim E.K. Lee H.B. Park K.D. Chung J.H. Yoo O.J. Biochem. Biophys. Res. Commun. 1996; 221: 631-635Crossref PubMed Scopus (29) Google Scholar). The introduction of a disulfide bond into the buried hydrophobic core of human carbonic anhydrase (A23C/L203C) markedly stabilizes this enzyme; the midpoint temperature of thermal unfolding (Tm) of the mutant is 10 °C higher than that of the wild-type protein (6Mårtensson L.-G. Karlsson M. Carlsson U. Biochemistry. 2002; 41: 15867-15875Crossref PubMed Scopus (34) Google Scholar). The engineered disulfide bonds in alkaline protease AprP (G199C/F236C) (11Ko J.H. Jang W.H. Kim E.K. Lee H.B. Park K.D. Chung J.H. Yoo O.J. Biochem. Biophys. Res. Commun. 1996; 221: 631-635Crossref PubMed Scopus (29) Google Scholar), xylanase (V98C/A152C) (7Wakarchuk W.W. Sung W.L. Campbell R.L. Cunningham A. Watson D.C. Yaguchi M. Protein Eng. 1994; 7: 1379-1386Crossref PubMed Scopus (150) Google Scholar), and manganese peroxidase (A48C/A63C) (10Reading N.S. Aust S.D. Biotechnol. Prog. 2000; 16: 326-333Crossref PubMed Scopus (45) Google Scholar) mutants increase their tolerance against heat inactivation. On the other hand, subtilisin BPN′ mutants (V26C/A232C and A29C/M119C) exhibit similar or slightly lower stability to irreversible thermal inactivation (8Mitchinson C. Wells J.A. Biochemistry. 1989; 28: 4807-4815Crossref PubMed Scopus (145) Google Scholar). Tolerance against heat denaturation is not directly correlated with conformational stability. Only the mutational effect on human carbonic anhydrase II (6Mårtensson L.-G. Karlsson M. Carlsson U. Biochemistry. 2002; 41: 15867-15875Crossref PubMed Scopus (34) Google Scholar) was examined in a reversible system. Little information is available about the thermodynamic effects of artificial disulfide bonds in the buried hydrophobic region.In a previous study (12Hagihara Y. Matsuda T. Yumoto N. J. Biol. Chem. 2005; 280: 24752-24758Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), we screened the amino acid pairs substituting for the disulfide bonds of four different immunoglobulin fold domains by a method based on the cellular quality control system (13Hagihara Y. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6619-6624Crossref PubMed Scopus (30) Google Scholar, 14Hagihara Y. Shiraki K. Nakamura T. Uegaki K. Takagi M. Imanaka T. Yumoto N. J. Biol. Chem. 2002; 277: 51043-51048Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The intradomain disulfide bonds in the immunoglobulin fold domains are buried and are important for stability (15Goto Y. Hamaguchi K. J. Biochem. (Tokyo). 1979; 86: 1433-1441Crossref PubMed Scopus (136) Google Scholar, 16Ohhashi Y. Hagihara Y. Kozhukh G. Hoshino M. Hasegawa K. Yamaguchi I. Naiki H. Goto Y. J. Biochem. (Tokyo). 2002; 131: 45-52Crossref PubMed Scopus (76) Google Scholar). At least two pairs combining Ala, Val, or both, i.e. Ala/Ala, Ala/Val, Val/Ala, and Val/Val, were selected in all four domains in the screening, and they were stably folded at 30 °C. These mutants found in three domains exhibited similar or higher stability compared with the corresponding reduced wild-type domains. The pairs using Ala and Ile, Gly and Leu, Gly and Phe, and Gly and Val were also selected in more than two of the four domains. Among those, the stability of one of the Ala/Ile mutants was higher than that of the corresponding reduced domains. This indicates that specific pairs of hydrophobic amino acids can replace the buried disulfide bond with a minimum loss of stability. Conversely, for construction of an artificial disulfide bond, the proximal pair of buried hydrophobic amino acids as reported by a previous study (12Hagihara Y. Matsuda T. Yumoto N. J. Biol. Chem. 2005; 280: 24752-24758Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) may be a good candidate for replacement with cystine.Here, we report the effects of an engineered disulfide bond in the hydrophobic core of the variable domain of the camelid heavy chain antibody (VHH), 2The abbreviations used are:VHHvariable domain of the camelid heavy chain antibodyhCGhuman chorionic gonadotropinDSCdifferential scanning calorimetrySPRsurface plasmon resonanceMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight. 2The abbreviations used are:VHHvariable domain of the camelid heavy chain antibodyhCGhuman chorionic gonadotropinDSCdifferential scanning calorimetrySPRsurface plasmon resonanceMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight. also known as the single-domain antibody. In the three-dimensional structure of llama VHH raised against the α-subunit of human chorionic gonadotropin (hCG) (17Spinelli S. Frenken L. Bourgeois D. de Ron L. Bos W. Verrips T. Anguille C. Cambillau C. Tegoni M. Nat. Struct. Biol. 1996; 3: 752-757Crossref PubMed Scopus (130) Google Scholar), Ala49 and Ile70 are buried in the domain, and the distance between β-carbons of these amino acids is within a normal distance of naturally occurring β-carbons of cystines. We replaced these two amino acids with Cys in the wild-type and mutant domains, substituting the native disulfide bond with the Trp/Ala amino acid pair. Then, the stability and antigen binding ability of the mutants created were studied by CD, differential scanning calorimetry (DSC), pulldown assay, and surface plasmon resonance (SPR) spectrometry.EXPERIMENTAL PROCEDURESPreparation of VHH Mutants—Construction of the expression vector containing wild-type VHH and the mutant lacking a disulfide bond was described previously (12Hagihara Y. Matsuda T. Yumoto N. J. Biol. Chem. 2005; 280: 24752-24758Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The mutations were introduced by PCR-based site-directed mutagenesis. The VHH mutants containing one or no disulfide bond were cloned into pAED4 (18Doering D.S. Matsudaira P. Biochemistry. 1996; 35: 12677-12685Crossref PubMed Scopus (100) Google Scholar), and mutant proteins were expressed in Escherichia coli strain BL21(DE3) pLysS (Stratagene, La Jolla, CA), where they accumulated in inclusion bodies. These inclusion bodies were dissolved with 6 m guanidine hydrochloride or 8 m urea, followed by overnight air oxidation at 4 °C. Proteins in the denaturant were refolded by 1:50 dilution with 10 mm sodium acetate (pH 5.3 or 4.7). A Resource S cation-exchange column (GE Healthcare) equilibrated with 10 mm sodium acetate (pH 5.3 or 4.7) was used to purify crude VHHs.The VHH gene with two disulfide bonds was cloned into pPIC11, which is a derivative of pPIC9 (Invitrogen) with the replication origin of the pUC vector. The constructed vector was digested with AatI (StuI) and transformed into Pichia pastoris GS115. The most efficient transformant was selected on the basis of expression efficiency in small-scale test tube culture. High cell density fermentation of the selected strain was carried out in a 2-liter fermentor (Mitsuwa Scientific Corp., Osaka, Japan) as described previously (19Hagihara Y. Enjyoji K. Omasa T. Katakura Y. Suga K. Igarashi M. Matsuura E. Kato H. Yoshimura T. Goto Y. J. Biochem. (Tokyo). 1997; 121: 128-137Crossref PubMed Scopus (23) Google Scholar). The culture was continued for 1 day after the induction of protein expression by the addition of methanol. The secreted protein was purified by SP-Sepharose FF (GE Healthcare) equilibrated with 10 mm sodium acetate (pH 4.7). The collected fraction was further purified by Superdex 75 (GE Healthcare) equilibrated with 10 mm sodium acetate (pH 5.3) and 50 mm NaCl. The purified protein was diluted to a concentration of 0.4 mg/ml in 20 mm Tris-HCl (pH 7.5). To complete the oxidative formation of the disulfide bond, the diluted sample was incubated at 65 °C and purified again by gel permeation chromatography under the same conditions as described above. Correct formation of the disulfide bonds was confirmed by tryptic digestion, followed by MALDI-TOF mass spectrometry with a Voyager DE STR-D1 (Applied Biosystems, Foster City, CA).The formation of disulfide bonds was examined by the method of Ellman (20Ellman G.L. Arch. Biochem. Biophys. 1958; 74: 443-450Crossref PubMed Scopus (915) Google Scholar) in 4 m guanidine hydrochloride, 0.1 m Tris-HCl (pH 8.5), and 1 mm 5,5′-dithiobis(nitrobenzoic acid) at a protein concentration of ∼20 μm, in which the fully reduced samples were expected to exhibit A412 nm = 0.5–1.0 with a UV-2500PC spectrophotometer (Shimadzu Corp., Kyoto, Japan). Free thiols of VHHs with one disulfide bond were undetectable under the experimental conditions used. MALDI-TOF mass spectrometry was then used to confirm that the molecular weights of the purified proteins were identical to the expected values calculated from their amino acid sequences (with an error of ±0.025%). The concentration of protein in the stock solution was determined by measuring the absorbance at 280 nm (21Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (2993) Google Scholar).Thermodynamic Analysis of VHH Unfolding—Thermal unfolding was monitored as the change in ellipticity at 235 nm at a protein concentration of 4 μm using a J-720 spectropolarimeter (Jasco, Tokyo, Japan) and a 10-mm cell. The buffers used for the experiments contained 20 mm sodium acetate (pH 4–5), glycine HCl (pH 2–4), or potassium phosphate (pH 7.1). In unfolding experiments using CD, >70% of the CD signals at 235 nm were retained at 10 °C after heat denaturation, except the unfolding of 0-SSVHH at pH 7.1, for which ∼40% of ellipticity at 235 nm was observed. We thus did not use the data of 0-SSVHH at neutral pH to calculate the thermodynamic parameters. DSC measurements of VHHs were made using a VP-DSC MicroCalorimeter (MicroCal, Northampton, MA) in buffer containing 20 mm sodium acetate (pH 4) or glycine HCl (pH 2–4). For both CD and DSC measurements, the heating rate was 1 °C/min. We examined the reversibility of the DSC measurements in each VHH in at least two different pH environments. More than 80% of the heat absorption was observed in the second run after the first heat denaturation. The CD and DSC results were analyzed on the basis of the two-state transition mechanism using IGOR Pro 4 (WaveMetrics, Inc., Lake Oswego, OR).In the analysis of the unfolding transitions detected by CD, the free energy change of unfolding at a given pH at temperature T was as shown in Equation 1, ΔGUpH(T)=(TmpH-T)TmpH-1(ΔHU,45-ΔCp,U(318.15-TmpH))-(TmpH-T)ΔCp,U+TΔCp,Uln(TmpHT-1)(Eq. 1) where TmpH and ΔHU,45 represent the Tm at a given pH and the enthalpy change from the native state to the unfolded state (ΔHU) at 45 °C, respectively. In this analysis, we assumed that the difference in heat capacity between the folded and unfolded states (ΔCp,U) was constant. Because ΔHU,45 and ΔCp,U are independent of pH, multiple CD unfolding curves at different pH values could be analyzed by global fitting using the same ΔHU,45 and ΔCp,U values.For DSC analysis, ΔCp,U was estimated to be the difference between the base-line values for the unfolded (BLU) and native (BLN) states. BLU and BLN are represented by BLU = BLU1T + BLU2 and BLN = BLN1T + BLN2, respectively. Thus, the ΔCp,U at temperature T is given by Equation 2. ΔCp,U(T)=(BLU1-BLN1)T+(BLU2-BLN2)(Eq. 2) Therefore, the ΔHU and entropy changes of unfolding (ΔSU) at temperature T are described by Equations 3 and 4, respectively. ΔHU(T)=ΔHU(Tm)-(0.5(BLU1-BLN1)(Tm2-T2)+(BLU2-BLN2)(Tm-T))(Eq. 3) ΔSU(T)=ΔHU(Tm)Tm-1-((BLU1-BLN1)(Tm-T)+(BLU2-BLN2)ln(TmT-1))(Eq. 4) The molar heat capacity (Cp(T)) is calculated by Equation 5, Cp(T)=ΔHU(T)2fU(1-fU)R-1T-2+(1-fU)(BLN1T-BLN2)+fU(BLU1T-BLU2)(Eq. 5) where fU is the fraction of molecules in the unfolded state calculated from the Gibbs-Helmholtz equation and Equations 3 and 4.Binding Assay of VHH with Antigen hCG—VHH mutants were immobilized on cyanogen bromide-activated Sepharose (GE Healthcare) according to the standard protocol supplied by the manufacturer. Aliquots of Sepharose beads (20 μl), on which ∼10 μg of VHHs had been immobilized, were incubated with 10 μg of hCG (Sigma) in 20 mm Tris-HCl (pH 8.0) containing 0.15 m NaCl at room temperature for 90 min. After the beads were washed three times with the same buffer, the samples were boiled for 15 min at 95 °C in standard SDS-PAGE sample buffer containing 2-mercaptoethanol and then applied to 10–20% gradient SDS-polyacrylamide gels. The gels were stained with Coomassie Brilliant Blue. The kinetics of binding of VHHs to antigen was examined by SPR spectroscopy. Antigen hCG (0.05 mg/ml) was coupled to a CM5 sensor chip (GE Healthcare) in 10 mm sodium acetate buffer (pH 6) by amine coupling according to the manufacturer's instructions. Analysis was performed on a Biacore 2000 instrument (GE Healthcare) in 10 mm HEPES (pH 7.4), 150 mm NaCl, 3 mm EDTA, and 0.005% surfactant P20 at 20 °C. Five serial dilutions (250 to 15 nm) of VHHs were injected into the cuvette, followed by regeneration with 20 mm HCl. As a control, binding of β2-microglubulin (500 to 125 nm) to hCG was also examined.RESULTSIntroduction of an Artificial Disulfide Bond into VHH—We found that llama VHH has a potential candidate for the introduction of the artificial disulfide bond in its hydrophobic amino acid pair: Ala49 and Ile70 (Fig. 1, A and B). Amino acid pairs matching the following criteria were searched for in the three-dimensional structure of VHH (Protein Data Bank code 1HCV) (17Spinelli S. Frenken L. Bourgeois D. de Ron L. Bos W. Verrips T. Anguille C. Cambillau C. Tegoni M. Nat. Struct. Biol. 1996; 3: 752-757Crossref PubMed Scopus (130) Google Scholar), for which the antigen is hCG. First, we selected pairs containing combinations of Ala, Val, Ala and Val, and Ala and Ile, which were reported previously to be stable at 30 °C in more than two different immunoglobulin fold domains (12Hagihara Y. Matsuda T. Yumoto N. J. Biol. Chem. 2005; 280: 24752-24758Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). We excluded pairs with Gly because it is hard to predict the orientation of the side chain of Cys substituted for Gly. Second, the solvent-exposed surface area of each amino acid needed to be <10 Å2, which corresponds to 5% of the total surface of Ala, because most naturally occurring disulfide bonds are buried (1Thornton J.M. J. Mol. Biol. 1981; 151: 261-287Crossref PubMed Scopus (668) Google Scholar, 2Nagano N. Ota M. Nishikawa K. FEBS Lett. 1999; 458: 69-71Crossref PubMed Scopus (114) Google Scholar, 3Rose G.D. Geselowitz A.R. Lesser G.J. Lee R.H. Zehfus M.H. Science. 1985; 229: 834-838Crossref PubMed Scopus (960) Google Scholar). The solvent-exposed surface area was calculated by MOLMOL (22Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6468) Google Scholar) considering a solvent radius of 1.4 Å. Usually, the distance between two β-carbons of cystine is ∼3.5–4.5 Å (5Dani V.S. Ramakrishnan C. Varadarajan R. Protein Eng. 2003; 16: 187-193Crossref PubMed Scopus (91) Google Scholar). Thus, a third criterion was that the distance between the β-carbons of the pair of amino acids needed to be <4.5 Å.The Ala49/Ile70 pair matched all of the above-mentioned criteria. The accessible surface areas of Ala49 and Ile70 are 3.4 and 7.5 Å2, respectively. These correspond to 1.6% (Ala49) and 2.7% (Ile70) of the total accessible surface areas of these amino acids alone. The distance between the β-carbons of Ala49 and Ile70 is 4.1 Å. We prepared two mutants in which we replaced these two amino acids with Cys (Fig. 1C). One was with a wild-type disulfide bond between Cys22 and Cys96, named 2-SSVHH. The other, 1-SSVHH, was constructed on the non-disulfide-bonded framework, where the wild-type disulfide bond was substituted with the Trp/Ala pair. In addition to these mutants with engineered disulfide bonds, we used wild-type VHH and a non-disulfide-bonded mutant (0-SSVHH) for comparison. Although the Trp/Ala pair was selected only once in a previous study, 0-SSVHH, in which the native disulfide bond was replaced with this pair, showed the highest thermal stability of the mutants that were found in the screening (12Hagihara Y. Matsuda T. Yumoto N. J. Biol. Chem. 2005; 280: 24752-24758Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Thus, we selected a mutant with Trp/Ala as the non-disulfide-bonded framework. It should be noted that the VHH mutants with Ala/Ala and Ala/Val exhibited the second and fourth highest Tm values of the selected mutants, respectively (12Hagihara Y. Matsuda T. Yumoto N. J. Biol. Chem. 2005; 280: 24752-24758Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar).The oxidative formation of the disulfide bond in the 1-SSVHH and wild-type domains can be completed by air oxidation in denaturant. However, in 2-SSVHH, the reproducibility of the renaturation of two disulfide bonds was low when we used E. coli as a host organism to express 2-SSVHH (data not shown). Thus, we selected the methylotrophic yeast P. pastoris to produce 2-SSVHH. After the expressed 2-SSVHH mutant was heated to complete the formation of disulfide bonds, the amount of free thiol was <5% of the amount of total thiol. Because the remaining free thiol may influence the thermodynamic parameters for the stability of 2-SSVHH, we simply analyzed 2-SSVHH in terms of Tm and antigen binding affinity, which are not sensitive to minor contamination with free thiols. For comparison among 1-SSVHH, 0-SSVHH, and wild-type domains, we precisely analyzed the thermodynamic effects of the engineered disulfide bond on the stability of VHH.Stability of VHH Mutants with an Artificial Disulfide Bond—The engineered disulfide bond significantly stabilized the VHHs. We measured the thermal unfolding curves of the mutant and wild-type VHHs at pH 7.1 following the change in ellipticity at 235 nm (Fig. 2). The Tm values of the 2-SSVHH, 1-SSVHH, 0-SSVHH, and wild-type domains were 74, 56, 46, and 64 °C, respectively. The most stable mutant, 2-SSVHH, was unfolded at a 10 °C higher Tm compared with the wild-type domain. The difference in Tm between 1-SSVHH and 0-SSVHH was 10 °C and was identical to that between 2-SSVHH and wild-type VHH.FIGURE 2Thermal unfolding curves of VHHs. Wild-type VHH (black), 0-SSVHH (red), 1-SSVHH (blue), and 2-SSVHH (green) were measured by the change in ellipticity at 235 nm using a 10-mm cell. Experiments were carried out at pH 7.1 (20 mm sodium phosphate) using a 10-mm cell. The protein concentration was 4 μm for all proteins. The Tm values of the 2-SSVHH, 1-SSVHH, 0-SSVHH, and wild-type domains were 74, 56, 46, and 64 °C, respectively. deg, degrees.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The thermal unfolding curves of 1-SSVHH, 0-SSVHH, and the wild-type domains at different pH values were measured by CD and DSC to further characterize the effects of the artificial disulfide bond (Fig. 3). The data from CD were analyzed by global fitting. We analyzed individually the heat absorption curves derived from the DSC measurements. Because we could measure the unfolding curves with Tm values of ∼45 °C in all three VHHs, we decided to compare the thermodynamic parameters at 45 °C.FIGURE 3Thermal denaturation of VHHs at different pH values. Shown are the unfolding curves of 1-SSVHH measured by CD at pH 3.9, 3.5, 3.2, 2.9, and 2.6 (A) and by DSC at pH 4.1, 3.6, 3.3, and 3.0 (B). Continuous red lines indicate the curves drawn by the parameters obtained by global (A) and individual (B) fitting methods. deg, degrees.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The ΔHU of 0-SSVHH was significantly higher than that of the 1-SSVHH and wild-type domains (Fig. 4 and Table 1). Thus, the introduction of a disulfide bond, both native and artificial, is enthalpically unfavorable for the stability of VHH. The linear extrapolation of the individual results of the DSC analysis to 45 °C showed that the ΔHU value of 0-SSVHH was 30 and 50 kJ/mol higher than those of the 1-SSVHH and wild-type domains, respectively. The analysis of the CD measurements showed the difference in ΔHU at 45 °C between 0-SSVHH and 1-SSVHH to be ∼50 kJ/mol and that between 0-SSVHH and wild-type VHH to be ∼80 kJ/mol.FIGURE 4Enthalpy of unfolding (ΔHU) of wild-type VHH (black), 0-SSVHH (red), and 1-SSVHH (blue). ΔHU values from individual analysis of DSC curves are indicated by open circles (wild-type VHH), open squares (0-SSVHH), and closed circles (1-SSVHH). Solid and dashed lines were obtained by linear fitting of individual ΔHU values from DSC and by global fitting of CD data, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Thermodynamic parameters of VHHsVHH/methodΔHU at 45 °CaΔHU and ΔCp,U were calculated by global fitting of CD data and linear fitting of ΔHU values obtained from individual DSC data, respectivelyΔCp,UaΔHU and ΔCp,U were calculated by global fitting of CD data and linear fitting of ΔHU values obtained from individual DSC data, respectivelyAverage difference in ΔSU at 45 °CbThe ΔSU values of wild-type and 1-SSVHH were subtracted from the ΔSU values of 0-SSVHH at the same pH values, and these values were averagedkJ/molkJ/molJ/mol0-SSVHH CD360 ± 75.4 ± 0.7 DSC342 ± 54.8 ± 0.4Wild-type VHH CD275 ± 65.3 ± 0.5-328 DSC295 ± 54.8 ± 0.3-2031-SSVHH CD309 ± 35.3 ± 0.7-205 DSC312 ± 55.2 ± 0.4-133a ΔHU and ΔCp,U were calculated by global fitting of CD data and linear fitting of ΔHU values obtained from individual DSC data, respectivelyb The ΔSU values of wild-type and 1-SSVHH were subtracted from the ΔSU values of 0-SSVHH at the same pH values, and these values were averaged Open table in a new tab ΔCp,U was estimated using both CD and DSC data (Table 1). With both techniques, estimated ΔCp,U values were identical within experimental errors, which indicates that the effect of disulfide introduction on ΔCp,U was negligible. ΔCp,U is considered to be related to the difference in the amount of hydration in the folded and unfolded states. Our data suggest that the introduction of a disulfide bond does not alter the extent of hydration upon unfolding. However, the error in estimated ΔCp,U was relatively high (∼10%), and uncertainty exists about the hydration effect based on this parameter.Both native and engineered disulfide bonds decreased ΔSU compared with 0-SSVHH (Table 1). We estimated the average difference in ΔSU at 45 °C between the 0-SSVHH and the domains with one disulfide bond at the same pH values. The ΔSU values of 0-SSVHH were 205 (CD) and 133 (DSC) J/mol larger than those of 1-SSVHH and 328 (CD) and 203 (DSC) J/mol larger than those of the wild-type domain. The effect of disulfide formation on ΔSU (ΔSU,loop) can be calculated using Equation 6, ΔSU,loop=-R(3/2lnN+A)(Eq. 6) where N is the number of residues in the loop-forming disulfide bond. Constant A differs in the literature. Poland and Scheraga (23Poland D.G. Scheraga H.A. Biopolymers. 1965; 3: 379-399Crossref Scopus (128) Google Scholar) and Pace et al. (24Pace C.N. Grimsley G.R. Thomson J.A. Barnett B.J. J. Biol. Chem. 1988; 263: 11820-11825Abstract Full Text PDF PubMed Google Scholar) assumed that constant A is 3.47 and 1.06, respectively. The calculated ΔSU,loop values using both values were -67 and -47 J/mol for 1-SSVHH and -83 and -63 J/mol for wild-type VHH. The differences in experimental ΔSU values of 1-SSVHH and wild-type VHH compared with 0-SSVHH deviated significantly from ΔSU,loop.Antigen Binding of VHH Mutants—All prepared VHH mutants bound to the antigen, the α-subunit of hCG. First, we carried out pulldown antigen experiments using immobilized 0-SSVHH, 1-SSVHH, 2-SSVHH, and wild-type domains (Fig. 5). Sepharose-immobilized VHHs were prepared and incubated with hCG at room temperature. After the residual proteins were washed, bound antigen was eluted by SDS. Then, each eluent was subjected to SDS-PAGE. The amounts of hCG bound to the immobilized mutants were similar compared with wild-type VHH. hCG did not interact with Sepharose alone (Fig. 5, lane 3).FIGURE 5Binding of antigen hCG to immobilized VHHs. Wild-type VHH (lane 4), 0-SSVHH (lane 5), 1-SSVHH (lane 7), and 2-SSVHH (lane 8) were immobilized on cyanogen bromide-activated Sepharose, and hCG was incubated with VHH-Sepharose at room temperature for 90 min. After washing and elution, bound hCG was applied to 10–20% gradient SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue. The antigen binding ability of non-disulfide-bonded VHH, in which the native disulfide bond was replaced with Ala/Ala (A/A*), is also shown for reference (lane 6).View Large Image Figure ViewerDownload Hi-res image" @default.
• W2043532306 created "2016-06-24" @default.
• W2043532306 creator A5050285161 @default.
• W2043532306 creator A5055364103 @default.
• W2043532306 creator A5074234453 @default.
• W2043532306 date "2007-12-01" @default.
• W2043532306 modified "2023-10-10" @default.
• W2043532306 title "Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region" @default.
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