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Conformational changes at the active site of adenylate kinase detected using a fluorescent probe and monoclonal antibody binding
Biochimie 84 (2002) 335–339
Conformational changes at the active site of adenylate kinase detected using a fluorescent probe and monoclonal antibody binding Tian-Hong Zhang, Jie Luo, Jun-Mei Zhou * National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China Received 14 September 2001; accepted 20 November 2001
Abstract A fluorescent probe, IAEDANS, was introduced into the active site of adenylate kinase (AK) by specifically modifying Cys-25. During modification, enzyme activity was greatly diminished. This probe allowed observation of conformational changes at the active site during denaturation that could not be detected directly in previous studies. The binding ability of modified AK with its monoclonal antibody (McAb3D3) was identical to that of native AK and the fluorescence of modified AK was quenched by interaction with McAb3D3. The relative fluorescence changes during the binding of modified AK with McAb3D3 in different concentrations of guanidine hydrochloride were monitored. The combination of this active site modification with the use of a conformation specific monoclonal antibody has potential for use in the study of the kinetics of folding of AK and in the detection of folding intermediates. © 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Adenylate kinase; Conformational change; Fluorescent probe; Fluorescence quenching; Monoclonal antibody
1. Introduction Adenylate kinase (AK, EC 2.7.4.3) is a small monomeric enzyme that catalyzes the phosphorylation of AMP by ATP to form two ADP molecules [1]. The rabbit muscle cytosolic enzyme contains 194 amino acid residues in a single peptide chain [2,3]. With two cysteines (Cys-25 and Cys-187) and without disulfide bonds, it is an ideal enzyme for studying protein folding. Previous work in this laboratory has involved the study of the activity and conformational changes of AK during thermal, guanidine and urea denaturation [4–7]. During denaturation, conformational changes at the enzyme active site cannot be detected due to lack of an appropriate probe. Therefore a probe introduced at the active site would provide useful information about the folding mechanism. Previously, monoclonal antibodies against AK were prepared in our laboratory [8]. This work has laid a foundation for further research to probe protein
* Corresponding author. Tel: +86-10-64889859; fax: +86-10-64872026. E-mail address: [email protected] (J.M. Zhou).
folding, as monoclonal antibodies, in particular conformation-dependent ones are a powerful tool for detecting and characterizing folding intermediates [9,10]. However, to be able to measure transient processes during folding, a suitable method to monitor the association of the antigen with the antibody must be designed. Signals related to fluorescence (quenching or energy transfer) provide a convenient probe of structural change if an appropriate fluorescent label can be introduced on either the antigen or the antibody. In this study, with the aim of monitoring directly conformational changes of AK at the active site and the interaction of AK with its monoclonal antibody McAb3D3, we introduced a fluorescent probe at the active site of AK by specifically modifying the SH group of Cys-25 with IAEDANS. Fluorescence changes during denaturation of the modified enzyme alone and in the presence of the antibody were monitored. The results are compared to previous studies on the relationship between structural changes and changes in enzymatic activity during denaturation.
© 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 3 8 4 - 6
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T.H. Zhang et al. / Biochimie 84 (2002) 335–339
2. Materials and methods Rabbit muscle AK was prepared as described previously [4]. The final preparation typically had a specific activity greater than 1600 unit mg–1 and showed only one peak in SDS electrophoresis. One unit is defined as the amount of enzyme catalyzing the formation of 1 µmol ATP generated from ADP per minute. The concentration of AK was determined by absorption at 280 nm with A1 mg/ml = 0.52. The monoclonal antibody McAb3D3 against AK was prepared as described [8]. Concentrations were measured spectrophotometrically at 280 nm by using A1 mg/ml = 1.4. Tris-HCl, ADP, NADP, hexokinase, glucose-6-phosphate dehydrogenase, BSA, ATP, AMP, and IAEDANS were Sigma products. GuHCl, ultrapure, was from ICN. Urea was a product of NACALA TESQUE, Inc. (Japan). Other reagents were local products of analytical grade. The urea solution was always freshly prepared immediately prior to use. The activity of AK was assayed by following the reduction of NADP in a coupled enzyme solution with hexokinase and glucose-6-phosphate dehydrogenase. The reaction mixture contained 2.5 mM ADP, 2.1 mM Mg(Ac)2, 6.7 mM NADP, 20 units hexokinase, and 10 units glucose6-phosphate dehydrogenase in 50 mM Tris-HCl buffer (pH 8.1). The rabbit muscle AK was labeled with IAEDANS using the method of Gardner and Matthews [11] with some modifications. The native AK (in 0.1 M Tris-HCl, pH 8.0, containing 1 mM EDTA) was incubated with a 50-fold molar excess of IAEDANS for 1 h at 4 °C in the dark. The reaction was stopped by addition of a 1000-fold molar excess of β-mercaptoethanol. The excess reagent was separated from the protein by dialyzing against four changes of dialyzing buffer (0.1 M Tris-HCl, pH 8.0, 1 mM EDTA, and 2 mM β-mercaptoethenol) at 4 °C overnight. The degree of labeling was determined from the absorption spectrum (A336 –1 cm–1 for IAEDANS) [12]. nm = 6100 M
2 M H2SO4. The change in A450 was recorded on a Bio-Rad 3550 microplate reader.
3. Results 3.1. Modification of AK by IAEDANS Native AK was modified according to the method described. The degree of labeling was found to be 1 mol of IAEDANS per mol of native AK. The activity of the modified AK was only 10% of native AK. The changes in fluorescence intensity and activity of this modified AK were measured during the modification and the results are shown in Fig. 1. An increase in the fluorescence signal accompanied by a decrease in enzyme activity took place during the modification, and both changed sharply within 15 min, after which there was little further change. This indicates that accompanying the SH group modification the activity of modified enzyme was greatly diminished. Modification was also carried out after denaturing native AK with 4 M GuHCl before addition of IAEDANS. In this case the labeling ratio was 2:1, and the activity was identical to that of the denatured enzyme, indicating that both cysteine residues can be labeled when the enzyme is unfolded. These results are very similar to those obtained previously in labeling the sulfhydryl groups of porcine muscle AK with fluorescent reagent NBD-chloride [13]. Cys-25 reacted with the reagent approximately 40-fold faster than Cys-187, and modification of Cys-25 alone led to inactivation of the enzyme. From X-ray analysis, it is known that the Cys-25 of porcine muscle is close to the presumed catalytic center [14]. The structure of rabbit muscle AK is expected to be identical to
A Hitachi-4500 fluorimeter was used for fluorescence measurements. Unless otherwise specified, an excitation wavelength of 336 nm was used, and the emission intensities were recorded at 490 nm at 25 °C, with both excitation and emission slits set at 5 nm. The affinity of AK binding with monoclonal antibody was measured by ELISA. Native AK (5 µg/ml, 100 µl) and modified AK (5 µg/ml, 100 µl) were first coated onto the microplates for 12 h at 4 °C. The remaining non-specific binding sites on the wells were blocked with 5% gelatin, then rinsed three times with PBST. One hundred microliters of McAb3D3 of different concentrations were then added to the wells in triplicate, and incubated for 1 h. After three rinses, 100 µl of ExtrAvidin Peroxidase (1:3000 dilution) were added and the mixture incubated for 1 h. After three rinses with PBST, 100 µl of TMB/H2O2 solution were added and the reaction stopped after 10 min with 100 µl of
Fig. 1. The changes of fluorescence intensity (squares) and enzyme activity (circles) of modified AK at various labeling times. The enzyme concentration was 1.0 µM in 0.1 M Tris-HCl pH 8.0, containing 1 mM EDTA. The fluorescence was measured at 336 nm excitation and at 490 nm emission at 25 °C.
T.H. Zhang et al. / Biochimie 84 (2002) 335–339
that of porcine muscle AK, as they have very similar amino acid sequences. It is therefore concluded that in the present work, the single labeled cysteine residue in native AK is Cys-25 and is located at the active site. This modified AK with labeling ratio 1:1 was used for further investigation. 3.2. The changes of fluorescence intensity of modified AK in GuHCl and urea The fluorescence intensity of modified AK in the presence of different concentrations of GuHCl and urea was determined. With increasing concentrations of GuHCl or urea, the fluorescence intensity of modified AK first increased and then gradually decreased (Fig. 2a,b). The fluorescence intensity of modified AK was enhanced about 1.8-fold in 0.2 M GuHCl, nearly 1.4-fold in 0.6 M urea, and no further fluorescence changes were observed above 1 M
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GuHCl or 2 M urea. These fluorescent changes closely resemble the pattern of activity changes during chemical denaturation of the unmodified enzyme [5]. Previous studies from this laboratory reported that the activity of the enzyme undergoes significant changes on addition of denaturant, showing a maximum increase of about 1.6-fold in 1 M urea or 0.25 M GuHCl, and then decreasing again at higher concentrations of denaturant. In contrast, the secondary and tertiary structures of AK were not noticeably perturbed at these concentrations of denaturant, as demonstrated by CD and UV measurements [15]. The results of this study demonstrate that addition of low concentrations of denaturant results in conformational changes at the active site of this enzyme. Taken together with the results of previous studies, it is clear that the conformational changes detected by the introduction of a fluorescent probe at the active site reflect the changes in activity that are observed on denaturation of the unmodified enzyme. It is clear that during denaturation of AK, conformational change at the active site occurs before unfolding of the rest of the molecule. This result supports the hypothesis proposed by Tsou, that the conformation at the active site of the enzyme is more flexible than the molecule as a whole [16–18]. 3.3. The binding of native AK and modified AK with McAb3D3 The ability of native AK and modified AK to bind to McAb3D3 was tested by ELISA as described in Section 2. It was apparent that the 1/A450 nm of native AK and modified AK are both proportional to the reciprocal of the antibody concentration, as shown in Fig. 3. The slopes of these two straight lines were the same, which shows that the binding of modified AK with McAb3D3 is identical to that of native AK. It can therefore be concluded that this chemical
Fig. 2. The changes of fluorescence intensity of modified AK in different concentrations of GuHCl (A) and urea (B). The enzyme concentration was 1.0 µM. The enzyme was incubated with GuHCl at 4 °C for 12 h before determination. Other details are as in the legend to Fig. 1. The changes were relative to the fluorescence intensity of modified AK in the absence of denaturant.
Fig. 3. Calibration curves of the binding of McAb3D3 to coated native AK (▲) and modified AK (▼) in the ELISA. The concentration range for McAb3D3 was 1.55 × 10–6 – 3.92 × 10–4 mg/ml. The coated amount of the two enzymes was 0.5 µg.
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modification does not interfere with the interaction between AK and McAb3D3. 3.4. The changes of fluorescence intensity during the interaction of modified AK with McAb3D3 The emission spectra of modified AK in the presence and absence of McAb3D3 when excited at 280 nm are shown in Fig. 4a. The mixture of modified AK and McAb3D3 (2:3 molar ratio) gave rise to a decrease in the emission intensity at 490 nm. No decrease was observed when BSA was used instead of McAb3D3 (Fig. 4b). This demonstrates that the decrease of the fluorescence intensity is due to the interaction of AK with McAb3D3. This specific signal was used to titrate the McAb3D3 preparation, and the result is shown in Fig. 5. From the curve obtained it can be calculated that
Fig. 5. Fluorescence titration of modified AK with McAb3D3. The enzyme concentration was 1.0 µM. Details are as in the legend of Fig. 1.
0.5 mol of McAb3D3 is saturated by 1 mol of modified AK, which corresponds to a titer of two binding sites for AK per molecule of McAb3D3. This fluorescent probe was also used to monitor the binding course of AK with McAb3D3, but the reaction was found to be complete within the dead time of manual mixing (12 s). 3.4. The interactions of modified AK with McAb3D3 in GuHCl monitored by the changes in fluorescence intensity
Fig. 4. Fluorescence quenching of modified AK in the presence of the monoclonal antibody McAb3D3 (A) or BSA control (B). In each case, the fluorescence emission spectra of McAb3D3 or BSA (curve 1), modified AK (curve 2), and modified AK mixed with McAb3D3 or BSA in a molar ratio of 2:3 (curve 3) is shown. The samples were in 0.1 M Tris-HCl pH 8.0, with 1 mM EDTA, and were excited at 280 nm.
The fluorescence intensities of modified AK with and without McAb3D3 were measured during the denaturation by GuHCl and the relative changes are shown in Fig. 6. The fluorescence quenching due to binding of McAb3D3 to modified AK varied with GuHCl concentration. The relative change in fluorescence of modified AK increased with increasing concentration of GuHCl and reached a maximum at 0.2 M GuHCl. This shows that partial unfolding of the antigen causes an increase in the antigen-antibody interaction. However, with further increase in the GuHCl concentration, the relative fluorescence decreased again to a level lower than non-denatured modified AK, indicating that the binding ratio of modified AK and McAb3D3 in higher concentrations of GuHCl is very low. From these results, it can be seen that binding of modified AK and the antibody varies greatly with GuHCl concentration, and is at a maximum at low concentration of GuHCl. This suggests that McAb3D3 preferentially binds to a more flexible or partially denatured conformation of AK. This is consistent with the finding that the affinity constant of McAb3D3 with coated AK is greater than that with free AK [8], which can be explained by partial denaturation of the protein when absorbed on the microplates.
T.H. Zhang et al. / Biochimie 84 (2002) 335–339
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Acknowledgements The present study was supported in part by Chinese Ministry of Science and Technology Grant G1999075608. The authors would like to thank Dr. S. Perrett of this laboratory for her critical reading of this paper and helpful suggestions.
References [1] [2]
[3] Fig. 6. The relative binding ability of modified AK mixed with McAb3D3 at various GuHCl concentrations. The enzyme concentration was 1.0 µM and the McAb3D3 concentration was 0.5 µM. Details are as in the legend of Fig. 2. The changes were related to the fluorescence intensity of modified enzyme in the absence of GuHCl.
[4]
[5]
4. Discussion [6]
The results presented in this study show that a fluorescent probe can be introduced at the active site of rabbit muscle AK using IAEDANS. Only Cys-25 was labeled when native AK was used, and modification led to enzyme activity greatly diminished. The changes of fluorescence intensity of modified AK mirrored the previously reported activity changes of native AK during denaturation [5]. This demonstrates that introduction of the fluorescent probe allows detection of conformational changes that occur at the active site of AK early in the process of denaturation. These conformational changes at the active site occur prior to global denaturation of the enzyme which is detected by changes in the intrinsic spectroscopic properties of the enzyme [15]. The modified AK is also shown to have the same affinity as native AK for the monoclonal antibody McAb3D3. Interaction of modified AK and McAb3D3 resulted in quenching of the fluorescence, and the relative changes varied with denaturant concentration. This observation shows that binding of AK and McAb3D3 may be conformation-dependent and that partially denatured AK is preferentially recognized by the antibody. We have demonstrated that introduction of the IAEDANS fluorescent probe at the active site of AK provides a useful method to monitor both conformational changes at the enzyme active site and interaction of AK with its monoclonal antibody during denaturation. The combination of this active site modification with the use of a conformation specific monoclonal antibody has potential for use in the study of the kinetics of folding of AK and particularly in the detection of folding intermediates.
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
[15]
[16] [17] [18]
L. Nado, Enzyme, in: P.D. Boyer (Ed.), 3rd ed., vol. 8, 1973, pp. 279–305. S.A. Kuby, R.H. Palmieri, A. Frischat, Studies on adenosine triphosphate transphorylases, amino acid sequence of rabbit muscle ATP-AMP transphosphorylase, Biochemistry 23 (1984) 2393–2399. M. Hamada, R.H. Palmieri, G.A. Russell, Studies on adenosine triphosphate transphosphorylases XIV, equilibrium binding properties of the crystalline rabbit and calf muscle ATP-AMP transphosphorylase (adenylate kinase) and derived peptide fragments, Arch. Biochem. Biophys. 195 (1979) 155–177. Y.L. Zhang, J.M. Zhou, C.L. Tsou, Inactivation precedes conformation change during thermal denaturation of adenylate kinase, Biochim. Biophys. Acta 1164 (1993) 61–67. Y.L. Zhang, J.M. Zhou, C.L. Tsou, Sequential unfolding of adenylate kinase during denaturation by guanidine hydrochloride, Biochim. Biophys. Acta 1295 (1996) 239–244. H.J. Zhang, X.R. Sheng, X.M. Pan, J.M. Zhou, Refolding of urea-denaturaed adenylate kinase, Biochem. J. 333 (1998) 401–405. H.J. Zhang, X.M. Pan, J.M. Zhou, H. Kihara, Activation and conformational changes of adenylate kinase in urea solution, Sci. China, Ser. C 41 (1998) 245–250. X.D. Wang, J.M. Zhou, Z.Q. Guo, Preparation and characterization of monoclonal antibodies against adenylate kinase, Sci. China, Ser. C 40 (6) (1997) 561–567. E. Katchalski-Katzir, D. Kenett, Use of monoclonal antibodies in the study of conformation and conformational alterations in proteins, Bull. Chem. Soc. Jpn. 61 (1988) 133–139. M.E. Goldberg, Investigating protein conformation, dynamics and folding with monoclonal antibodies, TIBS 16 (1991) 358–362. J.A. Gardner, K.S. Matthews, Energy transfer in lactose repressor protein modified with N-{[(iodoacetyl)amino]ethyl}– 5–naphthylamine–1-sulfonate, Biochemistry 30 (1991) 2707–2712. E.N. Hudson, G. Weber, Synthesis and characterization of two fluorescent sulfhydryl reagents, Biochemistry 12 (1973) 4154–4161. N.C. Price, M. Cohn, Fluorescent and spin label probes of the environments of the sulfhydryl groups of porcine muscle adenylate kinase, J. Biol. Chem. 250 (1975) 644–652. D. Dreusicke, P.A. Karplus, G.E. Schulz, Refined structure of porcine cytosolic adenylate kinase at 2.1 Å resolution, J. Mol. Biol. 199 (1988) 359–371. H.J. Zhang, X.R. Sheng, X.M. Pan, J.M. Zhou, Activation of adenylate kinase by denaturants is due to the increasing conformational flexibility at its active sites, Biochem. Biophys. Res. Comm. 238 (1997) 382–386. C.L. Tsou, Location of the active sites of some enzymes in limited and flexible molecular regions, TIBS 11 (1986) 427. C.L. Tsou, Conformational flexibility of enzyme active sites, Science 262 (1993) 380–381. C.L. Tsou, Inactivation precedes overall molecular conformation changes during enzyme denaturation, Biochim. Biophys. Acta 1253 (1995) 151–162.
Conformational changes at the active site of adenylate kinase detected using a fluorescent probe and monoclonal antibody binding Tian-Hong Zhang, Jie Luo, Jun-Mei Zhou * National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China Received 14 September 2001; accepted 20 November 2001
Abstract A fluorescent probe, IAEDANS, was introduced into the active site of adenylate kinase (AK) by specifically modifying Cys-25. During modification, enzyme activity was greatly diminished. This probe allowed observation of conformational changes at the active site during denaturation that could not be detected directly in previous studies. The binding ability of modified AK with its monoclonal antibody (McAb3D3) was identical to that of native AK and the fluorescence of modified AK was quenched by interaction with McAb3D3. The relative fluorescence changes during the binding of modified AK with McAb3D3 in different concentrations of guanidine hydrochloride were monitored. The combination of this active site modification with the use of a conformation specific monoclonal antibody has potential for use in the study of the kinetics of folding of AK and in the detection of folding intermediates. © 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Adenylate kinase; Conformational change; Fluorescent probe; Fluorescence quenching; Monoclonal antibody
1. Introduction Adenylate kinase (AK, EC 2.7.4.3) is a small monomeric enzyme that catalyzes the phosphorylation of AMP by ATP to form two ADP molecules [1]. The rabbit muscle cytosolic enzyme contains 194 amino acid residues in a single peptide chain [2,3]. With two cysteines (Cys-25 and Cys-187) and without disulfide bonds, it is an ideal enzyme for studying protein folding. Previous work in this laboratory has involved the study of the activity and conformational changes of AK during thermal, guanidine and urea denaturation [4–7]. During denaturation, conformational changes at the enzyme active site cannot be detected due to lack of an appropriate probe. Therefore a probe introduced at the active site would provide useful information about the folding mechanism. Previously, monoclonal antibodies against AK were prepared in our laboratory [8]. This work has laid a foundation for further research to probe protein
* Corresponding author. Tel: +86-10-64889859; fax: +86-10-64872026. E-mail address: [email protected] (J.M. Zhou).
folding, as monoclonal antibodies, in particular conformation-dependent ones are a powerful tool for detecting and characterizing folding intermediates [9,10]. However, to be able to measure transient processes during folding, a suitable method to monitor the association of the antigen with the antibody must be designed. Signals related to fluorescence (quenching or energy transfer) provide a convenient probe of structural change if an appropriate fluorescent label can be introduced on either the antigen or the antibody. In this study, with the aim of monitoring directly conformational changes of AK at the active site and the interaction of AK with its monoclonal antibody McAb3D3, we introduced a fluorescent probe at the active site of AK by specifically modifying the SH group of Cys-25 with IAEDANS. Fluorescence changes during denaturation of the modified enzyme alone and in the presence of the antibody were monitored. The results are compared to previous studies on the relationship between structural changes and changes in enzymatic activity during denaturation.
© 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 3 8 4 - 6
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T.H. Zhang et al. / Biochimie 84 (2002) 335–339
2. Materials and methods Rabbit muscle AK was prepared as described previously [4]. The final preparation typically had a specific activity greater than 1600 unit mg–1 and showed only one peak in SDS electrophoresis. One unit is defined as the amount of enzyme catalyzing the formation of 1 µmol ATP generated from ADP per minute. The concentration of AK was determined by absorption at 280 nm with A1 mg/ml = 0.52. The monoclonal antibody McAb3D3 against AK was prepared as described [8]. Concentrations were measured spectrophotometrically at 280 nm by using A1 mg/ml = 1.4. Tris-HCl, ADP, NADP, hexokinase, glucose-6-phosphate dehydrogenase, BSA, ATP, AMP, and IAEDANS were Sigma products. GuHCl, ultrapure, was from ICN. Urea was a product of NACALA TESQUE, Inc. (Japan). Other reagents were local products of analytical grade. The urea solution was always freshly prepared immediately prior to use. The activity of AK was assayed by following the reduction of NADP in a coupled enzyme solution with hexokinase and glucose-6-phosphate dehydrogenase. The reaction mixture contained 2.5 mM ADP, 2.1 mM Mg(Ac)2, 6.7 mM NADP, 20 units hexokinase, and 10 units glucose6-phosphate dehydrogenase in 50 mM Tris-HCl buffer (pH 8.1). The rabbit muscle AK was labeled with IAEDANS using the method of Gardner and Matthews [11] with some modifications. The native AK (in 0.1 M Tris-HCl, pH 8.0, containing 1 mM EDTA) was incubated with a 50-fold molar excess of IAEDANS for 1 h at 4 °C in the dark. The reaction was stopped by addition of a 1000-fold molar excess of β-mercaptoethanol. The excess reagent was separated from the protein by dialyzing against four changes of dialyzing buffer (0.1 M Tris-HCl, pH 8.0, 1 mM EDTA, and 2 mM β-mercaptoethenol) at 4 °C overnight. The degree of labeling was determined from the absorption spectrum (A336 –1 cm–1 for IAEDANS) [12]. nm = 6100 M
2 M H2SO4. The change in A450 was recorded on a Bio-Rad 3550 microplate reader.
3. Results 3.1. Modification of AK by IAEDANS Native AK was modified according to the method described. The degree of labeling was found to be 1 mol of IAEDANS per mol of native AK. The activity of the modified AK was only 10% of native AK. The changes in fluorescence intensity and activity of this modified AK were measured during the modification and the results are shown in Fig. 1. An increase in the fluorescence signal accompanied by a decrease in enzyme activity took place during the modification, and both changed sharply within 15 min, after which there was little further change. This indicates that accompanying the SH group modification the activity of modified enzyme was greatly diminished. Modification was also carried out after denaturing native AK with 4 M GuHCl before addition of IAEDANS. In this case the labeling ratio was 2:1, and the activity was identical to that of the denatured enzyme, indicating that both cysteine residues can be labeled when the enzyme is unfolded. These results are very similar to those obtained previously in labeling the sulfhydryl groups of porcine muscle AK with fluorescent reagent NBD-chloride [13]. Cys-25 reacted with the reagent approximately 40-fold faster than Cys-187, and modification of Cys-25 alone led to inactivation of the enzyme. From X-ray analysis, it is known that the Cys-25 of porcine muscle is close to the presumed catalytic center [14]. The structure of rabbit muscle AK is expected to be identical to
A Hitachi-4500 fluorimeter was used for fluorescence measurements. Unless otherwise specified, an excitation wavelength of 336 nm was used, and the emission intensities were recorded at 490 nm at 25 °C, with both excitation and emission slits set at 5 nm. The affinity of AK binding with monoclonal antibody was measured by ELISA. Native AK (5 µg/ml, 100 µl) and modified AK (5 µg/ml, 100 µl) were first coated onto the microplates for 12 h at 4 °C. The remaining non-specific binding sites on the wells were blocked with 5% gelatin, then rinsed three times with PBST. One hundred microliters of McAb3D3 of different concentrations were then added to the wells in triplicate, and incubated for 1 h. After three rinses, 100 µl of ExtrAvidin Peroxidase (1:3000 dilution) were added and the mixture incubated for 1 h. After three rinses with PBST, 100 µl of TMB/H2O2 solution were added and the reaction stopped after 10 min with 100 µl of
Fig. 1. The changes of fluorescence intensity (squares) and enzyme activity (circles) of modified AK at various labeling times. The enzyme concentration was 1.0 µM in 0.1 M Tris-HCl pH 8.0, containing 1 mM EDTA. The fluorescence was measured at 336 nm excitation and at 490 nm emission at 25 °C.
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that of porcine muscle AK, as they have very similar amino acid sequences. It is therefore concluded that in the present work, the single labeled cysteine residue in native AK is Cys-25 and is located at the active site. This modified AK with labeling ratio 1:1 was used for further investigation. 3.2. The changes of fluorescence intensity of modified AK in GuHCl and urea The fluorescence intensity of modified AK in the presence of different concentrations of GuHCl and urea was determined. With increasing concentrations of GuHCl or urea, the fluorescence intensity of modified AK first increased and then gradually decreased (Fig. 2a,b). The fluorescence intensity of modified AK was enhanced about 1.8-fold in 0.2 M GuHCl, nearly 1.4-fold in 0.6 M urea, and no further fluorescence changes were observed above 1 M
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GuHCl or 2 M urea. These fluorescent changes closely resemble the pattern of activity changes during chemical denaturation of the unmodified enzyme [5]. Previous studies from this laboratory reported that the activity of the enzyme undergoes significant changes on addition of denaturant, showing a maximum increase of about 1.6-fold in 1 M urea or 0.25 M GuHCl, and then decreasing again at higher concentrations of denaturant. In contrast, the secondary and tertiary structures of AK were not noticeably perturbed at these concentrations of denaturant, as demonstrated by CD and UV measurements [15]. The results of this study demonstrate that addition of low concentrations of denaturant results in conformational changes at the active site of this enzyme. Taken together with the results of previous studies, it is clear that the conformational changes detected by the introduction of a fluorescent probe at the active site reflect the changes in activity that are observed on denaturation of the unmodified enzyme. It is clear that during denaturation of AK, conformational change at the active site occurs before unfolding of the rest of the molecule. This result supports the hypothesis proposed by Tsou, that the conformation at the active site of the enzyme is more flexible than the molecule as a whole [16–18]. 3.3. The binding of native AK and modified AK with McAb3D3 The ability of native AK and modified AK to bind to McAb3D3 was tested by ELISA as described in Section 2. It was apparent that the 1/A450 nm of native AK and modified AK are both proportional to the reciprocal of the antibody concentration, as shown in Fig. 3. The slopes of these two straight lines were the same, which shows that the binding of modified AK with McAb3D3 is identical to that of native AK. It can therefore be concluded that this chemical
Fig. 2. The changes of fluorescence intensity of modified AK in different concentrations of GuHCl (A) and urea (B). The enzyme concentration was 1.0 µM. The enzyme was incubated with GuHCl at 4 °C for 12 h before determination. Other details are as in the legend to Fig. 1. The changes were relative to the fluorescence intensity of modified AK in the absence of denaturant.
Fig. 3. Calibration curves of the binding of McAb3D3 to coated native AK (▲) and modified AK (▼) in the ELISA. The concentration range for McAb3D3 was 1.55 × 10–6 – 3.92 × 10–4 mg/ml. The coated amount of the two enzymes was 0.5 µg.
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modification does not interfere with the interaction between AK and McAb3D3. 3.4. The changes of fluorescence intensity during the interaction of modified AK with McAb3D3 The emission spectra of modified AK in the presence and absence of McAb3D3 when excited at 280 nm are shown in Fig. 4a. The mixture of modified AK and McAb3D3 (2:3 molar ratio) gave rise to a decrease in the emission intensity at 490 nm. No decrease was observed when BSA was used instead of McAb3D3 (Fig. 4b). This demonstrates that the decrease of the fluorescence intensity is due to the interaction of AK with McAb3D3. This specific signal was used to titrate the McAb3D3 preparation, and the result is shown in Fig. 5. From the curve obtained it can be calculated that
Fig. 5. Fluorescence titration of modified AK with McAb3D3. The enzyme concentration was 1.0 µM. Details are as in the legend of Fig. 1.
0.5 mol of McAb3D3 is saturated by 1 mol of modified AK, which corresponds to a titer of two binding sites for AK per molecule of McAb3D3. This fluorescent probe was also used to monitor the binding course of AK with McAb3D3, but the reaction was found to be complete within the dead time of manual mixing (12 s). 3.4. The interactions of modified AK with McAb3D3 in GuHCl monitored by the changes in fluorescence intensity
Fig. 4. Fluorescence quenching of modified AK in the presence of the monoclonal antibody McAb3D3 (A) or BSA control (B). In each case, the fluorescence emission spectra of McAb3D3 or BSA (curve 1), modified AK (curve 2), and modified AK mixed with McAb3D3 or BSA in a molar ratio of 2:3 (curve 3) is shown. The samples were in 0.1 M Tris-HCl pH 8.0, with 1 mM EDTA, and were excited at 280 nm.
The fluorescence intensities of modified AK with and without McAb3D3 were measured during the denaturation by GuHCl and the relative changes are shown in Fig. 6. The fluorescence quenching due to binding of McAb3D3 to modified AK varied with GuHCl concentration. The relative change in fluorescence of modified AK increased with increasing concentration of GuHCl and reached a maximum at 0.2 M GuHCl. This shows that partial unfolding of the antigen causes an increase in the antigen-antibody interaction. However, with further increase in the GuHCl concentration, the relative fluorescence decreased again to a level lower than non-denatured modified AK, indicating that the binding ratio of modified AK and McAb3D3 in higher concentrations of GuHCl is very low. From these results, it can be seen that binding of modified AK and the antibody varies greatly with GuHCl concentration, and is at a maximum at low concentration of GuHCl. This suggests that McAb3D3 preferentially binds to a more flexible or partially denatured conformation of AK. This is consistent with the finding that the affinity constant of McAb3D3 with coated AK is greater than that with free AK [8], which can be explained by partial denaturation of the protein when absorbed on the microplates.
T.H. Zhang et al. / Biochimie 84 (2002) 335–339
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Acknowledgements The present study was supported in part by Chinese Ministry of Science and Technology Grant G1999075608. The authors would like to thank Dr. S. Perrett of this laboratory for her critical reading of this paper and helpful suggestions.
References [1] [2]
[3] Fig. 6. The relative binding ability of modified AK mixed with McAb3D3 at various GuHCl concentrations. The enzyme concentration was 1.0 µM and the McAb3D3 concentration was 0.5 µM. Details are as in the legend of Fig. 2. The changes were related to the fluorescence intensity of modified enzyme in the absence of GuHCl.
[4]
[5]
4. Discussion [6]
The results presented in this study show that a fluorescent probe can be introduced at the active site of rabbit muscle AK using IAEDANS. Only Cys-25 was labeled when native AK was used, and modification led to enzyme activity greatly diminished. The changes of fluorescence intensity of modified AK mirrored the previously reported activity changes of native AK during denaturation [5]. This demonstrates that introduction of the fluorescent probe allows detection of conformational changes that occur at the active site of AK early in the process of denaturation. These conformational changes at the active site occur prior to global denaturation of the enzyme which is detected by changes in the intrinsic spectroscopic properties of the enzyme [15]. The modified AK is also shown to have the same affinity as native AK for the monoclonal antibody McAb3D3. Interaction of modified AK and McAb3D3 resulted in quenching of the fluorescence, and the relative changes varied with denaturant concentration. This observation shows that binding of AK and McAb3D3 may be conformation-dependent and that partially denatured AK is preferentially recognized by the antibody. We have demonstrated that introduction of the IAEDANS fluorescent probe at the active site of AK provides a useful method to monitor both conformational changes at the enzyme active site and interaction of AK with its monoclonal antibody during denaturation. The combination of this active site modification with the use of a conformation specific monoclonal antibody has potential for use in the study of the kinetics of folding of AK and particularly in the detection of folding intermediates.
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
[15]
[16] [17] [18]
L. Nado, Enzyme, in: P.D. Boyer (Ed.), 3rd ed., vol. 8, 1973, pp. 279–305. S.A. Kuby, R.H. Palmieri, A. Frischat, Studies on adenosine triphosphate transphorylases, amino acid sequence of rabbit muscle ATP-AMP transphosphorylase, Biochemistry 23 (1984) 2393–2399. M. Hamada, R.H. Palmieri, G.A. Russell, Studies on adenosine triphosphate transphosphorylases XIV, equilibrium binding properties of the crystalline rabbit and calf muscle ATP-AMP transphosphorylase (adenylate kinase) and derived peptide fragments, Arch. Biochem. Biophys. 195 (1979) 155–177. Y.L. Zhang, J.M. Zhou, C.L. Tsou, Inactivation precedes conformation change during thermal denaturation of adenylate kinase, Biochim. Biophys. Acta 1164 (1993) 61–67. Y.L. Zhang, J.M. Zhou, C.L. Tsou, Sequential unfolding of adenylate kinase during denaturation by guanidine hydrochloride, Biochim. Biophys. Acta 1295 (1996) 239–244. H.J. Zhang, X.R. Sheng, X.M. Pan, J.M. Zhou, Refolding of urea-denaturaed adenylate kinase, Biochem. J. 333 (1998) 401–405. H.J. Zhang, X.M. Pan, J.M. Zhou, H. Kihara, Activation and conformational changes of adenylate kinase in urea solution, Sci. China, Ser. C 41 (1998) 245–250. X.D. Wang, J.M. Zhou, Z.Q. Guo, Preparation and characterization of monoclonal antibodies against adenylate kinase, Sci. China, Ser. C 40 (6) (1997) 561–567. E. Katchalski-Katzir, D. Kenett, Use of monoclonal antibodies in the study of conformation and conformational alterations in proteins, Bull. Chem. Soc. Jpn. 61 (1988) 133–139. M.E. Goldberg, Investigating protein conformation, dynamics and folding with monoclonal antibodies, TIBS 16 (1991) 358–362. J.A. Gardner, K.S. Matthews, Energy transfer in lactose repressor protein modified with N-{[(iodoacetyl)amino]ethyl}– 5–naphthylamine–1-sulfonate, Biochemistry 30 (1991) 2707–2712. E.N. Hudson, G. Weber, Synthesis and characterization of two fluorescent sulfhydryl reagents, Biochemistry 12 (1973) 4154–4161. N.C. Price, M. Cohn, Fluorescent and spin label probes of the environments of the sulfhydryl groups of porcine muscle adenylate kinase, J. Biol. Chem. 250 (1975) 644–652. D. Dreusicke, P.A. Karplus, G.E. Schulz, Refined structure of porcine cytosolic adenylate kinase at 2.1 Å resolution, J. Mol. Biol. 199 (1988) 359–371. H.J. Zhang, X.R. Sheng, X.M. Pan, J.M. Zhou, Activation of adenylate kinase by denaturants is due to the increasing conformational flexibility at its active sites, Biochem. Biophys. Res. Comm. 238 (1997) 382–386. C.L. Tsou, Location of the active sites of some enzymes in limited and flexible molecular regions, TIBS 11 (1986) 427. C.L. Tsou, Conformational flexibility of enzyme active sites, Science 262 (1993) 380–381. C.L. Tsou, Inactivation precedes overall molecular conformation changes during enzyme denaturation, Biochim. Biophys. Acta 1253 (1995) 151–162.