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Methylphenylmercury: A novel heavy atom reagent for protein crystallography
J. Mol. Biol. (1983) 170, 249-252
Methylphenylmercury: A Novel Heavy Atom Reagent for Protein Crystallography Methylphenylmercury reacts with two normally inaccessible cysteine residues in crystals of carbonmonoxyhaemoglobin, but not with the third, normally reactive one. It may, therefore, be useful in the preparation of new heavy atom derivatives for protein crystallography.
Crystallographic studies of binding of possible anti-sickling agents to haemoglobin (Abraham & Phillips, 1981), led to the discovery that methylphenylmercury (Freidlina et al., 1935) may have potential as a heavy atom reagent for buried sulphydryl groups, because it is soluble in the non-polar interior of proteins. We started from the following argument. Toluene binds to human H b t (Farnell & McMeekin, 1973; Novak et al., 1979), and induces crystallization of oxy- and carbonmonoxy-Hb (Hb02 and HbCO) (Drabkin, 1946). The aromatic nature of many known inhibitors of gelation of sickle-cell Hb (HbS), such as phenylalanine (Noguchi & Schechter, 1977), suggested a connection between this activity and a binding site for aromatic rings. The location of toluene binding sites in Hb might, therefore, aid in the design of new anti-sickling agents. However, bound toluene was not observed in the crystal structure of HbCO at 2-7 A resolution (Baldwin, 1980); so the covalent mercury compound MPM, which is equivalent to a toluene molecule with a mercury atom inserted between the ring and methyl group, was synthesized, not as a potential anti-sickling agent (it would be far too toxic), but merely as a heavy atom marker for what we had hoped would be the site of the aromatic ring. However, the outcome of our experiment was quite different and unexpected. Methylphenylmercury was prepared according to Freidlina et al. (1935). Methylmercuryiodide (10.2 g, 0.03 tool, Alfa Chemicals) was boiled in ethanol (100ml), but did not dissolve. Phenylboric acid (3.6g, 0-03mol, Aldrich Chemicals) in boiling ethanol (60 ml) was added, followed by 20°/o sodium hydroxide solution (15 ml). Water (360 ml) was added and an oil formed on the bottom of the flask, with some grey solid material around it. The oil was extracted with two portions (150 ml each) of diethyl ether, washed once with 25~/o sodium hydroxide (50 ml), and three times with water. The ether solution was dried over anhydrous calcium chloride, filtered, and the product obtained as a viscous liquid after rotary evaporation (yield 7-37 g, 84~o). The chemical constitution of this liquid was checked by nuclear magnetic resonance and by analysis of its decomposition products. Nuclear magnetic resonance of freshly prepared MPM gave a broad aromatic singlet at 5 7.3 and a t Abbreviations used: Hb, haemoglobin; MPM, methylphenylmercury. 0022-2836/83/290249-04 $03.00/0
249
© 1983 Academic Press Inc. (London) Ltd.
250
D. J. ABRAHAM E T AL.
methyl singlet at ~ 0-55 with a 2/1 ratio close to the expected proton ratio of 5/3 aromatic/methyl. Besides these peaks there was a slight shoulder at 7.35 on the aromatic singlet at 5 7-3 indicating the presence of Ph2Hg and two small singlets at 5 1.5 and 5 l-1. The reported analysis in the literature (Freidlina et al., 1935) for Hg gave: 68-52~/o calculated, 67"65~o found. This is not within the accepted value of 0.4% but expected, considering that the material was isolated without further purification, because this would have led to further decomposition of MPM. Koton & Bolshakova (1953) reported that MPM is rather unstable and best identified after conversion to MeHgC1. These authors also reacted MPM with p-chlorophenol to give 3-methylmercury-4-chlorophenyl with a melting point 116 to ll8°C after recrystallization from benzene. We have repeated this with the same results, 120 to 121°C, after recrystallization from ether. Analytical analysis of our compound confirms its composition. Calculated for C~H~CIHgO: C, 24.50; H, 2.06; Hg, 58-45; Cl, 10.32. Found: C, 24.46; H, 2-10; Hg, 58"83; Cl, 10.05. Rausch & van Wazer (1964) have studied the stability of MPM by nuclear magnetic resonance and found that at 100°C it decomposes to form Me2Hg and Phe2Hg. We have discovered that upon prolonged exposure to light decomposition took place to give a white solid which we suspected to be diphenylmercury (PhEHg). This was isolated by preparative thin layer chromatography using hexane as the developing solvent to give a white solid of melting point 110 to 111°C, which, after reerystallization from ether, gave a melting point of 123 to 125°C; (lit. 124 to 125°C). The other physical properties of this material, i.e. nuclear magnetic resonance, R F value, were also identical to that of an authentic sample of Ph2Hg. When freshly prepared MPM was stored under nitrogen in the dark, decomposition to Phe2Hg was observed only after several weeks. Crystallization of HbCO was carried out as described by Perutz (1968) in 2.05, 2.15 and 2.25 M-phosphate buffer. Two drops of MPM were added to each tube, but addition of toluene was still required for crystal formation. Crystals grew over a period of a few weeks with two apparently different morphologies, only one type resembling native HbCO. The latter form is isomorphous with native HbCO, but shows intensity changes in the X-ray diffraction pattern. Intensities were measured for one such crystal on a Nonius CAD4 diffractometer to 6 A resolution over a period of about 20 hours. Radiation damage was severe compared to the stable native crystals, with a 20~/o reduction in intensity during the course of data collection. A difference electron density map was calculated with coefficients (IFdl-IFnl) and phases c~¢, where F d and Fn are observed structure amplitudes for derivative and native crystals, and ac the calculated phases from the refined HbCO model (Baldwin, 1980). Sulphydryl groups were titrated with parachloromercuribenzoate (Sigma) using the method of Benesch & Benesch (1956). Human haemoglobin contains three pairs of cysteines, which lie at positions G l l (104)a, F9 (93)fl and G14 (ll2)fl. When mercurials are added to a solution of haemoglobin at or near neutral pH, CysF9fl always reacts, but neither of the other cysteines do because they are buried in the alfl 1 contact, which has a very low dissociation constant. Only after CysF9fl had been blocked with acetamide
LETTERS Hh4:O-Cy~elne
gltes
-
L
TO T H E E D I T O R
29-6-82
251
HbCO-Cystelne
sites
-
29-6-82
R
all~a-,-, •v - I C"
r-
"
.g
J,4-.
FIG. I. Stereo diagram of the HbCO tetramer viewed down the molecular dyad. Ca positions are shown joined by solid lines for fl, and broken lines for a subunits. Hg sites SI (Cys 104a) and $2 (Cys ll2fi) are shown as filled circles, and the exposed, but unmodified sites $3 (Cys93fi) as open circles.
did CysG14fl become reactive with HgC12 and both CysGlla and G14fl with CH3Hg + (Cullis et al., 1961; Muirhead et al., 1967). Our difference Fourier map shows spherical peaks, with heights, 0.09 and 0.08 e/A 3, close to the sulphur atoms of the two buried cysteines G1 la and G14fl. The standard deviation of the density is 0.01 e/A 3, as estimated from a known solvent area in the map. A centric refinement of the two observed mercury atoms, with temperature factors Biso set at 30 A 2, against the isomorphous differences, led to refined occupancies of 1.1 and 1-0_0.1, respectively, and an R factor of 0.55. We have found no density greater than 0-01 e/A 3 near the normally reactive cysteine F9fl. Both results are surprising. It is remarkable that MPM can wedge into the alfl 1 contact without apparently disrupting it, despite the many close van der Waals' interactions and hydrogen bonds between the two subunits, and that it should not have reacted with the free sulphydryl groups. Could reaction of CysF9fl with MPM have been obscured in the difference Fourier map by rotation about the Ca-Cfl and Cfl-S bonds? It is true that the sulphydryl group can oscillate between two alternative positions, an external and an internal one, but in all mercurial derivatives previously examined it lay in the external position because there is no room for a sulphydryl substituent in the internal one. Besides, even if the mercury atom were distributed over two alternative positions, the sensitivity of difference Fourier maps is such that its 80 electrons could hardly fail to produce density greater than 0.01 e/A 3 near CysF9fl. As a further check we titrated the free sulphydryl group 93fl in a solution of HbCO crystals that had been grown in the presence of MPM. In two experiments titration of native HbCO in 0.15 M-phosphate of pH 7.0 with parachloromercuribenzoate yielded an average of 2.37 sulphydryl groups per tetramer, in agreement with values found by other investigators. Crystals of HbCO grown in the presence of MPM and dissolved in 0-15 M-phosphate buffer of pH 7.0 yielded an average of
252
D. J. ABRAHAM ET AL.
2"38 sulphydryl groups per tetramer, the same as native HbCO, and consistent with the X - r a y evidence, which shows t h a t cysteines 93fl had not reacted with MPM. The mechanism of reaction of MPM with the buried sulphydryl groups is not known. I t might be asked whether the Phe2Hg t h a t forms on decomposition of MPM is the reactive species, but this is unlikely because Phe2Hg is much more stable than MPM. The latter molecule m a y bury itself in the hydrophobic interior of the protein, and then undergo a slow reaction with neighbouring sulphydryls. Its hydrophobicity and instability m a y render it an effective h e a v y atom reagent for normally unreactive sulphydryl groups in crystalline proteins. MPM should be handled with great care, because of its decomposition to volatile Me2Hg, its hydrophobic nature and the possible ability of both compounds to cross biological membranes. Experiments with it should be carried out with gloves in a fume hood. I t should be stored in the dark, and used material disposed of with caution. We thank Dr M. F. Perutz for continuous encouragement and helpful suggestions, Dr B. S. Shaanan for advice on crystallization of HbCO, Ms F. L. Williams for technical assistance, the H. J. Heinz Company, Thomas C. Usher Foundation, and National Institutes of Health grants no. NOI-HB-I-3001 (to D.J.A.) and NO1-AM-2-2205 (to S.E.V.P.) for financial support. 1Department of Medicinal Chemistry University of Pittsburgh Pittsburgh, PA 15261, U.S.A.
DONALD J. ABRAHAMI SIMON E. V. PHILLIPS2~" PAUL E. KENNEDY1
2Laboratory of Molecular Biology Medical Research Council Centre Cambridge CB2 2QH, England Received 9 July 1982 REFERENCES Abraham, D. J. & Phillips, S. E. V. (1981). International Symposium on Abnormal Hemoglobins: Genetics, Populations and Diseases, Jerusalem, Israel, Sept. 1981: Elsevier North-Holland, Amsterdam, in the press. Baldwin, J. M. (1980). J. Mol. Biol. 136, 103-128. Benesch, R. & Benesch, R. E. (1956). Methods Biochem. Anal. 10, 56-70. Cullis, A. F., Muirhead, H., Perutz, M. F. & Rossman, M. G. (1961). Proc. Roy. Soc. set. A, 265, 15-38. Drabkin, D. L. (1946). J. Biol. Chem. 164, 703-710. Farnell, K. J. & McMeekin, T. L. (1973). Arch. Biochem. Biophys. 158, 702-710. Freidlina, R. Ch., Nesmehanow, A. N. & Kozeschkow, K. A. (1935). Ber. Deut. Chem. Ges. 68, 565-569. Koton, M. M. & Bolshakova, A. A. (1953). Zhur. Obshchei Khim. 23, 2023-2027. l~uirhead, H., Cox, J. M., Mazzarella, L. & Perutz, M. F. (1967). J. Mol. Biol. 28, 117-156. Noguchi, C. T. & Scheehter, A. N. (I977). Biochem. Biophys. Res. Commun. 74, 637-642. Novak, R. F., Dershwitz, M. & Novak, F. C. (1979). Mol. Pharmacol. 16, 1046-1058. Perutz, M. F. (1968). J. Crystal Growth, 2, 54-56. Rausch, M. D. & Van Wazer, J. R. (1964). J. Inorg. Chem. 5, 761-766. Edited by R. Huber
Present address: D6partement d'Immunologie, In~titut Pasteur, 28 Rue du Dr. Roux, 75724 Paris, CEDEX 15, France.
Methylphenylmercury: A Novel Heavy Atom Reagent for Protein Crystallography Methylphenylmercury reacts with two normally inaccessible cysteine residues in crystals of carbonmonoxyhaemoglobin, but not with the third, normally reactive one. It may, therefore, be useful in the preparation of new heavy atom derivatives for protein crystallography.
Crystallographic studies of binding of possible anti-sickling agents to haemoglobin (Abraham & Phillips, 1981), led to the discovery that methylphenylmercury (Freidlina et al., 1935) may have potential as a heavy atom reagent for buried sulphydryl groups, because it is soluble in the non-polar interior of proteins. We started from the following argument. Toluene binds to human H b t (Farnell & McMeekin, 1973; Novak et al., 1979), and induces crystallization of oxy- and carbonmonoxy-Hb (Hb02 and HbCO) (Drabkin, 1946). The aromatic nature of many known inhibitors of gelation of sickle-cell Hb (HbS), such as phenylalanine (Noguchi & Schechter, 1977), suggested a connection between this activity and a binding site for aromatic rings. The location of toluene binding sites in Hb might, therefore, aid in the design of new anti-sickling agents. However, bound toluene was not observed in the crystal structure of HbCO at 2-7 A resolution (Baldwin, 1980); so the covalent mercury compound MPM, which is equivalent to a toluene molecule with a mercury atom inserted between the ring and methyl group, was synthesized, not as a potential anti-sickling agent (it would be far too toxic), but merely as a heavy atom marker for what we had hoped would be the site of the aromatic ring. However, the outcome of our experiment was quite different and unexpected. Methylphenylmercury was prepared according to Freidlina et al. (1935). Methylmercuryiodide (10.2 g, 0.03 tool, Alfa Chemicals) was boiled in ethanol (100ml), but did not dissolve. Phenylboric acid (3.6g, 0-03mol, Aldrich Chemicals) in boiling ethanol (60 ml) was added, followed by 20°/o sodium hydroxide solution (15 ml). Water (360 ml) was added and an oil formed on the bottom of the flask, with some grey solid material around it. The oil was extracted with two portions (150 ml each) of diethyl ether, washed once with 25~/o sodium hydroxide (50 ml), and three times with water. The ether solution was dried over anhydrous calcium chloride, filtered, and the product obtained as a viscous liquid after rotary evaporation (yield 7-37 g, 84~o). The chemical constitution of this liquid was checked by nuclear magnetic resonance and by analysis of its decomposition products. Nuclear magnetic resonance of freshly prepared MPM gave a broad aromatic singlet at 5 7.3 and a t Abbreviations used: Hb, haemoglobin; MPM, methylphenylmercury. 0022-2836/83/290249-04 $03.00/0
249
© 1983 Academic Press Inc. (London) Ltd.
250
D. J. ABRAHAM E T AL.
methyl singlet at ~ 0-55 with a 2/1 ratio close to the expected proton ratio of 5/3 aromatic/methyl. Besides these peaks there was a slight shoulder at 7.35 on the aromatic singlet at 5 7-3 indicating the presence of Ph2Hg and two small singlets at 5 1.5 and 5 l-1. The reported analysis in the literature (Freidlina et al., 1935) for Hg gave: 68-52~/o calculated, 67"65~o found. This is not within the accepted value of 0.4% but expected, considering that the material was isolated without further purification, because this would have led to further decomposition of MPM. Koton & Bolshakova (1953) reported that MPM is rather unstable and best identified after conversion to MeHgC1. These authors also reacted MPM with p-chlorophenol to give 3-methylmercury-4-chlorophenyl with a melting point 116 to ll8°C after recrystallization from benzene. We have repeated this with the same results, 120 to 121°C, after recrystallization from ether. Analytical analysis of our compound confirms its composition. Calculated for C~H~CIHgO: C, 24.50; H, 2.06; Hg, 58-45; Cl, 10.32. Found: C, 24.46; H, 2-10; Hg, 58"83; Cl, 10.05. Rausch & van Wazer (1964) have studied the stability of MPM by nuclear magnetic resonance and found that at 100°C it decomposes to form Me2Hg and Phe2Hg. We have discovered that upon prolonged exposure to light decomposition took place to give a white solid which we suspected to be diphenylmercury (PhEHg). This was isolated by preparative thin layer chromatography using hexane as the developing solvent to give a white solid of melting point 110 to 111°C, which, after reerystallization from ether, gave a melting point of 123 to 125°C; (lit. 124 to 125°C). The other physical properties of this material, i.e. nuclear magnetic resonance, R F value, were also identical to that of an authentic sample of Ph2Hg. When freshly prepared MPM was stored under nitrogen in the dark, decomposition to Phe2Hg was observed only after several weeks. Crystallization of HbCO was carried out as described by Perutz (1968) in 2.05, 2.15 and 2.25 M-phosphate buffer. Two drops of MPM were added to each tube, but addition of toluene was still required for crystal formation. Crystals grew over a period of a few weeks with two apparently different morphologies, only one type resembling native HbCO. The latter form is isomorphous with native HbCO, but shows intensity changes in the X-ray diffraction pattern. Intensities were measured for one such crystal on a Nonius CAD4 diffractometer to 6 A resolution over a period of about 20 hours. Radiation damage was severe compared to the stable native crystals, with a 20~/o reduction in intensity during the course of data collection. A difference electron density map was calculated with coefficients (IFdl-IFnl) and phases c~¢, where F d and Fn are observed structure amplitudes for derivative and native crystals, and ac the calculated phases from the refined HbCO model (Baldwin, 1980). Sulphydryl groups were titrated with parachloromercuribenzoate (Sigma) using the method of Benesch & Benesch (1956). Human haemoglobin contains three pairs of cysteines, which lie at positions G l l (104)a, F9 (93)fl and G14 (ll2)fl. When mercurials are added to a solution of haemoglobin at or near neutral pH, CysF9fl always reacts, but neither of the other cysteines do because they are buried in the alfl 1 contact, which has a very low dissociation constant. Only after CysF9fl had been blocked with acetamide
LETTERS Hh4:O-Cy~elne
gltes
-
L
TO T H E E D I T O R
29-6-82
251
HbCO-Cystelne
sites
-
29-6-82
R
all~a-,-, •v - I C"
r-
"
.g
J,4-.
FIG. I. Stereo diagram of the HbCO tetramer viewed down the molecular dyad. Ca positions are shown joined by solid lines for fl, and broken lines for a subunits. Hg sites SI (Cys 104a) and $2 (Cys ll2fi) are shown as filled circles, and the exposed, but unmodified sites $3 (Cys93fi) as open circles.
did CysG14fl become reactive with HgC12 and both CysGlla and G14fl with CH3Hg + (Cullis et al., 1961; Muirhead et al., 1967). Our difference Fourier map shows spherical peaks, with heights, 0.09 and 0.08 e/A 3, close to the sulphur atoms of the two buried cysteines G1 la and G14fl. The standard deviation of the density is 0.01 e/A 3, as estimated from a known solvent area in the map. A centric refinement of the two observed mercury atoms, with temperature factors Biso set at 30 A 2, against the isomorphous differences, led to refined occupancies of 1.1 and 1-0_0.1, respectively, and an R factor of 0.55. We have found no density greater than 0-01 e/A 3 near the normally reactive cysteine F9fl. Both results are surprising. It is remarkable that MPM can wedge into the alfl 1 contact without apparently disrupting it, despite the many close van der Waals' interactions and hydrogen bonds between the two subunits, and that it should not have reacted with the free sulphydryl groups. Could reaction of CysF9fl with MPM have been obscured in the difference Fourier map by rotation about the Ca-Cfl and Cfl-S bonds? It is true that the sulphydryl group can oscillate between two alternative positions, an external and an internal one, but in all mercurial derivatives previously examined it lay in the external position because there is no room for a sulphydryl substituent in the internal one. Besides, even if the mercury atom were distributed over two alternative positions, the sensitivity of difference Fourier maps is such that its 80 electrons could hardly fail to produce density greater than 0.01 e/A 3 near CysF9fl. As a further check we titrated the free sulphydryl group 93fl in a solution of HbCO crystals that had been grown in the presence of MPM. In two experiments titration of native HbCO in 0.15 M-phosphate of pH 7.0 with parachloromercuribenzoate yielded an average of 2.37 sulphydryl groups per tetramer, in agreement with values found by other investigators. Crystals of HbCO grown in the presence of MPM and dissolved in 0-15 M-phosphate buffer of pH 7.0 yielded an average of
252
D. J. ABRAHAM ET AL.
2"38 sulphydryl groups per tetramer, the same as native HbCO, and consistent with the X - r a y evidence, which shows t h a t cysteines 93fl had not reacted with MPM. The mechanism of reaction of MPM with the buried sulphydryl groups is not known. I t might be asked whether the Phe2Hg t h a t forms on decomposition of MPM is the reactive species, but this is unlikely because Phe2Hg is much more stable than MPM. The latter molecule m a y bury itself in the hydrophobic interior of the protein, and then undergo a slow reaction with neighbouring sulphydryls. Its hydrophobicity and instability m a y render it an effective h e a v y atom reagent for normally unreactive sulphydryl groups in crystalline proteins. MPM should be handled with great care, because of its decomposition to volatile Me2Hg, its hydrophobic nature and the possible ability of both compounds to cross biological membranes. Experiments with it should be carried out with gloves in a fume hood. I t should be stored in the dark, and used material disposed of with caution. We thank Dr M. F. Perutz for continuous encouragement and helpful suggestions, Dr B. S. Shaanan for advice on crystallization of HbCO, Ms F. L. Williams for technical assistance, the H. J. Heinz Company, Thomas C. Usher Foundation, and National Institutes of Health grants no. NOI-HB-I-3001 (to D.J.A.) and NO1-AM-2-2205 (to S.E.V.P.) for financial support. 1Department of Medicinal Chemistry University of Pittsburgh Pittsburgh, PA 15261, U.S.A.
DONALD J. ABRAHAMI SIMON E. V. PHILLIPS2~" PAUL E. KENNEDY1
2Laboratory of Molecular Biology Medical Research Council Centre Cambridge CB2 2QH, England Received 9 July 1982 REFERENCES Abraham, D. J. & Phillips, S. E. V. (1981). International Symposium on Abnormal Hemoglobins: Genetics, Populations and Diseases, Jerusalem, Israel, Sept. 1981: Elsevier North-Holland, Amsterdam, in the press. Baldwin, J. M. (1980). J. Mol. Biol. 136, 103-128. Benesch, R. & Benesch, R. E. (1956). Methods Biochem. Anal. 10, 56-70. Cullis, A. F., Muirhead, H., Perutz, M. F. & Rossman, M. G. (1961). Proc. Roy. Soc. set. A, 265, 15-38. Drabkin, D. L. (1946). J. Biol. Chem. 164, 703-710. Farnell, K. J. & McMeekin, T. L. (1973). Arch. Biochem. Biophys. 158, 702-710. Freidlina, R. Ch., Nesmehanow, A. N. & Kozeschkow, K. A. (1935). Ber. Deut. Chem. Ges. 68, 565-569. Koton, M. M. & Bolshakova, A. A. (1953). Zhur. Obshchei Khim. 23, 2023-2027. l~uirhead, H., Cox, J. M., Mazzarella, L. & Perutz, M. F. (1967). J. Mol. Biol. 28, 117-156. Noguchi, C. T. & Scheehter, A. N. (I977). Biochem. Biophys. Res. Commun. 74, 637-642. Novak, R. F., Dershwitz, M. & Novak, F. C. (1979). Mol. Pharmacol. 16, 1046-1058. Perutz, M. F. (1968). J. Crystal Growth, 2, 54-56. Rausch, M. D. & Van Wazer, J. R. (1964). J. Inorg. Chem. 5, 761-766. Edited by R. Huber
Present address: D6partement d'Immunologie, In~titut Pasteur, 28 Rue du Dr. Roux, 75724 Paris, CEDEX 15, France.