- Email: [email protected]
Comparison of DMSO-induced denaturation of hen egg-white lysozyme and bovine α-lactalbumin
PCS 1755
Journal of Physics and Chemistry of Solids 60 (1999) 1379–1381
Comparison of DMSO-induced denaturation of hen egg-white lysozyme and bovine a-lactalbumin H. Iwase a, M. Hirai a,*, S. Arai a, S. Mitsuya a, S. Shimizu b, T. Otomo c, M. Furusaka c a
Department of Physics, Gunma University, 4-2 Aramaki, Maebashi 371-8510, Japan College of Science and Technology, Nihon University, Chiyodaku 101-8308, Japan c High Energy Accelerator Research Organization, Tsukuba 305-0801, Japan
b
Abstract By using both wide-angle neutron and small-angle X-ray scattering techniques, we have studied the denaturation processes of hen egg-white lysozyme (HEWL) and bovine a-lactalbumin (BLA) in dimethyl sulfoxide (DMSO)/water mixtures. Although these proteins are known to be highly homologous, the present results indicate that the structure of BLA is liable to be destroyed by the addition of DMSO in comparison with the case of HEWL. DMSO-induced denaturation is suggested to relate to the collapse of hydration shell surrounding the protein surface. q 1999 Elsevier Science Ltd. All rights reserved. Keywords: A. Organic compounds; A. Polymers; A. Surfaces; C. Neutron scattering; D. Elastic properties
1. Introduction Under certain conditions of dimethyl sulfoxide (DMSO)/ water mixture solvents hen egg-white lysozyme (HEWL) was reported to take a partially-folded structure at equilibrium [1], which is regarded to be similar to that at an intermediate state in the previous refolding kinetic study [2]. However the structural stability and the partially-folded structure under the presence of DMSO have been still ambiguous. Small-angle X-ray scattering is a powerful technique for protein folding–unfolding study [3,4], however the observable q range in one measurement is rather limited owing to the use of monochromatized X-ray beam, where q is the magnitude of scattering vector defined by q 4p=lsinu (2u , the scattering angle; l , the wave length). Besides, it is difficult to discuss the X-ray scattering curve in the high-q region owing to the strong absorption of DMSO. On the other hand, pulsed-neutron scattering using time of flight method can cover a very wide q range and such an absorption effect by the presence of DMSO is negligibly small. Then, by using both wide-angle neutron and small-angle * Corresponding author. Tel.: 1 81-272-20-7554; fax: 1 81272-20-7551. E-mail address: [email protected] (M. Hirai)
X-ray scattering techniques, we have studied the structural properties of HEWL and bovine a-lactalbumin (BLA) in DMSO/water mixture, as the tertiary structures of these proteins are known to be highly homologous to each other.
2. Experimental HEWL and BLA were purchased from Sigma Chemical Co. The stock protein-solutions were prepared by dissolving the lyophilized powders of these proteins in 167 mM Hepes buffer at pH 7. The sample solutions used for the scattering experiments were obtained by the addition of DMSO (DMSO-d6 for the neutron scattering experiments) to the stock solutions. The final pH and the protein-concentration of the sample solutions were pH , 7 and 5% (w/v), respectively. The mixture of light and heavy water with 83.65% D2O was used for the neutron scattering to match out the scattering from DMSO-d6. Wide-angle neutron and small-angle X-ray scattering measurements were carried out by using the small-/ medium-angle neutron diffractometer (WINK) installed at the pulsed neutron source KENS and by using the smallangle X-ray scattering spectrometer (SAXES) installed at the Photon Factory at KEK, Tsukuba, Japan. WINK and SAXES ˚ 21 and 0.02–0.4 A ˚ 21 in can cover q ranges of 0.02–5.0 A
0022-3697/99/$ - see front matter q 1999 Elsevier Science Ltd. All rights reserved. PII: S0022-369 7(99)00123-7
1380
H. Iwase et al. / Journal of Physics and Chemistry of Solids 60 (1999) 1379–1381
Fig. 1. Variation of the X-ray scattering curves of (a) hen egg-white lysozyme, and (b) bovine a-lactalbumin, depending on DMSO concentration.
one measurement, respectively. All the scattering measurements were performed at room temperature.
3. Results and discussion Fig. 1 shows the X-ray scattering curves of the HEWL and BLA solutions depending on the DMSO concentration. In our experimental conditions, it was impossible to perform the scattering measurement of BLA above 50% (v/v) DMSO concentration, because the fluidity of the BLA solution was completely missing owing to the gelation of the solutions. In the case of HEWL, in Fig. 1(a), the broad peak around q , ˚ 21 at 0–60% (v/v) DMSO attributes to the repulsive 0.08 A interparticle interaction between the protein molecules. At 60–70% (v/v) DMSO this peak disappears and the scatter˚ 21 increases significantly, ing intensity below q , 0.05 A indicating the aggregation of the solute particles occurred with elevating the DMSO concentration. The HEWL solution at 70% (v/v) DMSO still maintains fluidity. On the other hand, the change of the scattering profile of BLA shows that such an aggregation starts from much lower DMSO concentration (20% (v/v)), namely the DMSO concentration of the start of aggregation of BLA remarkably differs from that of HEWL. Fig. 2 shows the neutron scattering curves of the HEWL and BLA solutions depending on the DMSO concentration.
Fig. 2. Variation of the neutron scattering curves of (a) hen eggwhite lysozyme, and (b) bovine a-lactalbumin, depending on DMSO concentration. The insets show the comparison of the scat˚ 21: (a) for hen egg-white lysozyme at tering profiles above q 0.1 A 0 and 70% (v/v) DMSO; (b) for bovine a-lactalbumin at 0 and 40% (v/v) DMSO.
The insets show the comparison of the neutron scattering ˚ 21, where (a) for HEWL at 0, 70% curves above q 0.1 A (v/v) DMSO; (b) for BLA at 0, 40% (v/v) DMSO. The changes of the neutron scattering curves of HEWL and BLA demonstrate similar changing tendencies observed by the X-ray scattering measurements in Fig. 1. As we described elsewhere [5], the scattering curves in the q ranges ˚ 21 (medium q range) and of 1–1.5 A ˚ 21 (high q of 0.3–0.6 A range) mostly reflect the domain-correlation and the polypeptide configuration within the protein molecule, respectively. As shown in the inset of Fig. 2(a), the evident difference of the scattering profile of HEWL is recognized in the medium q range, but not observed in the high q range. This suggests that HEWL is not completely denatured even at the highest DMSO concentration of 70% (v/v), whereas the scattering profile of BLA changes clearly in both q ranges. These results indicate that with the addition of DMSO the structure of BLA is liable to collapse in comparison with that of HEWL. Fig. 3(a) and (b) shows the apparent radius of gyration Rg
H. Iwase et al. / Journal of Physics and Chemistry of Solids 60 (1999) 1379–1381
Fig. 3. Dependence of the experimental structural parameters on DMSO concentration. The apparent radius of gyration Rg and the maximum diameter Dmax were estimated from the distance distribution functions obtained by the Fourier transform of the X-ray scattering curves in Fig. 1.
and the maximum diameter Dmax of both proteins as a function of DMSO concentration, respectively. The Rg and Dmax values were estimated from the distance distribution functions obtained by the Fourier transform of the X-ray scattering curves. The Rg and Dmax values of HEWL decrease slightly in the DMSO concentration of 0–50% (v/v). Those values of BLA also decrease slightly in 0–15% (v/v) DMSO concentration. On increasing the DMSO concentration further, the Rg and Dmax values of both the proteins start to increase gradually. Recently, Svergun et al. [6] developed a program named Crysol for calculating the solution scattering profile by taking account of the hydration shell surrounding the protein surface from the atomic coordinates data in the Brookhaven Protein Data Bank. They also showed that the hydration shell surrounding the protein surface has a higher density than that of the bulk water [7]. By using the Crysol program we calculated the X-ray
1381
scattering profile from the crystallographic structure of HEWL (PDB entry name of 6LYZ) with varying the scattering density of the hydration shell, which shows that the decrease of the scattering density of the hydration shell reduces the Rg value of the HEWL molecule to about ˚ . When we assume that the collapse of the hydration 1.1 A shell is induced by the addition of DMSO, the decrease of the Rg observed in the DMSO concentration of 0–50% (v/v) turns out to be comparable to the above simulated value. Previously, neutron diffraction and computer simulation studies of DMSO–water mixtures showed that water structure is broken down by the formation of strong hydrogen bonds between DMSO and water molecules [8,9], which strongly supports our present interpretation. The expansion of the protein structure, namely the collapse of the native tertiary structure, occurs above 50% (v/v) DMSO for HEWL and above 15% (v/v) DMSO for BLA. Although HEWL and BLA are well known to have homologous sequences and tertiary structures, we have found that DMSO-induced denaturation of both proteins are quite different, namely the structure of BLA is liable to be destroyed by adding DMSO in comparison with the case of HEWL. The slight decrease of the Rg values of the proteins occurs prior to the remarkable changes of the tertiary structures, suggesting that the collapse of the hydration shell of the protein proceeds at the initial stage to destabilize the tertiary structure. The difference in the structural stability between HEWL and BLA under the presence of DMSO indicates that the structure of BLA is destroyed more sensitively than that of HEWL by the collapse of the hydration shell.
References [1] A.A. Timchenko, M.D. Kirkitadze, D.A. Prokhorov, S.A. Potekhin, I.N. Serdyuk, Bioorg. Khim. 22 (1996) 420. [2] S.E. Radford, C.M. Dobson, P.A. Evans, Nature 358 (1992) 302. [3] E.E. Lattmann, Curr. Opn. Struct. Biol. 4 (1994) 87. [4] M. Hirai, S. Arai, H. Iwase, T. Takizawa, J. Phys. Chem. B 102 (1998) 1308. [5] M. Hirai, S. Arai, H. Iwase, T. Takizawa, S. Shimizu, M. Furusaka, Physica B 241–243 (1998) 1159. [6] D.I. Svergun, C. Barberato, M.J.H. Koch, J. Appl. Cryst. 28 (1995) 768. [7] D.I. Svergun, S. Richard, M.J.H. Koch, Z. Sayers, S. Kuprin, G. Zaccai, Proc. Natl. Acad. Sci. USA 95 (1998) 2267. [8] A.K. Soper, A. Luzar, J. Chem. Phys. 97 (1992) 1320. [9] A. Luzar, J. Chandler, J. Chem. Phys. 98 (1993) 8160.
Journal of Physics and Chemistry of Solids 60 (1999) 1379–1381
Comparison of DMSO-induced denaturation of hen egg-white lysozyme and bovine a-lactalbumin H. Iwase a, M. Hirai a,*, S. Arai a, S. Mitsuya a, S. Shimizu b, T. Otomo c, M. Furusaka c a
Department of Physics, Gunma University, 4-2 Aramaki, Maebashi 371-8510, Japan College of Science and Technology, Nihon University, Chiyodaku 101-8308, Japan c High Energy Accelerator Research Organization, Tsukuba 305-0801, Japan
b
Abstract By using both wide-angle neutron and small-angle X-ray scattering techniques, we have studied the denaturation processes of hen egg-white lysozyme (HEWL) and bovine a-lactalbumin (BLA) in dimethyl sulfoxide (DMSO)/water mixtures. Although these proteins are known to be highly homologous, the present results indicate that the structure of BLA is liable to be destroyed by the addition of DMSO in comparison with the case of HEWL. DMSO-induced denaturation is suggested to relate to the collapse of hydration shell surrounding the protein surface. q 1999 Elsevier Science Ltd. All rights reserved. Keywords: A. Organic compounds; A. Polymers; A. Surfaces; C. Neutron scattering; D. Elastic properties
1. Introduction Under certain conditions of dimethyl sulfoxide (DMSO)/ water mixture solvents hen egg-white lysozyme (HEWL) was reported to take a partially-folded structure at equilibrium [1], which is regarded to be similar to that at an intermediate state in the previous refolding kinetic study [2]. However the structural stability and the partially-folded structure under the presence of DMSO have been still ambiguous. Small-angle X-ray scattering is a powerful technique for protein folding–unfolding study [3,4], however the observable q range in one measurement is rather limited owing to the use of monochromatized X-ray beam, where q is the magnitude of scattering vector defined by q 4p=lsinu (2u , the scattering angle; l , the wave length). Besides, it is difficult to discuss the X-ray scattering curve in the high-q region owing to the strong absorption of DMSO. On the other hand, pulsed-neutron scattering using time of flight method can cover a very wide q range and such an absorption effect by the presence of DMSO is negligibly small. Then, by using both wide-angle neutron and small-angle * Corresponding author. Tel.: 1 81-272-20-7554; fax: 1 81272-20-7551. E-mail address: [email protected] (M. Hirai)
X-ray scattering techniques, we have studied the structural properties of HEWL and bovine a-lactalbumin (BLA) in DMSO/water mixture, as the tertiary structures of these proteins are known to be highly homologous to each other.
2. Experimental HEWL and BLA were purchased from Sigma Chemical Co. The stock protein-solutions were prepared by dissolving the lyophilized powders of these proteins in 167 mM Hepes buffer at pH 7. The sample solutions used for the scattering experiments were obtained by the addition of DMSO (DMSO-d6 for the neutron scattering experiments) to the stock solutions. The final pH and the protein-concentration of the sample solutions were pH , 7 and 5% (w/v), respectively. The mixture of light and heavy water with 83.65% D2O was used for the neutron scattering to match out the scattering from DMSO-d6. Wide-angle neutron and small-angle X-ray scattering measurements were carried out by using the small-/ medium-angle neutron diffractometer (WINK) installed at the pulsed neutron source KENS and by using the smallangle X-ray scattering spectrometer (SAXES) installed at the Photon Factory at KEK, Tsukuba, Japan. WINK and SAXES ˚ 21 and 0.02–0.4 A ˚ 21 in can cover q ranges of 0.02–5.0 A
0022-3697/99/$ - see front matter q 1999 Elsevier Science Ltd. All rights reserved. PII: S0022-369 7(99)00123-7
1380
H. Iwase et al. / Journal of Physics and Chemistry of Solids 60 (1999) 1379–1381
Fig. 1. Variation of the X-ray scattering curves of (a) hen egg-white lysozyme, and (b) bovine a-lactalbumin, depending on DMSO concentration.
one measurement, respectively. All the scattering measurements were performed at room temperature.
3. Results and discussion Fig. 1 shows the X-ray scattering curves of the HEWL and BLA solutions depending on the DMSO concentration. In our experimental conditions, it was impossible to perform the scattering measurement of BLA above 50% (v/v) DMSO concentration, because the fluidity of the BLA solution was completely missing owing to the gelation of the solutions. In the case of HEWL, in Fig. 1(a), the broad peak around q , ˚ 21 at 0–60% (v/v) DMSO attributes to the repulsive 0.08 A interparticle interaction between the protein molecules. At 60–70% (v/v) DMSO this peak disappears and the scatter˚ 21 increases significantly, ing intensity below q , 0.05 A indicating the aggregation of the solute particles occurred with elevating the DMSO concentration. The HEWL solution at 70% (v/v) DMSO still maintains fluidity. On the other hand, the change of the scattering profile of BLA shows that such an aggregation starts from much lower DMSO concentration (20% (v/v)), namely the DMSO concentration of the start of aggregation of BLA remarkably differs from that of HEWL. Fig. 2 shows the neutron scattering curves of the HEWL and BLA solutions depending on the DMSO concentration.
Fig. 2. Variation of the neutron scattering curves of (a) hen eggwhite lysozyme, and (b) bovine a-lactalbumin, depending on DMSO concentration. The insets show the comparison of the scat˚ 21: (a) for hen egg-white lysozyme at tering profiles above q 0.1 A 0 and 70% (v/v) DMSO; (b) for bovine a-lactalbumin at 0 and 40% (v/v) DMSO.
The insets show the comparison of the neutron scattering ˚ 21, where (a) for HEWL at 0, 70% curves above q 0.1 A (v/v) DMSO; (b) for BLA at 0, 40% (v/v) DMSO. The changes of the neutron scattering curves of HEWL and BLA demonstrate similar changing tendencies observed by the X-ray scattering measurements in Fig. 1. As we described elsewhere [5], the scattering curves in the q ranges ˚ 21 (medium q range) and of 1–1.5 A ˚ 21 (high q of 0.3–0.6 A range) mostly reflect the domain-correlation and the polypeptide configuration within the protein molecule, respectively. As shown in the inset of Fig. 2(a), the evident difference of the scattering profile of HEWL is recognized in the medium q range, but not observed in the high q range. This suggests that HEWL is not completely denatured even at the highest DMSO concentration of 70% (v/v), whereas the scattering profile of BLA changes clearly in both q ranges. These results indicate that with the addition of DMSO the structure of BLA is liable to collapse in comparison with that of HEWL. Fig. 3(a) and (b) shows the apparent radius of gyration Rg
H. Iwase et al. / Journal of Physics and Chemistry of Solids 60 (1999) 1379–1381
Fig. 3. Dependence of the experimental structural parameters on DMSO concentration. The apparent radius of gyration Rg and the maximum diameter Dmax were estimated from the distance distribution functions obtained by the Fourier transform of the X-ray scattering curves in Fig. 1.
and the maximum diameter Dmax of both proteins as a function of DMSO concentration, respectively. The Rg and Dmax values were estimated from the distance distribution functions obtained by the Fourier transform of the X-ray scattering curves. The Rg and Dmax values of HEWL decrease slightly in the DMSO concentration of 0–50% (v/v). Those values of BLA also decrease slightly in 0–15% (v/v) DMSO concentration. On increasing the DMSO concentration further, the Rg and Dmax values of both the proteins start to increase gradually. Recently, Svergun et al. [6] developed a program named Crysol for calculating the solution scattering profile by taking account of the hydration shell surrounding the protein surface from the atomic coordinates data in the Brookhaven Protein Data Bank. They also showed that the hydration shell surrounding the protein surface has a higher density than that of the bulk water [7]. By using the Crysol program we calculated the X-ray
1381
scattering profile from the crystallographic structure of HEWL (PDB entry name of 6LYZ) with varying the scattering density of the hydration shell, which shows that the decrease of the scattering density of the hydration shell reduces the Rg value of the HEWL molecule to about ˚ . When we assume that the collapse of the hydration 1.1 A shell is induced by the addition of DMSO, the decrease of the Rg observed in the DMSO concentration of 0–50% (v/v) turns out to be comparable to the above simulated value. Previously, neutron diffraction and computer simulation studies of DMSO–water mixtures showed that water structure is broken down by the formation of strong hydrogen bonds between DMSO and water molecules [8,9], which strongly supports our present interpretation. The expansion of the protein structure, namely the collapse of the native tertiary structure, occurs above 50% (v/v) DMSO for HEWL and above 15% (v/v) DMSO for BLA. Although HEWL and BLA are well known to have homologous sequences and tertiary structures, we have found that DMSO-induced denaturation of both proteins are quite different, namely the structure of BLA is liable to be destroyed by adding DMSO in comparison with the case of HEWL. The slight decrease of the Rg values of the proteins occurs prior to the remarkable changes of the tertiary structures, suggesting that the collapse of the hydration shell of the protein proceeds at the initial stage to destabilize the tertiary structure. The difference in the structural stability between HEWL and BLA under the presence of DMSO indicates that the structure of BLA is destroyed more sensitively than that of HEWL by the collapse of the hydration shell.
References [1] A.A. Timchenko, M.D. Kirkitadze, D.A. Prokhorov, S.A. Potekhin, I.N. Serdyuk, Bioorg. Khim. 22 (1996) 420. [2] S.E. Radford, C.M. Dobson, P.A. Evans, Nature 358 (1992) 302. [3] E.E. Lattmann, Curr. Opn. Struct. Biol. 4 (1994) 87. [4] M. Hirai, S. Arai, H. Iwase, T. Takizawa, J. Phys. Chem. B 102 (1998) 1308. [5] M. Hirai, S. Arai, H. Iwase, T. Takizawa, S. Shimizu, M. Furusaka, Physica B 241–243 (1998) 1159. [6] D.I. Svergun, C. Barberato, M.J.H. Koch, J. Appl. Cryst. 28 (1995) 768. [7] D.I. Svergun, S. Richard, M.J.H. Koch, Z. Sayers, S. Kuprin, G. Zaccai, Proc. Natl. Acad. Sci. USA 95 (1998) 2267. [8] A.K. Soper, A. Luzar, J. Chem. Phys. 97 (1992) 1320. [9] A. Luzar, J. Chandler, J. Chem. Phys. 98 (1993) 8160.