ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 297, No. 2, September, pp. 354-361,1992
Fragmentation of Dimyristoylphosphatidylcholine Vesicles by Apomyoglobin Jong Woo Lee and Hyoungman
Kim1
Department of Life Science, Korea Advanced Institute of Science and Technology, 373-l Kusong-Dong, Yusung-Gu, Taejon 305-701, Korea
Received November
27,1991, and in revised form March 31,1992
Previously we have reported results of a preliminary study on the micellization of phosphatidylcholine vesicles by apomyoglobin at pH 4 (J. W. Lee and H. Kim, 1988, FEBS Lett. 241, 181-184). The micellization study has been extended here to investigate the effect of the lipid to protein ratio, temperature, size of vesicles, and pH. The pH-dependent study indicated that micellization occurs when the protein assumes either a molten globular or random coil structure. Time-dependent hydrophobic labeling by 3-(trifluoromethyl)-3-(m-[1261]iodophenyl)diazirine showed that there is an initial increase in contact between the protein and hydrophobic acyl chain of lipid followed by a decrease in the interaction. This may be explained as the initial stage of vesicle aggregation which is subsequently superseded by the fragmentation. These reactions are discussed in term of protein unfolding at
10~
PH.
0 1992
Academic
press.
Inc.
It has been well established that the a-helix is a predominant structural motif for the membrane-spanning segments of integral proteins (1). The highly polar peptide bonds can avoid exposure to the hydrophobic acyl chains of the lipids in the membrane interior by forming intrachain hydrogen bonds. The a-helical structure is also very common for the proteins which are bound on the membrane surface despite the fact that segregation of peptide bonds from the hydrophobic phase of the membrane is not a prevailing concern (2). A similar situation also occurs when proteins cover the exposed peripheral acyl chains of lipid bilayer of a micellar complex as proposed first for the interaction of apolipoprotein A-I (apo A-U2 i To whom correspondence should be addressed. ’ Abbreviations used: apohlb, apomyoglobin; Mb, myoglobin; apo AI, apolipoprotein A-I; CNBr, cyanogen bromide; PC, phosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; MLV, multilamellar vesicles; LUV, large unilamellar vesicles; SUV, small unilamellar vesicles; L/P ratio, lipid/protein molar ratio; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; T,, phase transition temperature; [‘251]TID, 3-(trifluoromethyl)-3-(m-[‘25I]iodophenyl)diazirine. 354
with vesicles (3). The a-helical structures in these interface proteins are generally amphiphilic because they are in contact with the hydrophobic phase on one surface and the hydrophilic phase on the opposite surface. In an earlier preliminary study (4), we explored the possible micellization of phosphatidylcholine (PC) vesicles by apomyoglobin (apoMb) because its holoprotein contains some 80% amphiphilic a-helix. We found that this protein forms a micellar complex with PC only under the acidic condition. This suggests that acid-induced conformational change is important for the micellization process. Also, the physical state of the lipid bilayer is an important factor for this process as judged from the maximum micellization occurring at the phase transition temperature (T,) of the lipid bilayer (5). In order to address these and other problems, our previous investigation has been extended here to cover micellization under a wide range of conditions. MATERIALS
AND
METHODS
Materials. Dimyristoylphosphatidylcholine (DMPC), sperm whale myoglobin (Mb) and sodium phosphotungstate were purchased from Sigma. 3-(Trifluoromethyl)-3-(m-[‘25I]iodophenyl)diazirine ([‘r51]TID) was purchased from Amersham. All other chemicals were obtained in the highest purity available. The purity of phospholipid was ascertained by thin-layer chromatography. Phospholipid concentration was determined according to the method of Vaskovsky et al. (6). The removal of heme group from Mb was achieved by the 2-butanone extraction procedure of Teale (7). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a modified Laemmli discontinuous buffer system (8) showed that this apoMb preparation contained less than 1% minor protein components of unknown origin, About 0.2% Mb was estimated to be present in the apoMb preparation as determined from the ratio of optical density at 408 nm to that at 280 nm (9). apoMb concentration in the absence of phospholipid was obtained spectrophotometrically at 280 nm using the molar extinction coefficient of 1.59 X 10” (9) or by the Bradford method (10). In the presence of phospholipid, it was determined by using the modified Lowry method of Markwell et al. (11) with Mb as a standard. Buffers used in all experiments were made of 10 mM 2-{[2-hydroxy-l,lbis(hydroxymethyl)ethyl]aminojethanesulfonic acid (pH 7),4-morpholineethanesulfonic acid (pH 6), acetic acid (pH 5-4), glycine (pH 3), and hydrochloric acid (pH 2) with an appropriate amount of NaCl added. 0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
DIMYRISTOYLPHOSPHATIDYLCHOLINE
VESICLE
355
FRAGMENTATION
2
A
1.6 I
-i 1 t-
d
0.6
01 0
2
4
6
Time (min)
0
lo
4
6
Time (min)
s
” lo
I
0’ 0.
2
4
6
6
10
Time (min)
FIG. 1. Time courses of changes in light scattering after DMPC SUV was mixed with apoMb at pH 6 (A), pH 3 (B), and pH 2 (C). Solutions contained 0.1 M NaCl. a, L/P 220; b, L/P 300; c, L,‘P 400; d, L/P 600; e, L/P 1100; f, L/P 1500; g, L/P 2200; h, L/P 2500; i, L/P 3500; j, L/P 4000. I, intensity of scattered light; IO, intensity of scattered light by vesicles alone.
Vesicle preparation. Multilammelar vesicles (MLV) were prepared by first forming a thin lipid film on the inner wall of a vial and by vortexing after the addition of a buffer solution. Small unilamellar vesicles (SUV) were produced by sonicating MLV. Following sonication, the vesicle dispersion was centrifuged for 30 min at 100,OOOg(TLS-55.5 swing rotor) to remove any probe particles as well aj large multilamellar liposomes. Large unilamellar vesicles (LUV) were prepared by reversephase evaporation method (12). Light-scattering measurements. It was already established that the decrease in light scattering in the presence of apoMb is due to micellar complex formation 114).The micellization process was monitored by lightscattering measurements with a JASCO FP-770 spectrofluorometer at a wavelength of 400 nm, using 10.nm slit widths and a standard l-cm path length sample cell. The cell compartment was thermostatically controlled and the temperature of the solutions to be added into the cuvette was also maintained at the same temperature before mixing. The sample was continuously stirred by a small magnetic bar placed in the cuvette below the light path. The phospholipid solution was added to apoMb solution (
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 297, No. 2, September, pp. 354-361,1992
Fragmentation of Dimyristoylphosphatidylcholine Vesicles by Apomyoglobin Jong Woo Lee and Hyoungman
Kim1
Department of Life Science, Korea Advanced Institute of Science and Technology, 373-l Kusong-Dong, Yusung-Gu, Taejon 305-701, Korea
Received November
27,1991, and in revised form March 31,1992
Previously we have reported results of a preliminary study on the micellization of phosphatidylcholine vesicles by apomyoglobin at pH 4 (J. W. Lee and H. Kim, 1988, FEBS Lett. 241, 181-184). The micellization study has been extended here to investigate the effect of the lipid to protein ratio, temperature, size of vesicles, and pH. The pH-dependent study indicated that micellization occurs when the protein assumes either a molten globular or random coil structure. Time-dependent hydrophobic labeling by 3-(trifluoromethyl)-3-(m-[1261]iodophenyl)diazirine showed that there is an initial increase in contact between the protein and hydrophobic acyl chain of lipid followed by a decrease in the interaction. This may be explained as the initial stage of vesicle aggregation which is subsequently superseded by the fragmentation. These reactions are discussed in term of protein unfolding at
10~
PH.
0 1992
Academic
press.
Inc.
It has been well established that the a-helix is a predominant structural motif for the membrane-spanning segments of integral proteins (1). The highly polar peptide bonds can avoid exposure to the hydrophobic acyl chains of the lipids in the membrane interior by forming intrachain hydrogen bonds. The a-helical structure is also very common for the proteins which are bound on the membrane surface despite the fact that segregation of peptide bonds from the hydrophobic phase of the membrane is not a prevailing concern (2). A similar situation also occurs when proteins cover the exposed peripheral acyl chains of lipid bilayer of a micellar complex as proposed first for the interaction of apolipoprotein A-I (apo A-U2 i To whom correspondence should be addressed. ’ Abbreviations used: apohlb, apomyoglobin; Mb, myoglobin; apo AI, apolipoprotein A-I; CNBr, cyanogen bromide; PC, phosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; MLV, multilamellar vesicles; LUV, large unilamellar vesicles; SUV, small unilamellar vesicles; L/P ratio, lipid/protein molar ratio; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; T,, phase transition temperature; [‘251]TID, 3-(trifluoromethyl)-3-(m-[‘25I]iodophenyl)diazirine. 354
with vesicles (3). The a-helical structures in these interface proteins are generally amphiphilic because they are in contact with the hydrophobic phase on one surface and the hydrophilic phase on the opposite surface. In an earlier preliminary study (4), we explored the possible micellization of phosphatidylcholine (PC) vesicles by apomyoglobin (apoMb) because its holoprotein contains some 80% amphiphilic a-helix. We found that this protein forms a micellar complex with PC only under the acidic condition. This suggests that acid-induced conformational change is important for the micellization process. Also, the physical state of the lipid bilayer is an important factor for this process as judged from the maximum micellization occurring at the phase transition temperature (T,) of the lipid bilayer (5). In order to address these and other problems, our previous investigation has been extended here to cover micellization under a wide range of conditions. MATERIALS
AND
METHODS
Materials. Dimyristoylphosphatidylcholine (DMPC), sperm whale myoglobin (Mb) and sodium phosphotungstate were purchased from Sigma. 3-(Trifluoromethyl)-3-(m-[‘25I]iodophenyl)diazirine ([‘r51]TID) was purchased from Amersham. All other chemicals were obtained in the highest purity available. The purity of phospholipid was ascertained by thin-layer chromatography. Phospholipid concentration was determined according to the method of Vaskovsky et al. (6). The removal of heme group from Mb was achieved by the 2-butanone extraction procedure of Teale (7). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a modified Laemmli discontinuous buffer system (8) showed that this apoMb preparation contained less than 1% minor protein components of unknown origin, About 0.2% Mb was estimated to be present in the apoMb preparation as determined from the ratio of optical density at 408 nm to that at 280 nm (9). apoMb concentration in the absence of phospholipid was obtained spectrophotometrically at 280 nm using the molar extinction coefficient of 1.59 X 10” (9) or by the Bradford method (10). In the presence of phospholipid, it was determined by using the modified Lowry method of Markwell et al. (11) with Mb as a standard. Buffers used in all experiments were made of 10 mM 2-{[2-hydroxy-l,lbis(hydroxymethyl)ethyl]aminojethanesulfonic acid (pH 7),4-morpholineethanesulfonic acid (pH 6), acetic acid (pH 5-4), glycine (pH 3), and hydrochloric acid (pH 2) with an appropriate amount of NaCl added. 0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
DIMYRISTOYLPHOSPHATIDYLCHOLINE
VESICLE
355
FRAGMENTATION
2
A
1.6 I
-i 1 t-
d
0.6
01 0
2
4
6
Time (min)
0
lo
4
6
Time (min)
s
” lo
I
0’ 0.
2
4
6
6
10
Time (min)
FIG. 1. Time courses of changes in light scattering after DMPC SUV was mixed with apoMb at pH 6 (A), pH 3 (B), and pH 2 (C). Solutions contained 0.1 M NaCl. a, L/P 220; b, L/P 300; c, L,‘P 400; d, L/P 600; e, L/P 1100; f, L/P 1500; g, L/P 2200; h, L/P 2500; i, L/P 3500; j, L/P 4000. I, intensity of scattered light; IO, intensity of scattered light by vesicles alone.
Vesicle preparation. Multilammelar vesicles (MLV) were prepared by first forming a thin lipid film on the inner wall of a vial and by vortexing after the addition of a buffer solution. Small unilamellar vesicles (SUV) were produced by sonicating MLV. Following sonication, the vesicle dispersion was centrifuged for 30 min at 100,OOOg(TLS-55.5 swing rotor) to remove any probe particles as well aj large multilamellar liposomes. Large unilamellar vesicles (LUV) were prepared by reversephase evaporation method (12). Light-scattering measurements. It was already established that the decrease in light scattering in the presence of apoMb is due to micellar complex formation 114).The micellization process was monitored by lightscattering measurements with a JASCO FP-770 spectrofluorometer at a wavelength of 400 nm, using 10.nm slit widths and a standard l-cm path length sample cell. The cell compartment was thermostatically controlled and the temperature of the solutions to be added into the cuvette was also maintained at the same temperature before mixing. The sample was continuously stirred by a small magnetic bar placed in the cuvette below the light path. The phospholipid solution was added to apoMb solution (
