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[33] Monitoring of protein conformation changes during photocycle
[33]
265
PHOTOCYCLIC PROTEIN CONFORMATION CHANGES
Temperature Control. In all experiments carried out below room temperature, temperature was adjusted and controlled with equipment from L'Air Liquide (France). Electronics. The electronics for the single-beam spectrophotometer and the dual wavelength instrument (time constants 80 nsec, 15/zsec and 3 msec, respectively) were made in Dortmund (modified according to Oesterhelt and Hess 15and Hess et al. z4 They were used for absorption and fluorescence measurements under pulsed illumination. Output signals were recorded with transient recorders (Biomation 802, Nicolet 1090, 2090 and 1174). Fluorescence changes under continuous illumination were measured with a Keithley 153-/xV ammeter, connected through a Rockland electronic filter (10-Hz) with a two-channel Siemens recorder, simultaneously connected with the output of the dual wavelength instrument measuring the M4n intermediate (420-nm light path).
[33] Monitoring of
Protein Conformation Photocycle
Changes
during
By BRIAN BECHER
The absorption of light by the light-adapted purple membrane protein (BR) results in a photochemical reaction cycle through a series of intermediates. 1 Accompanying this cycle is the pumping of protons across the membrane .2 In order to study this relationship in terms of possible protein conformation changes, the use of low-temperature ultraviolet (320245 nm) absorption spectroscopy has proved valuable. ~ At specific low temperatures, certain intermediates of the bacteriorhodopsin photocycle can be "trapped" or prevented from thermally converting to the next intermediate in the sequence. This method offers a number of distinct advantages in the study of protein changes in the photocycle intermediates. First, relatively large percentages of the pigment can be converted to the first three intermediates (K, L, and M). Samples can be prepared with 28% K, 65% L, or 100% M. In addition, the only other species found in significant amounts in these pigment mixtures i s BR. This greatly simplifies analysis of the spectra of the intermediate. Furthermore, since the low-temperature technique traps the intermediates, the absorption measurements can be made carefully over rela1 R. Lozier, R. A. Bogomolni, and W. Stoeckenius, Biophys. J. 15, 955 (1975). D. Oesterhelt and W. Stoeckenius, Proc. Natl. Acad. Sci. U.S.A. 70, 2853 (1973). 3 B. Becher, F. Tokunaga, and T. G. Ebrey, Biochemistry 17, 2293 (1978).
METHODS IN ENZYMOLOGY, VOL. 88
Copyright © 1982by Academic Press, inc. All rights of reproduction in any form reserved. ISBN 0-12-181988-4
266
BACTERIORHODOPSIN
[33]
tively long periods of time. This is especially important in the measurement of proteins in the ultraviolet range where the signal-to-noise ratio is relatively low. Consequently, the low-temperature technique provides distinct advantages in terms of accuracy in ultraviolet absorption measurements of the bacteriorhodopsin protein. A major problem with all ultraviolet absorption measurements of particulate (membrane) systems is artifacts resulting from nonselective and selective light scattering of the measuring beam by the sample. 4 This problem is compounded in the case of low-temperature measurements owing to freeze-cracking of the sample. Freeze-cracking of the sample can easily increase light scattering to the extent that reliable ultraviolet absorption measurements are impossible. Both of these complications can be greatly alleviated by use of 67% (v/v) glycerol in the membrane sample. This concentration of glycerol significantly decreases light scattering by more closely matching the index of refraction of the media with that of the membrane. In addition, glycerol addition prevents serious cracking of the sample at low temperatures if the sample is gradually cooled. In order to monitor the protein changes during BR conversion to K and L, purple membrane is first suspended in a 2:1 glycerol-water (0.02 M phosphate buffer, pH 7.0) solution. For the study of the M conversion, purple membrane is first suspended in a 25% NaC1 solution titrated to pH 10 with 0.1 M NaOH and then mixed with 2 parts of glycerol. Under these conditions the lifetime of the M intermediate greatly increases, allowing accurate spectral measurements. ~ Low-temperature visible and ultraviolet absorption spectra of the K, L, and M intermediates are then recorded using a quartz Dewar flask. For the measurements of the conversion of BR to the K intermediate, purple membrane in the glycerol-buffer medium is placed in a stoppered 0.2-cm quartz cell, fully light adapted, and then, in the dark, lowered into an ethanol bath previously cooled to - 100° with liquid nitrogen. The cell is then removed from the ethanol bath and, after any ethanol remaining on the cell surface is rapidly blown off with a burst of nitrogen gas, lowered into the Dewar filled with liquid nitrogen. In this manner the absorption measurements of the sample are not significantly hampered by cracking of the sample on lowering its temperature to - 196°. Light-adapted purple membrane can be converted to the steady-state mixture of the K intermediate by 500-nm light (500-W slide projector light source and an interference filter). Measurement of the partial conversion of BR to the L intermediate at - 100° is done similarly except that the cell is placed in a Dewar flask 4 p. Latimer and E. Rabinowitch, Arch. Biochem. Biophys. 84, 428 (1959). 5 B. B e c h e r and T. G. Ebrey, Biophys. J. 17, 185 (1977).
[33]
PHOTOCYCLIC PROTEIN CONFORMATION CHANGES
267
filled with ethanol cooled to - 100°. A 640-nm interference filter is used in photoconverting the purple membrane to L, which remains stable at 100°. Complete conversion to M can be achieved by exposure of the NaCl-buffer-glycerol sample to 600-rim light at - 4 0 °. The near ultraviolet absorption spectrum (320-245 nm) of lightadapted BR includes distinct maxima or shoulders at 290, 280, and 274 nm that are primarily attributed to 7r-w* transitions of the amino acids tryptophan and tyrosine. (Cystine is not found in bacteriorhodopsin.) In addition, light scattering and minor transitions of the retinal chromophore contribute to the 320-245-nm spectrum. The secondary, tertiary, or quarternary structure of the purple membrane protein may also alter the environment of the rr-zr* transitions of the aromatic amino acids relative to free amino acids from which extinction coefficients are estimated. In particular, side chains "buried" within the nonpolar environment of the protein are expected to have appreciably higher extinctions than those of free amino acids. 6 In the case of BR conversion to K at - 196°, no significant change in the near ultraviolet spectrum is found, indicating little protein conformation change at - 196° (as expected). However, the conversions of BR to L and BR to M result in large decreases in near ultraviolet extinction. A conversion to 65% L results in a decrease in 280-nm extinction of 6000 + 1000 liters cm -1 mo1-1. Similarly, a 5000 + 1000 liter cm -1 mo1-1 decrease occurs on complete conversion to M. Most striking is the BR to L and BR to M difference spectra in the near ultraviolet that have shapes similar to the near ultraviolet absorption spectra of BR itself, including maxima or shoulders at 290, 280, 270 nm (Becher et al.2). These results strongly argue that a change in protein conformation occurs on conversion of BR to L or M. A conformation change in the protein could lead to the decreased absorbance at 280 nm by exposure of approximately 50% of the aromatic amino acids in the relatively nonpolar protein interior to the more polar (water) media. In addition, a conformational change could alter the relative orientation of originally interacting aromatic amino acid transitions, resulting in a loss of hyperchromism. Isomerization of the retinal chromophore is unlikely to contribute more than 2000 liters cm -~ mol -~ to the extinction decrease. It is likely that protein conformation changes in the intermediates of the bacteriorhodopsin photocycle are directly involved in the proton pumping accompanying the cycle. Low-temperature ultraviolet spectroscopy is an accurate, relatively uncomplicated technique to measure these protein changes. -
6 j. Donovan, in "Physical Principles and Techniques of Protein Chemistry" (S. J. Leach, ed.), Part A, p. 101. Academic Press, New York, 1969.
265
PHOTOCYCLIC PROTEIN CONFORMATION CHANGES
Temperature Control. In all experiments carried out below room temperature, temperature was adjusted and controlled with equipment from L'Air Liquide (France). Electronics. The electronics for the single-beam spectrophotometer and the dual wavelength instrument (time constants 80 nsec, 15/zsec and 3 msec, respectively) were made in Dortmund (modified according to Oesterhelt and Hess 15and Hess et al. z4 They were used for absorption and fluorescence measurements under pulsed illumination. Output signals were recorded with transient recorders (Biomation 802, Nicolet 1090, 2090 and 1174). Fluorescence changes under continuous illumination were measured with a Keithley 153-/xV ammeter, connected through a Rockland electronic filter (10-Hz) with a two-channel Siemens recorder, simultaneously connected with the output of the dual wavelength instrument measuring the M4n intermediate (420-nm light path).
[33] Monitoring of
Protein Conformation Photocycle
Changes
during
By BRIAN BECHER
The absorption of light by the light-adapted purple membrane protein (BR) results in a photochemical reaction cycle through a series of intermediates. 1 Accompanying this cycle is the pumping of protons across the membrane .2 In order to study this relationship in terms of possible protein conformation changes, the use of low-temperature ultraviolet (320245 nm) absorption spectroscopy has proved valuable. ~ At specific low temperatures, certain intermediates of the bacteriorhodopsin photocycle can be "trapped" or prevented from thermally converting to the next intermediate in the sequence. This method offers a number of distinct advantages in the study of protein changes in the photocycle intermediates. First, relatively large percentages of the pigment can be converted to the first three intermediates (K, L, and M). Samples can be prepared with 28% K, 65% L, or 100% M. In addition, the only other species found in significant amounts in these pigment mixtures i s BR. This greatly simplifies analysis of the spectra of the intermediate. Furthermore, since the low-temperature technique traps the intermediates, the absorption measurements can be made carefully over rela1 R. Lozier, R. A. Bogomolni, and W. Stoeckenius, Biophys. J. 15, 955 (1975). D. Oesterhelt and W. Stoeckenius, Proc. Natl. Acad. Sci. U.S.A. 70, 2853 (1973). 3 B. Becher, F. Tokunaga, and T. G. Ebrey, Biochemistry 17, 2293 (1978).
METHODS IN ENZYMOLOGY, VOL. 88
Copyright © 1982by Academic Press, inc. All rights of reproduction in any form reserved. ISBN 0-12-181988-4
266
BACTERIORHODOPSIN
[33]
tively long periods of time. This is especially important in the measurement of proteins in the ultraviolet range where the signal-to-noise ratio is relatively low. Consequently, the low-temperature technique provides distinct advantages in terms of accuracy in ultraviolet absorption measurements of the bacteriorhodopsin protein. A major problem with all ultraviolet absorption measurements of particulate (membrane) systems is artifacts resulting from nonselective and selective light scattering of the measuring beam by the sample. 4 This problem is compounded in the case of low-temperature measurements owing to freeze-cracking of the sample. Freeze-cracking of the sample can easily increase light scattering to the extent that reliable ultraviolet absorption measurements are impossible. Both of these complications can be greatly alleviated by use of 67% (v/v) glycerol in the membrane sample. This concentration of glycerol significantly decreases light scattering by more closely matching the index of refraction of the media with that of the membrane. In addition, glycerol addition prevents serious cracking of the sample at low temperatures if the sample is gradually cooled. In order to monitor the protein changes during BR conversion to K and L, purple membrane is first suspended in a 2:1 glycerol-water (0.02 M phosphate buffer, pH 7.0) solution. For the study of the M conversion, purple membrane is first suspended in a 25% NaC1 solution titrated to pH 10 with 0.1 M NaOH and then mixed with 2 parts of glycerol. Under these conditions the lifetime of the M intermediate greatly increases, allowing accurate spectral measurements. ~ Low-temperature visible and ultraviolet absorption spectra of the K, L, and M intermediates are then recorded using a quartz Dewar flask. For the measurements of the conversion of BR to the K intermediate, purple membrane in the glycerol-buffer medium is placed in a stoppered 0.2-cm quartz cell, fully light adapted, and then, in the dark, lowered into an ethanol bath previously cooled to - 100° with liquid nitrogen. The cell is then removed from the ethanol bath and, after any ethanol remaining on the cell surface is rapidly blown off with a burst of nitrogen gas, lowered into the Dewar filled with liquid nitrogen. In this manner the absorption measurements of the sample are not significantly hampered by cracking of the sample on lowering its temperature to - 196°. Light-adapted purple membrane can be converted to the steady-state mixture of the K intermediate by 500-nm light (500-W slide projector light source and an interference filter). Measurement of the partial conversion of BR to the L intermediate at - 100° is done similarly except that the cell is placed in a Dewar flask 4 p. Latimer and E. Rabinowitch, Arch. Biochem. Biophys. 84, 428 (1959). 5 B. B e c h e r and T. G. Ebrey, Biophys. J. 17, 185 (1977).
[33]
PHOTOCYCLIC PROTEIN CONFORMATION CHANGES
267
filled with ethanol cooled to - 100°. A 640-nm interference filter is used in photoconverting the purple membrane to L, which remains stable at 100°. Complete conversion to M can be achieved by exposure of the NaCl-buffer-glycerol sample to 600-rim light at - 4 0 °. The near ultraviolet absorption spectrum (320-245 nm) of lightadapted BR includes distinct maxima or shoulders at 290, 280, and 274 nm that are primarily attributed to 7r-w* transitions of the amino acids tryptophan and tyrosine. (Cystine is not found in bacteriorhodopsin.) In addition, light scattering and minor transitions of the retinal chromophore contribute to the 320-245-nm spectrum. The secondary, tertiary, or quarternary structure of the purple membrane protein may also alter the environment of the rr-zr* transitions of the aromatic amino acids relative to free amino acids from which extinction coefficients are estimated. In particular, side chains "buried" within the nonpolar environment of the protein are expected to have appreciably higher extinctions than those of free amino acids. 6 In the case of BR conversion to K at - 196°, no significant change in the near ultraviolet spectrum is found, indicating little protein conformation change at - 196° (as expected). However, the conversions of BR to L and BR to M result in large decreases in near ultraviolet extinction. A conversion to 65% L results in a decrease in 280-nm extinction of 6000 + 1000 liters cm -1 mo1-1. Similarly, a 5000 + 1000 liter cm -1 mo1-1 decrease occurs on complete conversion to M. Most striking is the BR to L and BR to M difference spectra in the near ultraviolet that have shapes similar to the near ultraviolet absorption spectra of BR itself, including maxima or shoulders at 290, 280, 270 nm (Becher et al.2). These results strongly argue that a change in protein conformation occurs on conversion of BR to L or M. A conformation change in the protein could lead to the decreased absorbance at 280 nm by exposure of approximately 50% of the aromatic amino acids in the relatively nonpolar protein interior to the more polar (water) media. In addition, a conformational change could alter the relative orientation of originally interacting aromatic amino acid transitions, resulting in a loss of hyperchromism. Isomerization of the retinal chromophore is unlikely to contribute more than 2000 liters cm -~ mol -~ to the extinction decrease. It is likely that protein conformation changes in the intermediates of the bacteriorhodopsin photocycle are directly involved in the proton pumping accompanying the cycle. Low-temperature ultraviolet spectroscopy is an accurate, relatively uncomplicated technique to measure these protein changes. -
6 j. Donovan, in "Physical Principles and Techniques of Protein Chemistry" (S. J. Leach, ed.), Part A, p. 101. Academic Press, New York, 1969.