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Site-directed isotope labeling of membrane proteins: A new tool for spectroscopists
Site-Directed Isotope Labeling of Membrane Proteins: A New Tool for Spectroscopists Sanjay Sonar1, Chan-Ping Lee§1, Cheryl F.C. Ludlam*, Xiao-Mei Liu*, Matthew Coleman1, Thomas Marti*2, Uttam L. RajBhandary§ and Kenneth J. Rothschild1* ^Physics Department and Molecular Biophysics Laboratory, Boston University, Boston MA 02215; department of Biology, and * Department of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
I. Introduction A key goal in understanding how an integral membrane protein works is to obtain a detailed picture of the structural changes which occur during function. For this purpose, Fourier Transform infrared (FTIR) difference spectroscopy has been increasingly used in conjunction with a variety of methods in biochemistry and molecular biology (for a recent review see (1) and references therein). The technique derives its usefulness from the ability of FTIR-difFerence spectroscopy to detect bands arising due to the vibrations of individual residues and chemical groups in a protein which undergo conformational and/or chemical change. However, a key problem in the use of FTIR-difference spectroscopy is the assignment of bands to individual amino acid residues. Only after such assignments are made can information be derived about changes in specific components of the protein. In the past, vibrational assignments have been made on the basis of two different approaches: uniform isotope labeling of amino acids and site-directed mutagenesis. While these methods led to the assignment of several specific bands (2-5), many of these assignments are still tentative, while most bands have not yet been assigned. The essential problems are: /) uniform isotope labeling can assign bands to particular amino acids but not to specific residues; and //') site-directed mutagenesis, especially of those amino acid residues in the active site of a protein, can alter the structure and function of a protein, thereby preventing unambiguous assignments of individual bands. We recently introduced a third method for assigning bands in FTIR-difference spectra (6) based on a technique which we have termed site-directed isotope labeling (SDIL). As summarized in Figure 1, a key element in SDIL is the use of a suppressor tRNA 1 2
Present Address: Tzu Chi College of Medicine, Hualien, Taiwan Present Address: Bernard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany Address correspondence to this author.
TECHNIQUES IN PROTEIN CHEMISTRY VII Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Figure 1: Major steps in site-directed isotope labeling of a protein: i) E. coli Tyr sup-tRNA is enzymatically aminoacylated with a deuterated tyrosine; ii) an amber codon is inserted in the gene at the position of the residue to be isotope labeled and iii) the mRNA is translated in a cell-free system in the presence of aminoacylated Tyr sup-tRNA. The resulting SDEL-analog is then purified and refolded. This illustration is reprinted from ref (6) with permission.
aminoacylated with an isotopically labeled amino acid. This tRNA is targeted to insert the isotopic amino acid at the proper position in the nascent protein by using an amber codon at the corresponding position in the gene. Cell-free synthesis (in vitro translation) and exogenous addition of the aminoacylated suppressor tRNA prevent aminoacylation of non-suppressor tRNAs with the isotopic amino acid, similar to the approach used for sitedirected non-native amino acid replacement (SNAAR) (7-9). We have chosen the integral membrane protein bacteriorhodopsin as a model system for demonstrating the application of SDIL-FTIR. Bacteriorhodopsin (bR) is a light-activatable proton pump containing an ?\\-trcms retinylidene chromophore and is found in the purple
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membrane of the extremely halophilic bacterium H. salinarium (10, 11). Due to its unusual properties, including a native 2-D crystalline lattice which has led to a 3-D structural model (12), stability at very high temperature (13), and a photocycle with distinct spectral intermediates (bR57o->K63o->L55o->M4i2->N55o->0640"->bR57o) (14), bR has become an important model of energy transduction and proton transport in membrane proteins. Several recent studies on bacteriorhodopsin have conclusively demonstrated that the FTIR-SDIL approach can probe the local environment and structural changes of specific residues and backbone carbonyl groups in a protein (6, 15, 16, 24).
II. Materials and Methods
Site-directed Mutagensis and In vitro Transcription The amber codon (TAG) was introduced into positions Tyr-57, Tyr-83, Tyr-147 and Tyr-185 in the synthetic bop gene using either cassette mutagenesis (17) or PCR based mutagenesis (18). mRNAs were obtained by in vitro transcription by placing these genes under control of an SP6 or T7 promoter (19).
Amber Suppression in Cell-free Protein Synthesis System Cell-free synthesis of bacterioopsin and its amber mutants were carried out in an mRNA-dependent wheat germ translation system as described (19) using 35S-methionine as the radioactive label. Aminoacylated E. coli tyrosine suppressor tRNA ([n«g-2H4] or [l-13C]Tyr-sup tRNA) was used for suppression of amber codons introduced in the bop gene at position Tyr-57, Tyr-83, Tyr-147 and Tyr-185. The suppressor tRNA was overexpressed in E. coli and isolated as total tRNA (20). Aminoacylation of the total tRNA was carried out using E. coli S-100 extract prepared as described (21). Synthesis of bOp and amber suppression was optimized with respect to the concentration of Mg2 + 3 K + and aminoacylated suppressor tRNA concentrations. The reaction mixtures were analyzed by gel electrophoresis and the extent of suppression was calculated using phosphorimager measurements (Molecular Dynamics) by comparing the band intensities of full length bOp and the truncated fragment (see Figure 2, Panel A, lanes 5 and 8). The amber suppressions were also carried out where the proteins synthesized were labeled with 3H-Tyr (100 mCi/ml; Amersham) present in the incubation mixture as free amino acid or as aminoacylated suppressor tRNA. SDS-PAGE was carried out in a 15% gel.
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Functional Refolding and Regeneration of SDIL-analogs Cell-free expressed bacterioopsin and its SDIL mutants were purified using procedures described earlier (19). Refolding in halobacterial lipids along with regeneration of bOp by exogenous addition of d^L-trans retinal results in formation of a purple complex absorbing at 560 nm, characteristic of dark-adapted bR (DR560). Light adaptation was carried out as described previously (22). Time-resolved absorption measurements of SDIL-analogs was carried out as described previously (19, 22).
FTIR-difference spectroscopy of SDIL-analogs FTIR-difTerence spectra were recorded at 2 cm"1 on a 740 Nicolet spectrometer equipped with an MCT-B detector. Approximately 25-30 micrograms of purple membrane or SDIL sample suspended in distilled water was placed on an AgCl window and was allowed to dry overnight in a dry-box with a -150 °C dewpoint. The window with the deposited sample was then placed in a specially designed cell, light-adapted and FTIR difference spectra for the bR-»K and bR-»M transitions measured at 80 K and 250 K, respectively, and at 2 cm"1 resolution using methods previously reported (23, 25).
ATR-FTIR difference spectroscopy of SDIL-analogs Sample solutions of SDIL-analogs of bR were prepared (1 mM NaPi, 1 raM KC1, 0.4 mM MgCl2, 0.6 mM CaCl2, and 50 mM NaCl), and 25 ul of each of these samples (2 uM bR) was dried onto a Ge-crystal internal reflection element as described (15). The sample film was equilibrated with a pH 9.0 solution (25 mM NaPi titrated with 0.5 M NaOH) at 3 °C, and Attenuted total reflection (ATR)-FTIR difference spectra were recorded as described earlier (15).
III. Results and Discussion
SDIL Analogs ofBacteriorhodopsin Seven SDIL analogs of bacteriorhodopsin, [n«g-2H4] Tyr-57, [n'«g-2H4] Tyr-147, [ring-m4] Tyr-185, [1-13C] Tyr-57, [1-13C] Tyr-83, [1-13C] Tyr-147 and [1-13C] Tyr-185 have been produced (6, 15, 16, 24). In contrast to earlier studies aimed at site-specific non-native amino acid replacement (SNAAR) in proteins (7-9), which relied on chemical aminoacylation of the suppressor tRNA involving several synthetic steps, we developed a highly efficient method employing a single step enzymatic aminoacylation for site-directed isotope labeling.
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bOp Tyr-185 Tyr-147
bOp Tyr-185 Tyr-147 ^n i
ii
1
12 3 4 5 6 7 8 mRNA Suppressor tRNA (Uncharged) [ring, 2 H 4 ] Tyr-Suppressor tRNA (Charged)
-+ + + ++ + + " - -
+ - "
- - - -
+ -
+ --
+
mRNA Suppressor tRNA (Uncharged) 3 H-Tyr-Suppressor tRNA (Charged) 3 H-Tyr + 19 other amino acids 20 amino acids
1 2 3 4 5 6 7 8 9 - ++ + ++ + + +
- - - -
+
- -
+
+ + +
+
+
+ +
+
+ +
+
46kDH
l tf lp : ' i l f l l 30kD-> .l «
21.5 kD->
|jf|
30 kD 21.5 kD
Iff |||•'fff *
Figure 2: Assays for amber suppression and site-directed isotope labeling using SDS-PAGE followed by autoradiography using 35S-methionine (A) and 3H-Tyr (B). The reaction composition of samples loaded in each lane is as indicated. The reaction mixtures were analyzed by SDS-PAGE in a 15% polyacrylamide gel. For (B), the reactions were similar as in (A) except that the proteins synthesized were labeled with 3H-Tyr (100 mCi/ml; Amersham) present in the incubation mixture as a free amino acid or as aminoacylated suppressor tRNA. Reprinted from ref. 6 with permission.
The results of cell-free expression of bR shown in Figures 2A and B establish that the aminoacylated E. coli Tyr suppressor tRNA efficiently suppresses the amber mutations and site-directed isotope labeling does not cause significant "scrambling" of the isotope label. Figure 2A and 2B show that: i) Cell-free translation of Y185am and Y147am mRNAs resulted in incomplete translation in the absence of the aminoacylated suppressor tRNAs, whereas full-length bacterioopsin was produced only in the presence of suppressor tRNAs aminoacylated with [ring-2H4] Tyr (Figure 2A). ii) Aminoacyl-linkage between the isotopic amino acid and the suppressor tRNA was not hydrolyzed and "scrambling" of [n«g-2H4] Tyr did not take place (lane 2, Fig 2B). ///) After incorporating [hng-2U4] Tyr at the amber codon, the free suppressor tRNA was not aminoacylated by the aminoacyl synthetases present in the cell-free extract, and thus neither "dilution" of the isotopic label nor insertion of normal amino acids occurred, iv) Efficiency of amber suppression is very high, typically 80-90%.
Functional Characterization After cell-free synthesis, the SDIL proteins were purified, refolded in halobacterial lipids and regenerated with exogenously added ail-trans retinal using the methods reported recently (19). Typically, 20-25 jig of refolded proteins were obtained per ml of cell-free translation reaction. We have recently reported various vector constructs that hold potential for further increasing the yields (18). Importantly, both SDIL proteins as well as the unlabeled cell-free expressed bR (cf-bR) exhibit a normal visible absorption spectra in the light and dark as compared to purple membrane (6, 22). Static and time-resolved
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visible absorption spectra indicate that the labeling, expression and reconstitution procedures yield SDIL analogs which are functionally similar to bR (6).
FTIR Analysis of SDIL Analogs An essential requirement for the successful application of SDIL is that high quality FTIR-difference spectra can be obtained that allow band assignments to particular molecular groups in a protein. This was demonstrated conclusively by us in several reports (6, 15, 16). As a first example, we focused on tyrosine residues, although with the help of chemical aminoacylation, this approach can be extended to any residues in bR. As seen in Figure 3, high signal-to-noise FTIR difference spectra can be obtained from SDIL samples. Isotope labeling facilitates band assignment by producing frequency shifts in bands that arise from vibrations of those chemical groups which contain the isotope. Thus, for example, perdeuteration of the tyrosine ring causes a downshift in the frequency of a tyrosinate mode near 1277 cm"1 which involves C-O" stretching (23). Figure 3 A shows the bR-^M difference spectra of bR, cell-free expressed bR (cf-bR), the three SDIL analogs of bR and bR containing uniform labeling at all tyrosines (L-[ring-2H4] all-Tyr). In the case of L-[ring-2H4] Tyr 57 and L-[ring-2H4] Tyr 147, all of the bands previously assigned to tyrosine vibrational modes on the basis of uniform isotope labeling remain unaltered. For example, a negative band at 833 cm"1 previously assigned to a characteristic Fermi resonance of tyrosinate (23), negative/positive bands at 1277/1271 cm'1 assigned to the CO' stretch of tyrosinate, a positive band at 1456 cm"1 assigned to a tyrosine vibrational mode and a negative band at 1590 cm"1 possibly due to a tyrosinate ring mode all appear (23, 25). In contrast to L-[ring-2H4] Tyr 57 and L-[ring-2H4] Tyr 147, the bR—»M difference spectrum of L-[ring-2H4] Tyr 185 exhibits significant changes compared to unlabeled bR (Figure 3A). Furthermore, these changes are very similar to those produced by uniform L-[ring-2H4] labeling of bR. In particular, bands characteristic of tyrosine or tyrosinate vibrations including the 1590, 1456, 1277 and 833 cm"1 bands disappear (Figures 3 and 4), whereas new bands appear in both spectra at 1575 cm"1, 1416 cm"1, and 1240-1250 cm"1, which were tentatively assigtied on the basis of model compound studies to L-[ring-2H4] Tyr (or tyrosinate) vibrational modes (23, 25). This is especially clear in the case of the negative/positive pair at 1277/1271 cm"1 (Figure 4) previously attributed to a change in the environment of a tyrosinate residue. This demonstrates that all of the spectral changes due to uniform [ring-2H4] Tyr labeling arise exclusively from the isotope labeling of Tyr-185. Thus, we conclude that Tyr-185 is the only tyrosine residue which is structurally active during the bR-»M transition of the bR photocycle. Since similar results were also obtained for the bR->K (6) bR-»L and bR-»N difference spectra (unpublished results, data not shown), it appears likely that Tyr185 is the only tyrosine which is structurally active during the entire bR photocycle.
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2.7X10 OD
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1600
1400 1200 Wavenumber (cm1)
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bR [1-"C]-Tyr57 n-1JCl-Tyr 147
[1- Cl-Tyrl85 aU-Ll-nCJ-TyrbR
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1700
1600 Wavenumber (cm"1)
1500
1400
Figure 3: FTIR Difference spectra of SDIL-analogs of bR: Top: FTIR Spectra corresponding to bR-»M transition recorded at 250 K (16). Bottom: ATR-FTIR spectra corresponding to the bR-»N transition at 276 K (15).
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Probing Local Structural Changes in a Protein Backbone It is also possible to use SDIL to incorporate isotope labels at specific positions in the peptide backbone of a protein and to use FTIR difference spectroscopy to probe for local conformational changes. In a recent study (24), four SDDL-analogs of bR containing L-[l13 C] Tyr at positions 57, 83, 147 and 185 were constructed (6, 15). Low temperature FTIR-difference spectra of cell-free produced unlabeled bR (WT) along with the four SDIL bR analogs reveal that the carbonyl group at position 185 is structurally active during the primary photochemical transition (bR-»K). For example, a downshift of a set of (+/-) bands from 1622 and 1618 cm"1 to 1586 and 1581 cm'1 is only found to occur in the [1-13C] Tyr 185 and the [1-13C] all-Tyr sample. On this basis we are able to unambiguously assign the tyrosine carbonyl group that is changing during the bR->K transition to Tyr 185. In addition, the similarity of the bR-»K difference spectra of [1-13C] Tyr 185 and [1-13C] all-Tyr samples indicates that no other tyrosine carbonyl groups significantly contribute to the bR-»K difference spectrum of wild type bR. Thus, we conclude that the peptide carbonyl group of Tyr 185 is the only one of the 11 tyrosines in bR which is significantly perturbed by chromophore isomerization during the primary phototransition of bR. In a second study, we used ATR-FTIR difference spectroscopy to measure the M-»N transition of SDIL-bR analogs (15). We have previously shown that this transition involves a major structural change involving membrane embedded a-helical structure (28). For example, as shown in Figure 3B, the most noticeable change is the drop in intensity and the small frequency shift of the 1670 cm"1 band in both [1-13C] Tyr 185 and uniform labeled all-[l-13C]Tyr sample. Importantly, this frequency shift is in accord with studies on synthetic polypeptides (26) and the membrane protein phospholamban (27). Changes are also found at 1680 (+), 1661 (+), 1657 (-) and 1647 cm"1 (+) and in the 1600-1635 cm'1 region. Our results show that this structural change involves alterations in the environment of the Tyr 185 carbonyl group. In contrast, no changes are observed in the carbonyl groups from all other tyrosines including the 6 that are embedded in the interior of membrane. On this basis and related experiments, we suggested that the Tyr 185-Pro 186 region serves as a hinge around which F-helix bends and could serve as a potential coupling point between chromophore isomerization and protein conformational changes.
Acknowledgments This work was supported by grants from the NIH (GM47527) and the NSF (MCB 9106017) to KJR, the Army Research Office (ARO) (DAAL03-92-G-0172) to KJR and ULR and the NIH (GM17151) to ULR. M. Coleman was supported by an NIH Training Grant in Molecular Biophysics (GM08291).
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References 1. Rothschild, K. J. (1992). J. Bioenerg. Biomembr. 24, 147-167. 2. Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G. & Rothschild, K. J. (1988). Biochemistry 27, 8516-8520. 3. Braiman, M. S., Mogi, T., Stern, L. J., Hackett, N. R, Chao, B. H., Khorana, H. G. & Rothschild, K. J. (1988). Proteins: Struct, Funct, Genet 3, 219-229. 4. Gerwert, K., Hess, B., Soppa, J. & Oesterhelt, D. (1989). Proc. Natl. Acad Sci. U. S. A. 86, 4943-4947. 5. Maeda, A., Sasaki, J., Shichida, Y., Yoshizawa, T., Chang, M., Ni, B., Needleman, R & Lanyi, J. K. (1992). Biochemistry 31, 4684-4690. 6. Sonar, S., Lee, C.-R, Coleman, M., Marti, T., Patel, N., Liu, X., Khorana, H. G., RajBhandary, U. L. & Rothschild, K. J. (1994). Nature Struct Biol. 1, 512-517. 7. Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C. & Schultz, P. G. (1989). Science 244, 182188. 8. Ellman, J. A., Mendel, D. & Schultz, P. G. (1992). Science 255, 197-200. 9. Bain, J. D., Diala, E. S., Glabe, C. G., Wacker, D. A., Lyttle, M. H., Dix, T. A. & Chamberlin, A. R (1991). Biochemistry 30, 5411-5421. 10. Stoeckenius, W. & Bogomolni, R. A. (1982). Annu. Rev. Biochem. 51, 587-616. 11. Rothschild, K. J. & Sonar, S. (1995) In CRC Handbook of Organic Photochemistry and Photobiology: Horspool, W. M. & Song, P-S., Eds., CRC Press Inc.: London, pp 1521-1544. 12. Henderson, R, Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckmann, E. & Downing, K. H. (1990). J. Mol Biol 213, 899-929. 13. Shen, Y., Safinya, C. R, Liang, K. S., Ruppert, A. F. & Rothschild, K. J. (1993). Nature 366, 48-50. 14. Lozier, R H., Xie, A., Hofrichter, J. & Clore, G. M. (1992). Proc. Natl. Acad. Sci. U. S. A. 89, 3610-3614. 15. Ludlam, C. F. C, Sonar, S., Lee, C.-P., Coleman, M., Herzfeld, J., RajBhandary, U. L. & Rothschild, K. J. (1995). Biochemistry 34, 2-6. 16. Liu, X.-M., Sonar, S., Lee, C.-P., Coleman, M., RajBhandary, U. L. & Rothschild, K. J. (1995). Biophys. Chem. 56,63-70. 17. Hackett, N. R, Stern, L. J., Chao, B. H., Kronis, K. A. & Khorana, H. G. (1987). J. Biol. Chem. 262, 9277-9284. 18. Coleman, M., Sonar, S., Patel, N. & Rothschild, K. J. (1994). in Dual-Use Technologies and Applications Conference: 4th Annual 1994 IEEE Mohawk Valley Section (SUNY Institute of Technology at Utica/Rome,)., May 23-26 Ed., Vol. 1, pp. 326-331. 19. Sonar, S. M., Patel, N. P., Fischer, W. & Rothschild, K. J. (1993). Biochemistry 32, 1377713781. 20. Lee, C. P., Seong, B. L. & RajBhandary, U. L. (1991). J. Biol. Chem. 266, 18012-18018. 21. RajBhandary, U. L. & Ghosh, H. P. (1969). J. Biol. Chem. 244, 1104-1113. 22. Sonar, S., Krebs, M. P., Khorana, H. G. & Rothschild, K. J. (1993). Biochemistry 32, 2263-2271. 23. Rothschild, K. J., Roepe, P., Ahl, P. L., Earnest, T. N., Bogomolni, R. A., Das Gupta, S. K., Mulliken, C. M. & Herzfeld, J. (1986). Proc. Natl. Acad. Sci. U. S. A. 83, 347-351. 24. Sonar, S., Liu, X.-M., Lee, C.-P., Coleman, M., He, Y.-W., Herzfeld, J., RajBhandary, U. L. & Rothschild, K. J. (1995). J. Amer. Chem. Soc. 117, In Press. 25. Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J. & Rothschild, K. J. (1987). Biochemistry 26, 6696-6707. 26. Martinez, G. V., Fiori, W. R. & Millhauser, G. (1994). Biophys. J. 66, A65. 27. Arkin, I. T., Rothman, M. S., Ludlam, C. F. C, Aimoto, S., Engelman, D. M., Rothschild, K. J. & Smith, S. O. (1995). J. Mol. Biol. 248, 824-834. 28. Rothschild, K. J., Marti, T., Sonar, S., He, Y. W., Rath, P., Fischer, W., Bousche, O. & Khorana, H. (1993). J. Biol. Chem. 268, 27046-27052.
I. Introduction A key goal in understanding how an integral membrane protein works is to obtain a detailed picture of the structural changes which occur during function. For this purpose, Fourier Transform infrared (FTIR) difference spectroscopy has been increasingly used in conjunction with a variety of methods in biochemistry and molecular biology (for a recent review see (1) and references therein). The technique derives its usefulness from the ability of FTIR-difFerence spectroscopy to detect bands arising due to the vibrations of individual residues and chemical groups in a protein which undergo conformational and/or chemical change. However, a key problem in the use of FTIR-difference spectroscopy is the assignment of bands to individual amino acid residues. Only after such assignments are made can information be derived about changes in specific components of the protein. In the past, vibrational assignments have been made on the basis of two different approaches: uniform isotope labeling of amino acids and site-directed mutagenesis. While these methods led to the assignment of several specific bands (2-5), many of these assignments are still tentative, while most bands have not yet been assigned. The essential problems are: /) uniform isotope labeling can assign bands to particular amino acids but not to specific residues; and //') site-directed mutagenesis, especially of those amino acid residues in the active site of a protein, can alter the structure and function of a protein, thereby preventing unambiguous assignments of individual bands. We recently introduced a third method for assigning bands in FTIR-difference spectra (6) based on a technique which we have termed site-directed isotope labeling (SDIL). As summarized in Figure 1, a key element in SDIL is the use of a suppressor tRNA 1 2
Present Address: Tzu Chi College of Medicine, Hualien, Taiwan Present Address: Bernard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany Address correspondence to this author.
TECHNIQUES IN PROTEIN CHEMISTRY VII Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Figure 1: Major steps in site-directed isotope labeling of a protein: i) E. coli Tyr sup-tRNA is enzymatically aminoacylated with a deuterated tyrosine; ii) an amber codon is inserted in the gene at the position of the residue to be isotope labeled and iii) the mRNA is translated in a cell-free system in the presence of aminoacylated Tyr sup-tRNA. The resulting SDEL-analog is then purified and refolded. This illustration is reprinted from ref (6) with permission.
aminoacylated with an isotopically labeled amino acid. This tRNA is targeted to insert the isotopic amino acid at the proper position in the nascent protein by using an amber codon at the corresponding position in the gene. Cell-free synthesis (in vitro translation) and exogenous addition of the aminoacylated suppressor tRNA prevent aminoacylation of non-suppressor tRNAs with the isotopic amino acid, similar to the approach used for sitedirected non-native amino acid replacement (SNAAR) (7-9). We have chosen the integral membrane protein bacteriorhodopsin as a model system for demonstrating the application of SDIL-FTIR. Bacteriorhodopsin (bR) is a light-activatable proton pump containing an ?\\-trcms retinylidene chromophore and is found in the purple
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membrane of the extremely halophilic bacterium H. salinarium (10, 11). Due to its unusual properties, including a native 2-D crystalline lattice which has led to a 3-D structural model (12), stability at very high temperature (13), and a photocycle with distinct spectral intermediates (bR57o->K63o->L55o->M4i2->N55o->0640"->bR57o) (14), bR has become an important model of energy transduction and proton transport in membrane proteins. Several recent studies on bacteriorhodopsin have conclusively demonstrated that the FTIR-SDIL approach can probe the local environment and structural changes of specific residues and backbone carbonyl groups in a protein (6, 15, 16, 24).
II. Materials and Methods
Site-directed Mutagensis and In vitro Transcription The amber codon (TAG) was introduced into positions Tyr-57, Tyr-83, Tyr-147 and Tyr-185 in the synthetic bop gene using either cassette mutagenesis (17) or PCR based mutagenesis (18). mRNAs were obtained by in vitro transcription by placing these genes under control of an SP6 or T7 promoter (19).
Amber Suppression in Cell-free Protein Synthesis System Cell-free synthesis of bacterioopsin and its amber mutants were carried out in an mRNA-dependent wheat germ translation system as described (19) using 35S-methionine as the radioactive label. Aminoacylated E. coli tyrosine suppressor tRNA ([n«g-2H4] or [l-13C]Tyr-sup tRNA) was used for suppression of amber codons introduced in the bop gene at position Tyr-57, Tyr-83, Tyr-147 and Tyr-185. The suppressor tRNA was overexpressed in E. coli and isolated as total tRNA (20). Aminoacylation of the total tRNA was carried out using E. coli S-100 extract prepared as described (21). Synthesis of bOp and amber suppression was optimized with respect to the concentration of Mg2 + 3 K + and aminoacylated suppressor tRNA concentrations. The reaction mixtures were analyzed by gel electrophoresis and the extent of suppression was calculated using phosphorimager measurements (Molecular Dynamics) by comparing the band intensities of full length bOp and the truncated fragment (see Figure 2, Panel A, lanes 5 and 8). The amber suppressions were also carried out where the proteins synthesized were labeled with 3H-Tyr (100 mCi/ml; Amersham) present in the incubation mixture as free amino acid or as aminoacylated suppressor tRNA. SDS-PAGE was carried out in a 15% gel.
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Functional Refolding and Regeneration of SDIL-analogs Cell-free expressed bacterioopsin and its SDIL mutants were purified using procedures described earlier (19). Refolding in halobacterial lipids along with regeneration of bOp by exogenous addition of d^L-trans retinal results in formation of a purple complex absorbing at 560 nm, characteristic of dark-adapted bR (DR560). Light adaptation was carried out as described previously (22). Time-resolved absorption measurements of SDIL-analogs was carried out as described previously (19, 22).
FTIR-difference spectroscopy of SDIL-analogs FTIR-difTerence spectra were recorded at 2 cm"1 on a 740 Nicolet spectrometer equipped with an MCT-B detector. Approximately 25-30 micrograms of purple membrane or SDIL sample suspended in distilled water was placed on an AgCl window and was allowed to dry overnight in a dry-box with a -150 °C dewpoint. The window with the deposited sample was then placed in a specially designed cell, light-adapted and FTIR difference spectra for the bR-»K and bR-»M transitions measured at 80 K and 250 K, respectively, and at 2 cm"1 resolution using methods previously reported (23, 25).
ATR-FTIR difference spectroscopy of SDIL-analogs Sample solutions of SDIL-analogs of bR were prepared (1 mM NaPi, 1 raM KC1, 0.4 mM MgCl2, 0.6 mM CaCl2, and 50 mM NaCl), and 25 ul of each of these samples (2 uM bR) was dried onto a Ge-crystal internal reflection element as described (15). The sample film was equilibrated with a pH 9.0 solution (25 mM NaPi titrated with 0.5 M NaOH) at 3 °C, and Attenuted total reflection (ATR)-FTIR difference spectra were recorded as described earlier (15).
III. Results and Discussion
SDIL Analogs ofBacteriorhodopsin Seven SDIL analogs of bacteriorhodopsin, [n«g-2H4] Tyr-57, [n'«g-2H4] Tyr-147, [ring-m4] Tyr-185, [1-13C] Tyr-57, [1-13C] Tyr-83, [1-13C] Tyr-147 and [1-13C] Tyr-185 have been produced (6, 15, 16, 24). In contrast to earlier studies aimed at site-specific non-native amino acid replacement (SNAAR) in proteins (7-9), which relied on chemical aminoacylation of the suppressor tRNA involving several synthetic steps, we developed a highly efficient method employing a single step enzymatic aminoacylation for site-directed isotope labeling.
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bOp Tyr-185 Tyr-147
bOp Tyr-185 Tyr-147 ^n i
ii
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12 3 4 5 6 7 8 mRNA Suppressor tRNA (Uncharged) [ring, 2 H 4 ] Tyr-Suppressor tRNA (Charged)
-+ + + ++ + + " - -
+ - "
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+ -
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+
mRNA Suppressor tRNA (Uncharged) 3 H-Tyr-Suppressor tRNA (Charged) 3 H-Tyr + 19 other amino acids 20 amino acids
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Figure 2: Assays for amber suppression and site-directed isotope labeling using SDS-PAGE followed by autoradiography using 35S-methionine (A) and 3H-Tyr (B). The reaction composition of samples loaded in each lane is as indicated. The reaction mixtures were analyzed by SDS-PAGE in a 15% polyacrylamide gel. For (B), the reactions were similar as in (A) except that the proteins synthesized were labeled with 3H-Tyr (100 mCi/ml; Amersham) present in the incubation mixture as a free amino acid or as aminoacylated suppressor tRNA. Reprinted from ref. 6 with permission.
The results of cell-free expression of bR shown in Figures 2A and B establish that the aminoacylated E. coli Tyr suppressor tRNA efficiently suppresses the amber mutations and site-directed isotope labeling does not cause significant "scrambling" of the isotope label. Figure 2A and 2B show that: i) Cell-free translation of Y185am and Y147am mRNAs resulted in incomplete translation in the absence of the aminoacylated suppressor tRNAs, whereas full-length bacterioopsin was produced only in the presence of suppressor tRNAs aminoacylated with [ring-2H4] Tyr (Figure 2A). ii) Aminoacyl-linkage between the isotopic amino acid and the suppressor tRNA was not hydrolyzed and "scrambling" of [n«g-2H4] Tyr did not take place (lane 2, Fig 2B). ///) After incorporating [hng-2U4] Tyr at the amber codon, the free suppressor tRNA was not aminoacylated by the aminoacyl synthetases present in the cell-free extract, and thus neither "dilution" of the isotopic label nor insertion of normal amino acids occurred, iv) Efficiency of amber suppression is very high, typically 80-90%.
Functional Characterization After cell-free synthesis, the SDIL proteins were purified, refolded in halobacterial lipids and regenerated with exogenously added ail-trans retinal using the methods reported recently (19). Typically, 20-25 jig of refolded proteins were obtained per ml of cell-free translation reaction. We have recently reported various vector constructs that hold potential for further increasing the yields (18). Importantly, both SDIL proteins as well as the unlabeled cell-free expressed bR (cf-bR) exhibit a normal visible absorption spectra in the light and dark as compared to purple membrane (6, 22). Static and time-resolved
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visible absorption spectra indicate that the labeling, expression and reconstitution procedures yield SDIL analogs which are functionally similar to bR (6).
FTIR Analysis of SDIL Analogs An essential requirement for the successful application of SDIL is that high quality FTIR-difference spectra can be obtained that allow band assignments to particular molecular groups in a protein. This was demonstrated conclusively by us in several reports (6, 15, 16). As a first example, we focused on tyrosine residues, although with the help of chemical aminoacylation, this approach can be extended to any residues in bR. As seen in Figure 3, high signal-to-noise FTIR difference spectra can be obtained from SDIL samples. Isotope labeling facilitates band assignment by producing frequency shifts in bands that arise from vibrations of those chemical groups which contain the isotope. Thus, for example, perdeuteration of the tyrosine ring causes a downshift in the frequency of a tyrosinate mode near 1277 cm"1 which involves C-O" stretching (23). Figure 3 A shows the bR-^M difference spectra of bR, cell-free expressed bR (cf-bR), the three SDIL analogs of bR and bR containing uniform labeling at all tyrosines (L-[ring-2H4] all-Tyr). In the case of L-[ring-2H4] Tyr 57 and L-[ring-2H4] Tyr 147, all of the bands previously assigned to tyrosine vibrational modes on the basis of uniform isotope labeling remain unaltered. For example, a negative band at 833 cm"1 previously assigned to a characteristic Fermi resonance of tyrosinate (23), negative/positive bands at 1277/1271 cm'1 assigned to the CO' stretch of tyrosinate, a positive band at 1456 cm"1 assigned to a tyrosine vibrational mode and a negative band at 1590 cm"1 possibly due to a tyrosinate ring mode all appear (23, 25). In contrast to L-[ring-2H4] Tyr 57 and L-[ring-2H4] Tyr 147, the bR—»M difference spectrum of L-[ring-2H4] Tyr 185 exhibits significant changes compared to unlabeled bR (Figure 3A). Furthermore, these changes are very similar to those produced by uniform L-[ring-2H4] labeling of bR. In particular, bands characteristic of tyrosine or tyrosinate vibrations including the 1590, 1456, 1277 and 833 cm"1 bands disappear (Figures 3 and 4), whereas new bands appear in both spectra at 1575 cm"1, 1416 cm"1, and 1240-1250 cm"1, which were tentatively assigtied on the basis of model compound studies to L-[ring-2H4] Tyr (or tyrosinate) vibrational modes (23, 25). This is especially clear in the case of the negative/positive pair at 1277/1271 cm"1 (Figure 4) previously attributed to a change in the environment of a tyrosinate residue. This demonstrates that all of the spectral changes due to uniform [ring-2H4] Tyr labeling arise exclusively from the isotope labeling of Tyr-185. Thus, we conclude that Tyr-185 is the only tyrosine residue which is structurally active during the bR-»M transition of the bR photocycle. Since similar results were also obtained for the bR->K (6) bR-»L and bR-»N difference spectra (unpublished results, data not shown), it appears likely that Tyr185 is the only tyrosine which is structurally active during the entire bR photocycle.
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2.7X10 OD
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bR [1-"C]-Tyr57 n-1JCl-Tyr 147
[1- Cl-Tyrl85 aU-Ll-nCJ-TyrbR
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Figure 3: FTIR Difference spectra of SDIL-analogs of bR: Top: FTIR Spectra corresponding to bR-»M transition recorded at 250 K (16). Bottom: ATR-FTIR spectra corresponding to the bR-»N transition at 276 K (15).
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Probing Local Structural Changes in a Protein Backbone It is also possible to use SDIL to incorporate isotope labels at specific positions in the peptide backbone of a protein and to use FTIR difference spectroscopy to probe for local conformational changes. In a recent study (24), four SDDL-analogs of bR containing L-[l13 C] Tyr at positions 57, 83, 147 and 185 were constructed (6, 15). Low temperature FTIR-difference spectra of cell-free produced unlabeled bR (WT) along with the four SDIL bR analogs reveal that the carbonyl group at position 185 is structurally active during the primary photochemical transition (bR-»K). For example, a downshift of a set of (+/-) bands from 1622 and 1618 cm"1 to 1586 and 1581 cm'1 is only found to occur in the [1-13C] Tyr 185 and the [1-13C] all-Tyr sample. On this basis we are able to unambiguously assign the tyrosine carbonyl group that is changing during the bR->K transition to Tyr 185. In addition, the similarity of the bR-»K difference spectra of [1-13C] Tyr 185 and [1-13C] all-Tyr samples indicates that no other tyrosine carbonyl groups significantly contribute to the bR-»K difference spectrum of wild type bR. Thus, we conclude that the peptide carbonyl group of Tyr 185 is the only one of the 11 tyrosines in bR which is significantly perturbed by chromophore isomerization during the primary phototransition of bR. In a second study, we used ATR-FTIR difference spectroscopy to measure the M-»N transition of SDIL-bR analogs (15). We have previously shown that this transition involves a major structural change involving membrane embedded a-helical structure (28). For example, as shown in Figure 3B, the most noticeable change is the drop in intensity and the small frequency shift of the 1670 cm"1 band in both [1-13C] Tyr 185 and uniform labeled all-[l-13C]Tyr sample. Importantly, this frequency shift is in accord with studies on synthetic polypeptides (26) and the membrane protein phospholamban (27). Changes are also found at 1680 (+), 1661 (+), 1657 (-) and 1647 cm"1 (+) and in the 1600-1635 cm'1 region. Our results show that this structural change involves alterations in the environment of the Tyr 185 carbonyl group. In contrast, no changes are observed in the carbonyl groups from all other tyrosines including the 6 that are embedded in the interior of membrane. On this basis and related experiments, we suggested that the Tyr 185-Pro 186 region serves as a hinge around which F-helix bends and could serve as a potential coupling point between chromophore isomerization and protein conformational changes.
Acknowledgments This work was supported by grants from the NIH (GM47527) and the NSF (MCB 9106017) to KJR, the Army Research Office (ARO) (DAAL03-92-G-0172) to KJR and ULR and the NIH (GM17151) to ULR. M. Coleman was supported by an NIH Training Grant in Molecular Biophysics (GM08291).
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