Summary
Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated3H leucine from the digested membranes.
During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.
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References
Argos, P., Rao, J.K.M., Hargrave, P.A. 1982. Structural prediction of membrane-bound proteins.Eur. J. Biochem. 128:565–575
Blaurock, A.E. 1975. The structure of purple membrane fromHalobacterium halobium: Analysis of the X-ray diffraction patterns.J. Mol. Biol. 93:139–158
Brunner, J., Richards, F.M. 1980. Analysis of membranes photolabeled with lipid analogs.J. Biol. Chem. 255:3319–3329
Dittmer, J.C., Wells, M.A. 1969. Quantitative and qualitative analysis of lipids and lipid components.Methods Enzymol. 14:482–530
Dumont, M.E., Richards, F.M. 1984. Insertion of apocytochromec into lipid vesicles.J. Biol. Chem. 259:4147–4156
Engelman, D.M., Goldman, A., Steitz, T. 1982. The identification of helical segments in the polypeptide chain of bacteriorhodopsinMethods Enzymol. 88:81–88
Engelman, D.M., Steitz, T.A. 1981. The spontaneous insertion of proteins into membranes.Cell 23:411–422
Engelman, D.M., Steitz, T.A. 1984. On the folding and insertion of globular membrane proteins.In: The Protein Folding Problem. D.B. Wetlaufer, editor. pp. 87–113. AAAS Selected Symposium 89. Westview Press, Boulder, Colo.
Engelman, D.M., Zaccai, G. 1980. Bacteriorhodopsin is an inside out protein.Proc. Nat'l. Acad. Sci. USA 77:5894–5898
Gerber, G.E., Anderegg, R.J., Herlihy, W.C., Gray, C.P., Biemann, K., Khorana, H.G. 1979. Partial primary structure of bacteriorhodopsin: Sequencing methods for membrane proteins.Proc. Nat'l. Acad. Sci. USA 76:227–231
Gerber, G.E., Gray, C.P., Wildenauer, D., Khorana, H.G. 1977. Orientation of bacteriorhodopsin inHalobacterium halobium as studied by selective proteolysis.Proc. Nat'l. Acad. Sci. USA 74:5426–5430
Govindjee, R., Ebrey, T.G., Crofts, A.R. 1980. The quantum efficiency of proton pumping of the purple membrane ofHalobacterium Halobium.Biophys. J. 30:231–242
Henderson, R. 1975. The structure of the purple membrane fromHalobacterium halobium: Analysis of the diffraction pattern.J. Mol. Biol. 93:123–138
Henderson, R., Unwin, P.N.T. 1975. Three dimensional model of purple membrane obtained by electron microscopy.Nature (London) 257:28–32
Hess, G.P. 1971. Chymotrypsin-chemical properties and catalysis.In: The Enzymes (3rd Ed.) Vol. 3, pp. 213–248. Paul D. Boyer, editor. Academic, New York
Huang, K.-S., Bayley, H., Liao, M.-J., London, E., Khorana, H.G. 1981. Refolding of an integral membrane protein: Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin.J. Biol. Chem. 256:3802–3809
Jahnig, F. 1983. Thermodynamics and kinetics of protein incorporation into membranes.Proc. Nat'l. Acad. Sci. USA 80:3691–3695
Jap, B.K., Maestre, M.F., Hayward, S.B., Glaeser, R.M. 1983. Peptide chain secondary structure of bacteriorhodopsin.Biophys. J. 43:81–89
Katre, N.V., Stroud, R.M. 1981. A probable linking sequence between two transmembrane components of bacteriorhodopsin.FEBS Lett. 136:170–174
Khorana, H.G., Gerber, G.E., Herlihy, W.C., Gray, C.P., Anderegg, R.J., Nihei, K., Biemann, K. 1979. Amino acid sequence of bacteriorhodopsin.Proc. Nat'l. Acad. Sci. USA 76:5046–5050
Kyte, J., Doolittle, R.F. 1982. A simple method for displaying the hydrophobic character of a protein.J. Mol. Biol. 157:105–132
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–685
Lemke, H.-D., Bergmeyer, J., Oesterhelt, D. 1982. Determination of modified positions in the polypeptide chain of bacteriorhodopsin.Methods Enzymol. 88:89–98
Liao, M.-J., Huang, K.-S., Khorana, H.G. 1984. Regeneration of native bacteriorhodopsin structure from fragments.J. Biol. Chem. 259:4200–4204
Liao, M.-J., Khorana, H.G. 1984. Removal of the carboxyl-terminal peptide does not affect refolding or function of bacteriorhodopsin as a light-dependent proton pump.J. Biol. Chem. 259:4194–4199
Mao, D., Wallace, B.A. 1984. Differential light scattering and absorption flattening effects are minimal in the circular dichroism spectra of small unilamellar vesicles.Biochemistry 23:2667–2673
Michel-Villaz, M., Saibil, H.R., Chabre, M. 1979. Orientation of rhodopsin alpha helices in retinal rod outer segment membranes.Proc. Nat'l. Acad. Sci. USA 76:4405–4408
Michel, H., Oesterhelt, D., Henderson, R. 1980. Orthorhombic two-dimensional crystal form of purple membrane.Proc. Nat'l. Acad. Sci. USA 77:338–342
Moore, W.M., Holladay, L.A., Puett, D., Brady, R.N. 1974. On the conformation of the acetylcholine receptor protein fromTorpedo nobiliana.FEBS Lett. 45:145–149
Oesterhelt, D., Stoeckenius, W. 1971. Rhodopsin-like protein from the purple membrane ofHalobacterium halobium.Nature New Biol. 233:149–152
Ort, D.R., Parson, W.W. 1979. Enthalpy changes during the photo-chemical cycle of bacteriorhodopsin.Biophys. 25:341–354
Ovchinnikov, Yu.A., Abdulaev, N.G., Feigina, M.Y., Kiselev, A.V., Lobanov, N.A. 1979. The structural basis of the functioning of bacteriorhodopsion: An overview.FEBS Lett. 100:219–224
Popot, J.-L., Trewhella, J., Gerchman, S.E., Engelman, D.M. 1984. Two-dimensional lattice of hybrid bacteriorhodopsin molecules.In: Proceedings of the 8th International Biophysics Congress, Bristol, U.K. p. 51
Racker, E., Stoeckenius, W. 1974. Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphate formation.J. Biol. Chem. 249:662–663
Rohorek, M., Heyn, M.P. 1979. Binding of all-trans retinal to the purple membrane: Evidence for cooperativity and determination of the extinction coefficient.Biochemistry 18:4977–4983
Rosenheck, K., Brith-Lindner, M., Lindner, P., Zakaria, A., Caplan, S.R. 1978. Proteolysis and flash photolysis of bacteriorhodopsin in purple membrane fragments.Biophys. Struct. Mechan. 4:301–313
Ross, A.H., Radhakrishnan, R., Robson, R.J., Khorana 1982. Glycophorin as studied using photoactivatable phospholipids.J. Biol. Chem. 257:4152–4161
Stubbs, G.W., Smith, H.G., Jr., Litman, B.J. 1976. Alkyl glucosides as effective solubilizing agents for bovine rhodopsin: A comparison with several commonly used detergents.Biochim. Biophys. Acta 425:46–56
Swank, R.T., Munkres, K.D. 1971. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium docecyl sulfate.Anal. Biochem. 39:462–477
Tanford, C. 1980. The Hydrophobic Effect. John Wiley & Sons, New York
Tarr, G.E., Crabb, J.W. 1983. Reverse phase high performance liquid chromatography of hydrophobic proteins and fragments thereof.Anal. Biochem. 39:99–107
Trewhella, J., Anderson, S., Fox, R., Gogol, E., Khan, S., Engelman, D.M. 1983. Assignment of segments of the bacteriorhodopsin sequence to positions in the structural map.Biophys. J. 42:233–241
Unwin, P.N.T., Henderson, R. 1975. Molecular structure determination by electron microscopy of unstained crystalline specimens.J. Mol. Biol. 94:425–440
Von Heijne, G., Blomberg, C. 1979. Trans-membrane translocation of proteins: The direct transfer model.Eur. J. Biochem. 97:175–181
Walker, J.E., Carne, A.F., Schmitt, H.W. 1979. Topography of the purple membrane.Nature (London) 278:653–654
Wallace, B.A., Henderson, R. 1982. Location of the carboxyl terminal of bacteriorhodopsin in purple membrane.Biophys. J. 39:233–239
Zwizinski, C., Wickner, W. 1980. Purification and characterization of the leader (signal) peptidase fromEscherichia coli.J. Biol. Chem. 255:7973–7977
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Dumont, M.E., Trewhella, J., Engelman, D.M. et al. Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis. J. Membrain Biol. 88, 233–247 (1985). https://doi.org/10.1007/BF01871088
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DOI: https://doi.org/10.1007/BF01871088