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. 1993 Jul;2(7):1161–1170. doi: 10.1002/pro.5560020711

Refolding and proton pumping activity of a polyethylene glycol-bacteriorhodopsin water-soluble conjugate.

G Sirokmán 1, G D Fasman 1
PMCID: PMC2142423  PMID: 8358299

Abstract

Bacteriorhodopsin (BR), from the purple membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol (m-PEG; molecular weight = 5,000 Da) succinimidyl carbonate. The polyethylene glycol-bacteriorhodopsin (m-PEG-SC-BR33) conjugate, containing one polyethylene glycol chain, was water soluble. The secondary structure of the conjugate in water appeared partially denatured, but was shown to contain alpha-helical segments by circular dichroism spectroscopy. The isolated bacteriorhodopsin conjugate, with added retinal, was refolded in a mixed detergent-lipid micelle and had an absorption maximum at 555 nm. The refolded conjugate was transferred into vesicles that pumped protons, upon illumination, as efficiently as did native BR. Modification of the PM with m-PEG did not alter the native structure or inhibit proton pumping, and therefore it is suggested that the glycol polymer is present as a moiety covalently linked to residues unnecessary for proton pumping and proper folding. The site of attachment of m-PEG was determined to be at either Lys 129 or Lys 159, with position Lys 129 the most probable site of attachment. The m-PEG-SC-BR33 could be stepwise refolded to the native conformation by the addition of trifluoroethanol to lower the dielectric constant, simulating the insertion of the BR into the phospholipid bilayer.

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Selected References

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  1. Amons R., Schrier P. I. Removal of sodium dodecyl sulfate from proteins and peptides by gel filtration. Anal Biochem. 1981 Sep 15;116(2):439–443. doi: 10.1016/0003-2697(81)90385-7. [DOI] [PubMed] [Google Scholar]
  2. Arakawa T., Timasheff S. N. Mechanism of poly(ethylene glycol) interaction with proteins. Biochemistry. 1985 Nov 19;24(24):6756–6762. doi: 10.1021/bi00345a005. [DOI] [PubMed] [Google Scholar]
  3. Arnold K., Zschoernig O., Barthel D., Herold W. Exclusion of poly(ethylene glycol) from liposome surfaces. Biochim Biophys Acta. 1990 Mar;1022(3):303–310. doi: 10.1016/0005-2736(90)90278-v. [DOI] [PubMed] [Google Scholar]
  4. Braiman M. S., Stern L. J., Chao B. H., Khorana H. G. Structure-function studies on bacteriorhodopsin. IV. Purification and renaturation of bacterio-opsin polypeptide expressed in Escherichia coli. J Biol Chem. 1987 Jul 5;262(19):9271–9276. [PubMed] [Google Scholar]
  5. Cleland J. L., Randolph T. W. Mechanism of polyethylene glycol interaction with the molten globule folding intermediate of bovine carbonic anhydrase B. J Biol Chem. 1992 Feb 15;267(5):3147–3153. [PubMed] [Google Scholar]
  6. Gerber G. E., Anderegg R. J., Herlihy W. C., Gray C. P., Biemann K., Khorana H. G. Partial primary structure of bacteriorhodopsin: sequencing methods for membrane proteins. Proc Natl Acad Sci U S A. 1979 Jan;76(1):227–231. doi: 10.1073/pnas.76.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Henderson R., Baldwin J. M., Ceska T. A., Zemlin F., Beckmann E., Downing K. H. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol. 1990 Jun 20;213(4):899–929. doi: 10.1016/S0022-2836(05)80271-2. [DOI] [PubMed] [Google Scholar]
  8. Henderson R., Jubb J. S., Whytock S. Specific labelling of the protein and lipid on the extracellular surface of purple membrane. J Mol Biol. 1978 Aug 5;123(2):259–274. doi: 10.1016/0022-2836(78)90325-x. [DOI] [PubMed] [Google Scholar]
  9. Huang K. S., Bayley H., Liao M. J., London E., Khorana H. G. Refolding of an integral membrane protein. Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin and two proteolytic fragments. J Biol Chem. 1981 Apr 25;256(8):3802–3809. [PubMed] [Google Scholar]
  10. Liao M. J., London E., Khorana H. G. Regeneration of the native bacteriorhodopsin structure from two chymotryptic fragments. J Biol Chem. 1983 Aug 25;258(16):9949–9955. [PubMed] [Google Scholar]
  11. McIntosh T. J., Magid A. D., Simon S. A. Steric repulsion between phosphatidylcholine bilayers. Biochemistry. 1987 Nov 17;26(23):7325–7332. doi: 10.1021/bi00397a020. [DOI] [PubMed] [Google Scholar]
  12. Nelson C. A. The binding of detergents to proteins. I. The maximum amount of dodecyl sulfate bound to proteins and the resistance to binding of several proteins. J Biol Chem. 1971 Jun 25;246(12):3895–3901. [PubMed] [Google Scholar]
  13. Oesterhelt D., Stoeckenius W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 1974;31:667–678. doi: 10.1016/0076-6879(74)31072-5. [DOI] [PubMed] [Google Scholar]
  14. Popot J. L., Gerchman S. E., Engelman D. M. Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process. J Mol Biol. 1987 Dec 20;198(4):655–676. doi: 10.1016/0022-2836(87)90208-7. [DOI] [PubMed] [Google Scholar]
  15. Prevelige P. E., Jr, Fasman G. D. Structural studies of acetylated and control inner core histones. Biochemistry. 1987 May 19;26(10):2944–2955. doi: 10.1021/bi00384a041. [DOI] [PubMed] [Google Scholar]
  16. Racker E., Stoeckenius W. Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphate formation. J Biol Chem. 1974 Jan 25;249(2):662–663. [PubMed] [Google Scholar]
  17. Sigrist H., Allegrini P. R., Stauffer K., Schaller J., Abdulaev N. G., Rickli E. E., Zahler P. Group-directed modification of bacteriorhodopsin by arylisothiocyanates. Labeling, identification of the binding site and topology. J Mol Biol. 1984 Feb 15;173(1):93–108. doi: 10.1016/0022-2836(84)90405-4. [DOI] [PubMed] [Google Scholar]
  18. Sigrist H., Wenger R. H., Kislig E., Wüthrich M. Refolding of bacteriorhodopsin. Protease V8 fragmentation and chromophore reconstitution from proteolytic V8 fragments. Eur J Biochem. 1988 Oct 15;177(1):125–133. doi: 10.1111/j.1432-1033.1988.tb14352.x. [DOI] [PubMed] [Google Scholar]
  19. Stern L. J., Khorana H. G. Structure-function studies on bacteriorhodopsin. X. Individual substitutions of arginine residues by glutamine affect chromophore formation, photocycle, and proton translocation. J Biol Chem. 1989 Aug 25;264(24):14202–14208. [PubMed] [Google Scholar]
  20. Subramaniam S., Marti T., Khorana H. G. Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: the blue form is inactive in proton translocation. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1013–1017. doi: 10.1073/pnas.87.3.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tilcock C. P., Fisher D. The interaction of phospholipid membranes with poly(ethylene glycol). Vesicle aggregation and lipid exchange. Biochim Biophys Acta. 1982 Jun 14;688(2):645–652. doi: 10.1016/0005-2736(82)90375-3. [DOI] [PubMed] [Google Scholar]
  22. Wuethrich M., Sigrist H. Peptide building blocks from bacteriorhodopsin: isolation and physicochemical characterization of two individual transmembrane segments. J Protein Chem. 1990 Apr;9(2):201–207. doi: 10.1007/BF01025310. [DOI] [PubMed] [Google Scholar]

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