A poly(ethylene glycol) water-soluble conjugate of porin: refolding to the native state.
Porin, from Rhodabacter capsulatus, was chemically modified with methoxypoly(ethylene glycol) (m-PEG; molecular mass = 5000 Da) succinimidyl carbonate to yield methoxypoly(ethylene glycol)-porin (m-PEG-SC-Porin), as previously reported for bacteriorhodopsin [Sirokman, G., & Fasman, G. D. (1993) Protein Sci. 3, 1101-1170]. The m-poly(ethylene glycol)-porin (m-PEG-SC-Porin 50) conjugate, containing one poly(ethylene glycol) chain, was water soluble. The secondary structure of the conjugate in water was mainly random coil. Circular dichroism spectroscopy showed it was predominantly in the beta-pleated sheet structure in 0.6% octyltetraoxyethylene and 0.3 M LiCl, as was porin. A proteoliposome, containing the isolated porin conjugate, was prepared to measure permeability of the sugar stachyose. The m-PEG-SC-Porin 50 proteoliposome of porin maintained the permeability for the sugar, as did the proteoliposome of porin. The swelling rate of the conjugate versus the sugar was lower than it was for porin. This indicated that a pore in the conjugate exists but perhaps with a slightly different pore size. The refolding of the conjugate was studied by stepwise addition of trifluoroethanol (TFE) to lower the dielectric constant, simulating the insertion of porin into the membrane. An alpha-helical structure that did not exist in the native porin was formed with the m-PEG-SC-Porin 50, upon the addition of TFE, and the helicity increased with increasing concentrations of TFE. The m-PEG-SC-Porin 50 could be stepwise refolded to the native conformation, predominantly in the beta-sheet conformation, by the addition of hexafluoro-2-propanol in the 5-10% concentration range.(ABSTRACT TRUNCATED AT 250 WORDS)
Wei
J
J
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Fasman
G D
GD
D002463:Cell Membrane Permeability; D002473:Cell Wall / Q000737:chemistry; D002942:Circular Dichroism; D011092:Polyethylene Glycols; D018272:Porins / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000737:chemistry; D016354:Rhodobacter capsulatus; D012995:Solubility; D014270:Trifluoroethanol / Q000737:chemistry; D014867:Water
D018272:Porins; D011510:Proteolipids; C020485:proteoliposomes; D014867:Water; D011092:Polyethylene Glycols; D014270:Trifluoroethanol