Cell-free synthesis, functional refolding, and spectroscopic characterization of bacteriorhodopsin, an integral membrane protein.
Bacteriorhodopsin (bR) is an integral membrane protein which functions as a light-driven proton pump in Halobacterium halobium (also known as Halobacterium salinarium). The cell-free synthesis of bR in quantities sufficient for FTIR and NMR spectroscopy and the ability to selectively isotope label bR using aminoacylated suppressor tRNAs would provide a powerful approach for studying the role of specific amino acid residues. However, no integral membrane protein has yet been expressed in a cell-free system in quantities sufficient for such biophysical studies. We report the cell-free synthesis of bacterioopsin, its purification, its refolding in polar lipids from H. halobium, and its regeneration with all-trans-retinal to yield bacteriorhodopsin in a form functionally similar to bR in purple membrane. Importantly, the yields obtained from in vitro and in vivo expression are comparable. Functionality of the cell-free expressed bR is established using static and time-resolved absorption spectroscopy and FTIR difference spectroscopy.
Sonar
S
S
Physics Department, Boston University, Massachusetts 02215.
Patel
N
N
Fischer
W
W
Rothschild
K J
KJ
D001436:Bacteriorhodopsins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D002474:Cell-Free System; D019617:Halobacterium salinarum / Q000378:metabolism; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding; D017550:Spectroscopy, Fourier Transform Infrared
D001436:Bacteriorhodopsins